WO2014005076A2 - Methods and biomarkers for detection of kidney disorders - Google Patents

Methods and biomarkers for detection of kidney disorders

Info

Publication number
WO2014005076A2
WO2014005076A2 PCT/US2013/048688 US2013048688W WO2014005076A2 WO 2014005076 A2 WO2014005076 A2 WO 2014005076A2 US 2013048688 W US2013048688 W US 2013048688W WO 2014005076 A2 WO2014005076 A2 WO 2014005076A2
Authority
WO
Grant status
Application
Patent type
Prior art keywords
mutation
gt
gene
method
kit
Prior art date
Application number
PCT/US2013/048688
Other languages
French (fr)
Other versions
WO2014005076A9 (en )
WO2014005076A3 (en )
Inventor
Friedhelm Hildebrandt
Original Assignee
The Regents Of The University Of Michigan
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/156Polymorphic or mutational markers
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/34Genitourinary disorders
    • G01N2800/347Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy

Abstract

Provided herein are compositions, kits, methods and biomarkers for detection and characterization and diagnosis of kidney disorders (e.g., one or more of nephronophthisis-related ciliopathies (NPHP-RC), congenital abnormalities of the kidney and urinary tract (CAKUT)), and karyomegalic interstitial nephritis (KIN)) in biological samples (e.g., tissue samples, blood samples, plasma samples, cell samples, serum samples).

Description

METHODS AND BIOMARKERS FOR DETECTION OF KIDNEY DISORDERS

This application claims priority to provisional applications 61/666,423, filed June 29, 2012 and 61/672,916, filed July 18, 2012, each of which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

Provided herein are compositions, kits, methods and biomarkers for detection and characterization and diagnosis of kidney disorders (e.g., one or more of nephronophthisis- related ciliopathies (NPHP-RC), congenital abnormalities of the kidney and urinary tract (CAKUT)), and karyomegalic interstitial nephritis (KIN)) in biological samples (e.g., tissue samples, blood samples, plasma samples, cell samples, serum samples).

BACKGROUND OF THE INVENTION

Nephronophthisis (NPHP) is a recessive cystic kidney disease that represents the most frequent genetic cause of end-stage kidney disease in the first three decades of life. NPHP- related ciliopathies (NPHP-RC) are single-gene recessive disorders that affect kidney, retina, brain and liver by prenatalonset dysplasia or by organ degeneration and fibrosis in early adulthood. Identification of recessive mutations in more than 10 different genes (NPHP1- NPHP10) (Attanasio et al, Nat Genet 39, 1018-1024 2007; Delous et al, Nat Genet 39, 875- 881 2007; Hildebrandt et al, Nat Genet 17, 149-153 1997; Mollet et al, Nat Genet 32, 300- 305 2002; Olbrich et al, Nat Genet 34, 455-459 2003; Otto et al, Am J Hum Genet 71, 1167-1171 2002; Otto, Nat Genet 37, 282-288 2005; Otto et al, Nat Genet 42, 840-850 2010a; Otto et al, Nat Genet 34, 413-420 2003; Otto et al, J. Am Soc Nephrol 19, 587-592 2008; Sayer et al, Nat Genet 38, 674-681 2006; Valente et al, Nat Genet 38, 623-625 2006) revealed that their gene products share localization at the primary cilia-centrosomes complex and mitotic spindle poles in a cell cycle dependent manner, characterizing them as retinal- renal "ciliopathies" (Ansley et al, Nature 425, 628-633 2003; Hildebrandt et al, N Engl J Med 364, 1533-1543 2011). Multiple signaling pathways downstream of cilia have been implicated in the disease mechanisms of NPHP-RC, including Wnt signaling (Germino, Nat Genet 37, 455-457 2005; Simons et al, Nat Genet 37, 537-543 2005) and Shh signaling (Huangfu and Anderson, Proc Natl Acad Sci U S A 102, 11325-11330 2005; Huangfu et al, Nature 426, 83-87 2003). However, despite convergence of ciliopathy pathogenesis at cilia and centrosomes it remains largely unknown what signaling pathways downstream of cilia and centrosome function operate in the disease mechanisms that generate the NPHP-RC phenotypes.

SUMMARY OF THE INVENTION

Provided herein are compositions, kits, methods and biomarkers for detection and characterization and diagnosis of kidney disorders (e.g., one or more of nephronophthisis- related ciliopathies (NPHP-RC), congenital abnormalities of the kidney and urinary tract (CAKUT)), and karyomegalic interstitial nephritis (KIN)) in biological samples (e.g., tissue samples, blood samples, plasma samples, cell samples, serum samples).

Embodiments of the present invention provide a kit for detecting gene variants associated with nephronophthisis-related ciliopathies (NPHP-RC) in a subject, comprising (e.g., consisting essentially of): a) a first NPHP-RC informative detection reagent for identification of one or more variants in a first gene selected from, for example, meiotic recombination 11 homo log (MRE11), zinc finger protein 423 (ZNF423), or centrosomal protein 164kDa (CEP 164); and b) a second NPHP-RC informative detection reagent for identification of one or more variants in a second gene selected from, for example, MRE11, ZNF423, CEP 164, wherein the second gene is different than the first gene. In some embodiments, the variations result in loss of function mutations in the one or more genes. In some embodiments, the MRE11 mutation is c. l897C>T (p.R633X), the ZNF423 mutation is selected from, for example, c.2738C>T, cl518delC, or c.3829C>T (p.P913L, p.P506fzX43, or p.H1277Y)), and the CEP164 mutations are selected from, for example, c.32A>C,

C.2770T, C.15730T, C.17260T, or c.4383A>G (p.Q11P, p.R93W, p.Q525X, p.R576X, and p.X1460W). In some embodiments, the determining comprises detecting variant nucleic acids or polypeptides (e.g., using a nucleic acid detection method selected from, for example, sequencing, amplification or hybridization). In some embodiments, the first gene is MREl 1 and the second gene is ZNF423 or CEP 164. In some embodiments, the first gene is CEP 164 and the second gene is ZNF423. In some embodiments, kits, uses and methods provide a third reagent that identifies variants in a third distinct gene. For example, in some embodiments, reagents identify MREl 1, ZNF423, and CEP164. In some embodiments, reagents identify one or more mutations (e.g., 1, 2, 3, 4, 5 or more) in the above described genes. In some embodiments, the first and second reagents are nucleotide probes that specifically bind to the variants. In some embodiments, the first and second reagents are antibodies that specifically bind to polypeptides encoded by the variants. In some embodiments, the first reagent is a pair of primers for amplifying the first gene and the second reagent is a pair of primers for amplifying the second gene. In some embodiments, the first and second reagents are sequence primers for sequencing the first and second genes.

Further embodiments provide methods and uses (e.g., utilizing the kits described herein) for detecting gene variants associated with nephronophthisis-related ciliopathies (NPHP-RC) in a subject; and b) diagnosing the subject with NPHP-RC when the variants are present in the sample. In some embodiments, the sample is, for example, a tissue sample, a cell sample, or a blood sample. In some embodiments, the determining comprises a computer implemented method. In some embodiments, the computer implemented method comprises analyzing variant information and displaying the information to a user. In some

embodiments, the method further comprises the step of treating the subject for NPHP-RC (e.g., under condition such that at least one symptom of NPHP-RC is diminished or eliminated).

Additional embodiments of the present invention provide a kit for detecting gene variants associated with congenital abnormalities of the kidney and urinary tract (CAKUT) in a subject, comprising (e.g., consisting essentially of): a) a first CAKUT informative detection reagent for identification of one or more variants in a first gene selected from, for example, fraser syndrome 1 (FRASl), FRASl related extracellular matrix protein 2 (FREM2), Ret proto-oncogene, (RET), or bone morphogenetic protein 4 (BMP4); and b) a second CAKUT informative detection reagent for identification of one or more variants in a second gene selected from, for example, FRASl, FREM1, RET, or BMP4, wherein the second gene is different than the first gene. In some embodiments, the FRASl mutation is c.776T>G, c.299G>T, c.981G>A, c.7861C>T, or C.112680A (p.L259R, p.D998Y, p.R3273H, p.R2621X, or p.H3757Q), the FREM2 mutation is C.7013C>T (p.T2338I), the RET mutation is c.2110G>T (p.V704F), and the BMP4 mutation is c.362A>G (p.H121R). In some embodiments, the first gene is FRASl and the second gene is FREM1, RET or BMP4. In some embodiments, the first gene is FREMl and the second gene is RET or BMP4. In some embodiments, the first gene is RET and the second gene is BMP4. In some embodiments, kits further comprise a third reagent that identifies variants in a third distinct gene and optionally a fourth reagent that identifies variants in a fourth distinct gene. For example, in some embodiments, reagents identify FRAS 1 , FREM2, and RET; FRAS 1 , FREM2, and BMP4; FREM2, RET, and BMP4; or FRASl, FREM2, RET and BMP4. In some embodiments, reagents identify one or more mutations (e.g., 1, 2, 3, 4, 5 or more) in the above described genes. In some embodiments, the present invention provides methods and uses for detecting gene variants associated with CAKUT, comprising a) contacting a sample from a subject with a variant detection assay (e.g., the above described kits), under conditions that the presence of a variant associated with CAKUT is determined; and b) diagnosing the subject with CAKUT when the variants are present in the sample.

In certain embodiments, the present invention provides kits, methods, and uses for identifying KIN in a subject. In some embodiments, the kits, methods and uses utilize a first KIN informative detection reagent for identification of a first mutation in a KAN gene; and optionally b) a second KIN informative detection reagent for identification of a second mutation in a KANl gene, wherein the second mutation is different than the first mutation (e.g., to identify, treat, or characterize KIN in a subject). In some embodiments, the first and second mutations are, for example, c. l234+2T>A, c.2036_7delGA, c.2245C>T,

C.1375+1G>A, c.2616delA, C.1606C>T, c.2878G>A, c.2120G>A, c.2786A>C, c.2611T>C, c.2774_5delTT, or c.2810G>A (e.g., c.l234+2T>A and c.2036_7delGA; c.l234+2T>A and C.22450T; C.1375+1G>A and c.2616delA; C.1606C>T and c.2786A>C; C.1606C>T and c.2878G>A; c.2611T>C and c.2878G>A; or c.2774_5delTT and c.2810G>A). In some embodiments, the first and second mutations encode a polypeptide selected from, for example, p.Arg679Thrfs*5, p.Arg749*, p.Asp873Thrfs* 17, p.Arg536*, p.Cys871Arg, p.Trp707*, p.Leu925Profs*25, p.Gln929Pro, p.Gly937Asp, p.Asp960Asn, or and a splice site mutation (e.g., a splice site mutation and p.Arg679Thrfs*5; a splice site mutation and p.Arg749*; a splice site mutation and p.Asp873Thrfs* 17; p.Arg536* and p.Gln929Pro;

p.Arg536* and p.Asp960Asn; p.Cys871Arg and p.Asp960Asn; or p.Leu925Profs*25 and p.Gly937Asp).

Additional embodiments will be apparent to persons skilled in the relevant art based on the teachings contained herein.

DESCRIPTION OF THE DRAWINGS

Figure 1 shows identification of recessive mutations in MRE11, ZNF423 and CEP 164 in NPHP-RC using homozygosity mapping and WER. Data regarding homozygosity mapping and mutations are shown for family A3471 with MRE11 mutation (A-B), family F874 with ZNF423 mutation (C-D), and family KKESHOOl with CEP 164 mutation (E-F). (A, C, E) Non-parametric lod scores (NPL) are plotted across the human genome in 3 families (A3471, F874 and KKESHOOl) with NPHP-RC. (B, D, F) Homozygous mutations detected in families with NPHP-RC. Family number (underlined), mutation (arrowheads) and predicted translational changes (in parenthesis) are indicated.

Figure 2 shows two ZNF423 mutations have dominant negative characteristics (A-D), ZNF423 mutation abrogates interaction with PARP1 (E), and ZNF423 directly interacts with the NPHPRC protein CEP290/NPHP6 (F-G). (A) Amino acid residues altered by NPHP-RC mutations in ZNF423 are drawn in relation to functional annotation of its 30 Zn-fingers. (B- D) S-phase index assay (fraction of transfected cells incorporating BrdU) for P19 cells transfected with either wildtype or mutant ZNF423. (B) Representative field of cells transfected with wildtype ZNF423 shows high frequency of BrdU+ FLAG+ double-positive cells. (C) ZNF423-H1277Y transfected cells exhibits fewer FLAG-positive cells and a lower proportion that are double positive. (D) S-phase index measured in duplicate transfections for each of three DNA preparations per construct. (E) ZNF423 interacts with PARPl . (F-G) ZNF423 directly interacts with CEP290/NPHP6. (F) A human fetal brain yeast two-hybrid library screened with human CEP290/NPHP6 (JAS2; aa 1917-2479) fused to the DNA- binding domain of GAL4 (pDEST32) identified ZNF423 as a direct interaction partner of CEP290/NPHP6. The interaction was confirmed using direct yeast two-hybrid assay where 1 and 2 represent colony growth of CEP290 bait with ZNF423 prey. (G) HEK293T were cotransfected with human V5 -tagged partial human V5-CEP290 clone and GFPtagged full-length human ZNF423 clone.

Figure 3 shows (A-D) expression of mutant CEP 164 in renal epithelial cells abrogates localization to centrosomes. (A) Immunofluorescence using a-SDCCAG8/NPHP10-CG antibody, labels both centrioles, whereas a-CEP164-ENR antibody demonstrates CEP 164 staining at the mother centriole only. (B) Inducible overexpression of N-terminally GFPtagged human full-length CEP 164 isoform 1 (NGFPCEP 164-WT) in IMCD3 cells demonstrates, in addition to a cytoplasmic expression pattern, localization at one of the two centrioles (inset, arrow heads). (C) In contrast, the centrosomal signal is abrogated upon overexpression of an N-terminally GFP-tagged truncated CEP 164 construct representing the mutation p.Q525X. (D) The number of centrosomes positive for CEP 164 is reduced upon overexpression of C-terminally GFPtagged human full-length CEP 164 isoform 1 (CGFP- hCEP164-WT), which mimics the mutation p.X1460fs57 that causes a read-through of the stop-codon X1460, adding 57 aberrant amino acid residues to the Cterminus of CEP164. (E- H) Knockdown of Cepl64 disrupts ciliary frequency. (E) Depletion of Cepl64 by siRNA (F) causes a ciliary defect in 3D spheroid growth assays. (G) Nuclei and cilia were scored within a single spheroid to generate ciliary frequencies. siCepl64 transfected cells manifest lower cilia frequencies (33%) compared to control transfected IMCD3 cells (49%). (H) Ciliary frequency is not rescued by mutant CEP 164. Ciliary frequencies are reduced in siCepl64 transfected IMCD3 cells (39%) compared to control siCtrl transfected IMCD3 cells (54%).

Figure 4 shows (A-P) colocalization upon immunofluorescence of the NPHP-RC proteins SDCCAG8/NPHP 10, ZNF423 and CEP 164 to nuclear foci that are positive for the DDR signaling proteins SC35, TIP60 and Chkl in hTERT-RPE cells. (A-G) Colocalization of NPHP-RC proteins with SC35 in nuclear foci. SDCCAG8/NPHP 10 (A-C) and ZNF423 (D) fully colocalize to nuclear foci with SC35, and (E) CEP164 partially colocalizes with SC35. SDCCAG8/ NPHP 10 also colocalizes with the newly identified NPHP-RC proteins ZNF423 (F) and CEP 164 (G). (H-J) Colocalization of NPHP-RC proteins with the DDR protein TIP60 I nuclear foci. (H) TIP60 fully colocalizes with SC35. (I) TIP60 partially colocalizes with CEP 164. (J) Chkl fully colocalizes with SC35/ SRSF2. DNA is stained in blue with DAPI. (K-P) Colocalization of DDR and NPHP proteins upon induction of DDR by UV radiation in HeLa cells. (K) Following irradiation of HeLa cells with UV light at 20 J/m2 a strong immunofluorescence signal of an

Figure imgf000007_0001
antibody indicates activation of DDR. (L-M) Upon irradiation with UV light, CEP164-positive nuclear foci condense and colocalize with newly appearing TIP60 foci of similar size. (N-O) In untreated cells (N) a pattern of broad CEP 164 speckles, which are CHK1 -negative and locate to DAPI-negative domains, changes to a pattern of multiple smaller foci (O) that are doublepositive for both, CEP 164- Ni l and CHK1. (P) p317-CHKl fully colocalizes with TIP60 to nuclear foci and to the centrosome.

Figure 5 shows knockdown of Cepl64 causes anaphase lag and retarded cell growth. (A-B) Knockdown of CEP 164 causes anaphase lag. (B). Bars represent SEM, p values (student T-test) are indicated above the bar graph. (C-D) Transient knockdown of Cepl64 inhibits proliferation, which is rescued by wild type but not mutant CEP 164. (C) IMCD3 cells depleted of murine Cepl64 grew more slowly (siRNA) than non-depleted cells (control) or the non-depleted cells induced to express human wild type CEP 164 (Dox). Expression of WT Cepl64 in siRNA-depleted cells rescued the slow growth phenotype of Cepl64 depletion (siRNA+dox). (D) As in C, except mutant Cepl64 cDNA (CEP164-Q525X) was expressed under doxycyclin control. (E-H) The effect of roscovitine on UV-induced DDR. (E-F)

Immunofluorescence analysis showed that roscovitine triggered uniform nuclear distribution of (activated H2AX phosphorylated at Serl39) in non UVirradiated cells showing

Figure imgf000007_0002

partial DDR activation (E). UV radiation caused enhanced γΗ2Αx staining with a prominent nuclear foci pattern, characteristic of strong DDR activation (F). (G-H) The effect of roscovitine on UV-triggered subcellular localization of CEP 164 and Chkl .

Figure 6 shows that knockdown of cepl64 in zebrafish embryos results in ciliopathy phenotypes, and knockdown of Cepl64 or Zfp423(Znf423) causes sensitivity to DNA damage. (A-E) Whereas p53 MO injection (n=67) did not produce any phenotype (A), coinjection of cepl64 MO at 28 hpf caused the mild ciliopathy phenotype of ventral body axis curvature in 48% of embryos (60/125) (B). 50%> of embryos (62/125) showed severe cell death throughout the body as judged by grey-appearing cells in the head region (C). Embryos with severe cell death also showed increased expression of phosphorylated γΗ2ΑΧ (D) compared to p53 MO control (E). Most embryos with massive cell death did not survive beyond 48 hpf. (F-I) At 48 hpf, surviving cepl64 morphants displayed the ciliopathy phenotype of laterality defects. Whereas p53 MO did not cause any abnormal heart looping (F,G), cepl64 MO caused inverted heart looping (H) or ambiguous heart looping (I). (A, atrium; L, left; V, ventricle). (J-M) At 72 hpf, embryos developed further ciliopathy phenotypes. When compared to p53 MO controls (J), pronephric tubules (arrow heads) exhibited cystic dilation (K, asterisks) in 25% (7/28) of embryos. In addition, when compared to p53 MO controls (L), 0% (0/67) of embryos showed kidney cysts, hydrocephalus

(asterisk), or retinal dysplasia (brackets) (M). (N) At 0.2 mM, cepl64 MO knockdown effectively altered mRNA processing as revealed by RT-PCR. (O) Quantification of γ-Η2ΑΧ levels in cepl64 MO morphants. (P-Q) Cepl64-deficient IMCD3 cells exhibit radiation sensitivity. (R-T) Zfp423(Znf423)-deficient P19 cells exhibit radiation sensitivity.

Figure 7 shows links of newly identified nephronophthisis-related ciliopathy (NPHP- RC) proteins to DNA damage response (DDR) signaling and cell cycle control. Columns depict two pathways of DDR signaling, the ATM (ataxia-telangiectasia mutated) pathway (A), and the ATR (ATM-and-Rad-related) pathway (C). Rows depict stages of DDR signaling including DNA damage sensing and repair (A, C), as well as outcomes regarding checkpoint activation, cell cycle arrest, and apoptosis (D).

Figure 8 shows alternative transcripts, knockdown targets, and human mutations of CEP 164 with interacting domains, interaction partners, and antigens of the Cepl64 protein. (A) The CEP 164 gene extends over 85.4 kb, contains 33 exons (vertical hatches) and an alternative exon 5 a used in isoform 2. (B) Exon structure of human CEP 164 cDNA. Positions of start codon (ATG) and of stop codon (TGA) are indicated. (C) Domain structure of the Cepl64 protein. (D) Minimal segment to which CEP 164 the interaction partner ATRIP has been mapped. (E) Extent of antigens used for a-CEP164 antibody production. (F) CEP 164 mutations detected in 4 families with NPHP-RC.

Figure 9 shows subcellular localization of CEP 164. (A) Characterization of anti- CEP164 antibodies by immunob lotting. (B) Characterization of a-CEP164 antibodies by overexpression of N-terminally GFP-labeled human isoform 1 wild type construct EGFP- CEP164-WT and immunofluorescence in cell lines. (C) CEP 164 in GFP-Centrin-2 mouse photoreceptors. (D-G) Expression of mutant CEP 164 in hTERT-RPE cells abrogates localization to centrosomes. (D) Doxycyclin (Dox)-inducible overexpression of N-terminally GFP-tagged human full-length CEP 164 wild type isoform 1 (NGFP-hCEP164-WT) in hTERT-RPE (human retinal pigment epithelium) cells demonstrates, in addition to a cytoplasmic expression pattern, localization at centrosomes. (E) In contrast, the signal at centrosomes is abrogated upon overexpression of an N-terminally GFP-tagged truncated CEP 164 construct (NGFP-CEP164-Q525X) representing the mutation p.Q525X occurring in NPHP-RC family F59. (F) Expression of GFP alone as a negative control yielded no centrosomal expression in IMCD3 or hTERTRPE cells. (G) Immunob lot of IMCD3 and hTERT-RPE cells expressing inducible GFP-CEP164 constructs confirmed doxycyclin- inducible NGFP-CEP164 fusion protein expression (~191 kDa) both with a-CEP164-NR and a-GFP antibody blotting (arrow heads).

Figure 10 shows subcellular localization of CEP 164 and SDCCAG8 by

immunofluorescence. (A) CEP 164 co localizes with the mother centriole (labeled with γ- tubulin), the mitotic spindle poles (actetylated tubulin), and the midbody throughout the cell cycle in hTERT-RPE cells. (B-D) Upon immunofluorescence (IF) in hTERT-RPE cells antibody a-SDCCAG8-CG recognizes nuclear foci that are absent upon transient

SDCCAG8/NPHP 10 knockdown. (B) hTERT cells transiently transfected with GFP-labeled negative control shRNA construct exhibit nuclear foci upon IF with a-SDCCAG8-CG (arrow heads in right panel), whereas in cells transfected with GFP-labeled SDCCAG8 shRNA knockdown constructs pGIPZ-259547 (C) or pGIPZ-90858 (D) nuclear foci are absent (asterisks in right panels of C and D), demonstrating specificity of the nuclear foci signal detected by a-SDCCAG8-CG.

Figure 11 shows (A-B) Immuo fluorescence imaging of the NPHP-RC protein

SDCC AG8/NPHP 10 with other proteins of nuclear foci in hTERT-RPE cells, and (C-J) identification of CCDC92 and TTKB2 as direct interaction partners of CEP 164. (A-B) SDCCAG8 labeled with antibody a-SDCCAG8-CG exhibits a similar number and size of nuclear foci in comparison to signals from antibodies against the nucler foci markers promyelocytic leukemia protein (PML) (A) and centromere protein C (CENP-C) (B). (C-J) Identification of CCDC92 and TTKB2 as direct interaction partners of CEP 164. (C) Four baits, BD-CEP164fi, BD-CEP1641-550, BD-CEP164551-1100, and BD-CEP1641101-1640 were used to screen two retinal cDNA libraries, a human oligo-dT primed library and a bovine randomly primed library, employing a GAL-4 based yeast two-hybrid system. (D) Lysates from COS-1 cells transfected with 3xFlag-CCDC92fl or 3xFlag-TTBK2fl were used in a pulldown assay of GST fusion proteins GST-CEP164fl (191kDa), GST-CEPl-550 (87kDa), GST-CEP164551-1100 (92kDa), and GST-CEP1641101-1640 (68kDa). (E-F) For co-immunoprecipitation 3xHA-CEP164fl in combination with 3xFlag-CCDC92fl were overexpressed in COS-1 cells (10% protein input shown). (G-H) For co-immunoprecipitation 3xHA-CEP164fl in combination with 3xFlag-TTBK2fl were overexpressed in COS-1 cells (10% protein input shown). (I- J) (I) The a-CCDC92 antibody signal fully colocalizes with a - CEP164-M26 at the mother centriole upon immunofluorescence imaging in hTERT-RPE cells. (J) TTBK2 weakly colocalizes with a -CEP164-M26 at one of the centrioles, but yields a strong signal at the mid body in dividing hTERT-RPE cells.

Figure 12 shows molecular interaction of NPHP-RC gene products with DDR proteins. (A-B) Interaction of NPHP3 with CEP164. (C-D) Interaction of NPHP2 with DDB1. (E-H) Cepl64 and Dvl3 are in a precipitable complex.

Figure 13 shows sequence chromatograms of 11 confirmed variants identified in 11 individuals with unilateral renal agenesis.

Figure 14 shows renal histology in individuals with KIN. (a-c) Renal histology of individuals with FAN1 mutation shows the characteristic triad of nephronophthisis, with cystic dilation of renal tubules (asterisks), interstitial infiltrations (dotted circle in a), and widespread fibrosis (b). Karyomegaly is observed (arrowheads in b and c) in tubules that have lost epithelial cells at their circumference (black arrowheads in b and c). The tubular basement membrane is thickened (double arrows in c) as well as attenuated (black arrow in c). a and b are from individual A4393-21; c is from individual A4466-21.

Figure 15 shows phenotypes of FAN 1 -mutant cells, (a) Protein blot analysis with antibody raised against the N-terminal 90 aa of FAN1. (b) Examples of metaphase spreads of the indicated cell lines after exposure to 50 nM MMC. (c,d) Sensitivity to ICL of the indicated FAN 1 -mutant (mut) cell lines in comparison to FANCA-mutant and wild-type cell lines. Primary fibroblasts (c) or LCLs (d) were treated in triplicate with increasing concentrations of MMC (left) or DEB (right), (e) Cell cycle analysis of the indicated fibroblast cell lines after treatment with 100 nM MMC or 0.1 μg/ml of DEB. Figure 16 shows complementation of FAN1 -mutant cells with FAN1 cDNA and epistasis analysis with genes implicated in ICL resistance, (a) Complementation of MMC sensitivity in fibroblasts of individual Al 170-22 with KIN. (b) Immunoblot showing expression of FANl mutants in Al 170-22 fibroblasts used in the MMC sensitivity assay in a. (c) MMC sensitivity in Al 170-22 fibroblasts transfected with the indicated small interfering RNAs (siRNAs) (d) Immunoblots of expression of the indicated proteins after siRNA- mediated knockdown in Al 170-22 fibroblasts from the experiment in c.

Figure 17 shows phenotypes caused by loss of fanl function in zebrafish. (a-c) Knockdown of fanl in zebrafish causes developmental abnormalities. (d,e) Knockdown of fanl in zebrafish induces widespread apoptosis. (f-h) Knockdown of fan1 on the background of p53 morphants reveals pronephric cysts.

Figure 18 shows differential expression of FANCD2 and FANl in human tissues and greater DDR in CKD. (a,b) Expression of FANCD2 and FAN1 in 25 different human tissues, (c-h) DDR is pronounced in CKD.

DEFINITIONS

To facilitate an understanding of the present invention, a number of terms and phrases are defined below:

As used herein, the term "sensitivity" is defined as a statistical measure of

performance of an assay (e.g., method, test), calculated by dividing the number of true positives by the sum of the true positives and the false negatives.

As used herein, the term "specificity" is defined as a statistical measure of

performance of an assay (e.g., method, test), calculated by dividing the number of true negatives by the sum of true negatives and false positives.

As used herein, the term "informative" or "informativeness" refers to a quality of a marker or panel of markers, and specifically to the likelihood of finding a marker (or panel of markers) in a positive sample.

As used herein, the terms "NPHP-RC informative reagent" "KIN informative reagent" or "CAKUT informative reagent" refers to reagents that are informative for identification of variants or mutations in one or more of the markers described herein. In some embodiments, reagents are primers, probes or antibodies for detection of mutant or variant alleles of the markers described herein.

As used herein, the term "adverse outcome" refers to an undesirable outcome in a patient diagnosed with NPHP-RC, KIN, or CAKUT. In some embodiments, the patient is undergoing or has undergone treatment for NPHP-RC, KIN, or CAKUT. Examples of adverse outcome include but are not limited to, recurrence of NPHP-RC, KIN or CAKUT, progression of disease, disability, or death.

As used herein, the term "amplicon" refers to a nucleic acid generated using one or more primers (e.g., two primers). The amplicon is typically single-stranded DNA (e.g., the result of asymmetric amplification), however, it may be RNA or dsDNA.

The term "amplifying" or "amplification" in the context of nucleic acids refers to the production of multiple copies of a polynucleotide, or a portion of the polynucleotide, typically starting from a small amount of the polynucleotide (e.g. , a single polynucleotide molecule), where the amplification products or amplicons are generally detectable.

As used herein, the term "primer" refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product that is complementary to a nucleic acid strand is induced (e.g., in the presence of nucleotides and an inducing agent such as a biocatalyst (e.g., a DNA polymerase or the like) and at a suitable temperature and pH). The primer is typically single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is generally first treated to separate its strands before being used to prepare extension products. In some embodiments, the primer is an

oligodeoxyribonucleotide. The primer is sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method. In certain embodiments, the primer is a capture primer.

A "sequence" of a biopolymer refers to the order and identity of monomer units (e.g., nucleotides, etc.) in the biopolymer. The sequence (e.g., base sequence) of a nucleic acid is typically read in the 5' to 3' direction.

As used herein, the term "subject" refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

As used herein, the term "non-human animals" refers to all non-human animals including, but are not limited to, vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc. The term "locus" as used herein refers to a nucleic acid sequence on a chromosome or on a linkage map and includes the coding sequence as well as 5 ' and 3 ' sequences involved in regulation of the gene. DETAILED DESCRIPTION OF THE INVENTION

Provided herein are compositions, kits, methods and biomarkers for detection and characterization and diagnosis of kidney disorders (e.g., one or more of nephronophthisis- related ciliopathies (NPHP-RC), congenital abnormalities of the kidney and urinary tract (CAKUT)), and karyomegalic interstitial nephritis (KIN)) in biological samples (e.g., tissue samples, blood samples, plasma samples, cell samples, serum samples).

Recessive null mutations in certain NPHP genes cause severe, congenital -onset phenotypes (Meckel syndrome) of dysplasia and malformation in kidney (polycystic dysplastic kidneys), eye (coloboma/microphthalmia), cerebellum (vermis hypoplasia in Joubert syndrome), and liver (cysts, ductal plate malformation), whereas hypomorphic mutations in the same gene cause late-onset, degenerative phenotypes such as renal tubular degeneration with fibrosis (nephronophthisis), retinal degeneration (Senior-Loken syndrome; SLSN), and liver fibrosis (Chaki et al, Kidney Int 2011; Hildebrandt et al, N Engl J Med 364, 1533-1543 2011). The more than 10 known NPHP genes explain less than 50% of all cases with NPHP-RC, and that many of the single-gene causes of NPHP-RC are still unknown (Otto et al, J. Med Genet 2010b). Some of the recently identified genetic causes of NPHP-RC are exceedingly rare (Attanasio et al, Nat Genet 39, 1018-1024 2007).

Embodiments of the present invention utilized homozygosity mapping with whole exome resequencing (WER) (Otto et al, Nat Genet 42, 840-850 2010a) to identify multiple different causes of NPHP-RC within a short time frame. Experiments described herein demonstrate that NPHP-RC proteins play a role in DDR signaling by demonstrating, i) mutation of MREl 1, ZNF423 and CEP 164 in NPHP-RC individuals; ii) abrogation by an NPHP-RC mutation of the ZNF423 interaction with the DDR protein PARP1; iii) colocalization of the centrosomal proteins ZNF423, CEP 164, and SDCC AG8/NPHP 10 to nuclear foci with the DDR protein TIP60; iv) cellular sensitivity to DNA damaging agents upon knockdown of ZNF423 or CEP 164; and v) occurrence of dysregulated DDR and an NPHP-RC phenotype in zebrafish upon knockdown of cepl64.

Further experiments conducted during the course of development of embodiments of the present invention identified seven different mutations of four different genes in 7 out of 40 patients with CAKUT, using massively parallel exon resequencing. Two heterozygous mutations were novel mutations in known human CAKUT-causing genes: BMP4 and RET. Five heterozygous mutations were in two candidate genes that have never been reported in nonsyndromic CAKUT: FRAS1 and FREM2. McGregor et al. (Nat Genet 2003; 34: 203- 208) and Vrontou et al. (Nat Genet 2003; 34: 209-214) identified recessive mutations in FRAS 1 using linkage analysis in families that were affected with Fraser syndrome, a rare multi-organ disorder characterized by cryptophthalmos, cutaneous syndactyly, and renal agenesis.

Frasl encodes Frasl, a protein containing repeats of the chondroitin sulfate proteoglycan core domain, the function of which is to maintain epithelial cell integrity. Frasl protein was detected in several developing tissues such as limb, lung, gut, and kidney. In metanephros, Frasl was detected in the extracellular matrix underlying the basal surface of outgrowing ureter and the basement membrane of collecting tubule. All FRASl mutations that have been reported in Fraser syndrome individuals were either homozygous or compound heterozygous, indicating a recessive genetic mechanism. Most mutations were truncating (McGregor et al, supra; Slavotinek et al, J Med Genet 2002; 39: 623-633; van Haelst et al, Am J Med Genet A 2008; 146A: 2252-2257; van Haelst et al., Am J Med Genet A 2007; 143A: 3194-3203).

Frem2, a FRASl -related extracellular matrix protein 2 gene, was identified as segregating in the mouse myUcl strain that showed phenotypes similar to that of

Frasl/mutant mice. Frem2 was expressed in mesonephric and metanephric epithelium, especially in the ureteric bud (Jadeja et al, Nat Genet 2005; 37: 520-52). Frem2 shares several structural domains with Frasl, including the core region of 12 chondroitin sulfate proteoglycans. Jadeja et al. (supra) detected a homozygous mutation in FREM2 in two families with Fraser syndrome not linked to FRASl . To date, there have been only two different homozygous mutations of FREM2 reported, representing three unrelated families with Fraser syndrome (Jadeja et al., supra; Shafeghati et al, Am J Med Genet A 2008; 146A: 529-531).

Nephronophthisis (NPHP)-related ciliopathies are a heterogeneous group of recessive diseases that cause CKD through chronic fibrosis and cyst development in the kidney

(Hildebrandt et al, N. Engl. J. Med. 364, 1533-1543 (2011)). To identify additional causative genes for NPHP, experiments described herein of homozygosity mapping (Hildebrandt, F. et al. PLoS Genet. 5, el000353 (2009)) and exome sequencing (Otto, E.A. et al.Nat. Genet. 42, 840-850 (2010)) in two siblings (from family Al 170) of Maori descent (Table 3) showed that renal histology in these individuals was indistinguishable from that associated with NPHP, except for the presence of enlarged nuclei, as seen in karyomegalic interstitial nephritis (KIN) (Fig. 14). Variants in FANCD2/FANCI-associated nuclease 1 (KANl) were identified as associated with KIN. KIN is a kidney disease with renal tubular degeneration of unknown origin that was first described in 1974 (Burry, J. Pathol. 113, 147-150 (1974)). KIN causes CKD through renal histological changes that are characteristic of NPHP2, including tubular basement membrane degeneration, atrophic tubules, tubular microcysts, interstitial infiltrations and pronounced fibrosis (Fig. 14a) (Zollinger, H.U. et al. Helv. Paediatr. Acta 35, 509-530 (1980)). The main feature that distinguishes KIN from NPHP is the presence of karyomegaly (Fig. 14b,c), which can also be present in the lung, liver and brain (Spoendlin, M. et al. Karyomegalic interstitial nephritis: further support for a distinct entity and evidence for a genetic defect. Am. J. Kidney Dis. 25, 242-252 (1995)). To date, only 12 families with KIN have been described (Palmer et al., Diagn. Cytopathol. 35, 179-182 (2007); Baba et al., Pathol. Res. Pract. 202, 555-559 (2006)), which is compatible with an autosomal recessive mode of inheritance. Variants of KANl include, but are not limited to, p.Trp707*, p.Gln929Pro, p.Gly937Asp and p.Asp960Asn.

Diagnostic and Screening Applications

Embodiments of the present invention provide diagnostic, prognostic, and screening compositions, kits, and methods. In some embodiments, compositions, kits, and methods characterize and diagnose NPHP-RC and/or CAKUT. Exemplary, non-limiting reagents and methods vof identifying mutations associated with NPHP-RC and/or CAKUT are described below.

A. Mutations

Embodiments of the present invention provide compositions and methods for detecting mutations in one or more genes (e.g., to identify or diagnose NPHP-RC and/or CAKUT). The present invention is not limited to particular mutations. In some embodiments, mutations are loss of function mutations (e.g., truncation, nonsense, missense, or frameshift mutations).

Exemplary mutations include, but are not limited to, mutations in MREl 1, ZNF423, and CEP 164 associated with HPHP-RC, FRAS1, FREM2, RET and BMP4 associated with CAKUT, and KANl associated with KIN. For example, in some embodiments, the MREl 1 mutation is c.1897C>T (p.R633X), the ZNF423 mutation is selected from, for example,

C.27380T, c1518delC, or c.3829C>T (p.P913L, p.P506fzX43, or p.H1277Y)), and the CEP164 mutations are selected from, for example, c.32A>C, c.277C>T, c.1573C>T,

C.17260T, or c.4383A>G (p.Q11P, p.R93W, p.Q525X, p.R576X, and p.X1460W), the FRASl mutation is c.776T>G, c.299G>T, c.981G>A, c.7861C>T, or C.112680A (p.L259R, p.D998Y, p.R3273H, p.R2621X, or p.H3757Q), the FREM2 mutation is C.7013C>T

(p.T2338I), the RET mutation is c.2110G>T (p.V704F), and the BMP4 mutation is c.362A>G (p.H121R). In some embodiments, the KANl variants include, but are not limited to, p.Trp707*, p.Gln929Pro, p.Gly937Asp and p.Asp960Asn.

While the present invention exemplifies several markers specific for detecting one or more of NPHP-RC, KIN, and CAKUT, any marker that is correlated with the presence or absence or prognosis of one or more of NPHP-RC, KIN, and CAKUT may be used. A marker, as used herein, includes, for example, nucleic acid(s) whose production or mutation or lack of production is characteristic of one or more of NPHP-RC, KIN, and CAKUT and mutations that cause the same effect (e.g., deletions, truncations, etc).

In some embodiments, one or more (e.g., 2, 3, 4, or all) of the above-described mutations are identified in order to diagnose or characterize one or more of NPHP-RC, KIN, and CAKUT. In some embodiments, mutations are identified in combination with one or more additional markers of one or more of NPHP-RC, KIN, and CAKUT. In some embodiments, multiple markers are detected in a panel or multiplex format.

For example, in some embodiments, one or more of the following combinations is utilized to identify NPHP-RC: a) one or more mutations in MREl 1 and one or more mutations in ZNF423, one or more mutations in MREl 1 in combination with one or more mutations in CEP 164, one or more mutations in ZNF423 in combination with one or more mutations in CEP 164, and one or more mutations in MREl 1 in combination with one or more mutations in ZNF423 and one or more mutations in CEP 164.

In some embodiments, one or more of the following combinations is utilized to identify CAKUT: a) one or more mutations in FRASl and one or more mutations in FREM2, one or more mutations in FRASl in combination with one or more mutations in RET, one or more mutations in FRAS 1 in combination with one or more mutations in BMP4, one or more mutations in FREM2 and one or more mutations in RET, one or more mutations in FREM2 in combination with one or more mutations in BMP4, one or more mutations in RET in combination with one or more mutations in BMP4 , one or more mutations in FRAS 1 in combination with one or more mutations in FREM2 and one or more mutations in RET, one or more mutations in FRAS 1 in combination with one or more mutations in FREM2 and one or more mutations in BMP4, one or more mutations in BMP4 in combination with one or more mutations in FREM2 and one or more mutations in RET, and one or more mutations in FRAS1 in combination with one or more mutations in FREM2, one or more mutations in RET, and one or more mutations in BMP4. One or more (e.g., 1, 2, 3, 4, 5, or all) mutations are assayed in each gene, alone or in combination.

In some embodiments, one or more (e.g., 1, 2, 3, 4, or all) KAN1 variants including, but are not limited to, c. l234+2T>A, c.2036_7delGA, c.2245C>T, C.1375+1G>A, c.2616delA, C.1606C>T, c.2878G>A, c.2120G>A, c.2786A>C, c.2611T>C, c.2774_5delTT, or c.2810G>A (e.g., c. l234+2T>A and c.2036_7delGA; c.l234+2T>A and c.2245C>T; C.1375+1G>A and c.2616delA; C.1606C>T and c.2786A>C; C.1606C>T and c.2878G>A; C.2611T>C and c.2878G>A; or c.2774_5delTT and c.2810G>A). In some embodiments, the varaints encode a polypeptide selected from, for example, p.Arg679Thrfs*5, p.Arg749*, p.Asp873Thrfs* 17, p.Arg536*, p.Cys871Arg, p.Trp707*, p.Leu925Profs*25, p.Gln929Pro, p.Gly937Asp, p.Asp960Asn, or and a splice site mutation (e.g., a splice site mutation and p.Arg679Thrfs*5; a splice site mutation and p.Arg749*; a splice site mutation and p.Asp873Thrfs* 17; p.Arg536* and p.Gln929Pro; p.Arg536* and p.Asp960Asn; p.Cys871Arg and p.Asp960Asn; or p.Leu925Profs*25 and p.Gly937Asp). In some embodiments, one or more of the KANl variants in Table 3 is used to identify KIN.

Particular combinations of markers may be used that show optimal function with different ethnic groups or sex, different geographic distributions, different stages of disease, different degrees of specificity or different degrees of sensitivity. Particular combinations may also be developed which are particularly sensitive to the effect of therapeutic regimens on disease progression. Subjects may be monitored after a therapy and/or course of action to determine the effectiveness of that specific therapy and/or course of action. B. Detection of Variant Alleles

In some embodiments, the present invention provides methods of detecting the presence of wild type or variant (e.g., mutant or polymorphic) nucleic acids or polypeptides. The detection of variant alleles finds use in the diagnosis of disease (e.g., one or more of NPHP-RC, KIN, and CAKUT), research, and selection of appropriate treatment and/or monitoring regimens.

Accordingly, the present invention provides compositions, kits, and methods for determining whether a patient has a mutation profile associated with one or more of NPHP- RC, KIN, and CAKUT. A number of methods maybe be used for analysis of variant (e.g., mutant or polymorphic) nucleic acid sequences. Assays for detecting variants (e.g., polymorphisms or mutations) fall into several categories, including, but not limited to direct sequencing assays, fragment polymorphism assays, hybridization assays, and computer based data analysis. In some embodiments, assays are performed in combination or in hybrid (e.g., different reagents or technologies from several assays are combined to yield one assay). The following assays are useful in the present invention.

Any patient sample containing nucleic acids or polypeptides may be tested according to the methods of the present invention. By way of non-limiting examples, the sample may be tissue, blood, urine, semen, or a fraction thereof (e.g., plasma, serum, whole blood, cells, etc.).

The patient sample may undergo preliminary processing designed to isolate or enrich the sample for the variant nucleic acids or polypeptides or cells that contain the variant nucleic acids. Centrifugation; immunocapture; cell lysis; sequence capture; and, nucleic acid target capture maybe used for such purposes. i. DNA and RNA Detection

The variants of the present invention may be detected as genomic DNA or mRNA with techniques including but not limited to: nucleic acid sequencing; nucleic acid hybridization; and, nucleic acid amplification.

1. Sequencing

Illustrative non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. Those of ordinary skill in the art will recognize that because RNA is less stable in the cell and more prone to nuclease attack experimentally RNA is usually reverse transcribed to DNA before sequencing.

Chain terminator sequencing uses sequence-specific termination of a DNA synthesis reaction using modified nucleotide substrates. Extension is initiated at a specific site on the template DNA by using a short radioactive, fluorescent or other labeled, oligonucleotide primer complementary to the template at that region. The oligonucleotide primer is extended using a DNA polymerase, standard four deoxynucleotide bases, and a low concentration of one chain terminating nucleotide, most commonly a di-deoxynucleotide. This reaction is repeated in four separate tubes with each of the bases taking turns as the di-deoxynucleotide. Limited incorporation of the chain terminating nucleotide by the DNA polymerase results in a series of related DNA fragments that are terminated only at positions where that particular di-deoxynucleotide is used. For each reaction tube, the fragments are size-separated by electrophoresis in a slab polyacrylamide gel or a capillary tube filled with a viscous polymer. The sequence is determined by reading which lane produces a visualized mark from the labeled primer as you scan from the top of the gel to the bottom.

Dye terminator sequencing alternatively labels the terminators. Complete sequencing can be performed in a single reaction by labeling each of the di-deoxynucleotide chain- terminators with a separate fluorescent dye, which fluoresces at a different wavelength.

Some embodiments of the present invention utilize next generation or high- throughput sequencing. A variety of nucleic acid sequencing methods are contemplated for use in the methods of the present disclosure including, for example, chain terminator (Sanger) sequencing, dye terminator sequencing, and high-throughput sequencing methods. See, e.g., Sanger et al, Proc. Natl. Acad. Sci. USA 74:5463-5467 (1997); Maxam et al, Proc. Natl. Acad. Sci. USA 74:560-564 (1977); Drmanac, et al, Nat. Biotechnol. 16:54-58 (1998); Kato, Int. J. Clin. Exp. Med. 2: 193-202 (2009); Ronaghi et al, Anal. Biochem. 242:84-89 (1996); Margulies et al, Nature 437:376-380 (2005); Ruparel et al, Proc. Natl. Acad. Sci. USA 102:5932-5937 (2005), and Harris et al, Science 320: 106-109 (2008); Levene et al, Science 299:682-686 (2003); Korlach et al, Proc. Natl. Acad. Sci. USA 105: 1176-1181 (2008);

Branton et al, Nat. Biotechnol. 26(10): 1146-53 (2008); Eid et al, Science 323: 133-138 (2009); each of which is herein incorporated by reference in its entirety.

In some embodiments, sequencing technology including, but not limited to, pyrosequencing, sequencing-by-ligation, single molecule sequencing, sequence-by-synthesis (SBS), massive parallel clonal, massive parallel single molecule SBS, massive parallel single molecule real-time, massive parallel single molecule real-time nanopore technology, etc. Morozova and Marra provide a review of some such technologies in Genomics, 92: 255 (2008), herein incorporated by reference in its entirety. Those of ordinary skill in the art will recognize that because RNA is less stable in the cell and more prone to nuclease attack experimentally RNA is usually reverse transcribed to DNA before sequencing.

A number of DNA sequencing techniques may be utilized, including fluorescence- based sequencing methodologies (See, e.g., Birren et al, Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor, N.Y.; herein incorporated by reference in its entirety). In some embodiments, the sequence techniques are parallel sequencing of partitioned amplicons (PCT Publication No: WO2006084132 to Kevin McKernan et al., herein incorporated by reference in its entirety). In some embodiments, the technology finds use in DNA sequencing by parallel oligonucleotide extension (See, e.g., U.S. Pat. No. 5,750,341 to Macevicz et al, and U.S. Pat. No. 6,306,597 to Macevicz et al., both of which are herein incorporated by reference in their entireties). Additional examples of sequencing techniques in which the technology finds use include the Church polony technology (Mitra et al., 2003, Analytical Biochemistry 320, 55-65; Shendure et al, 2005 Science 309, 1728-1732; U.S. Pat. No.

6,432,360, U.S. Pat. No. 6,485,944, U.S. Pat. No. 6,511,803; herein incorporated by reference in their entireties), the 454 picotiter pyrosequencing technology (Margulies et al, 2005 Nature 437, 376-380; US 20050130173; herein incorporated by reference in their entireties), the Solexa single base addition technology (Bennett et al, 2005, Pharmacogenomics, 6, 373- 382; U.S. Pat. No. 6,787,308; U.S. Pat. No. 6,833,246; herein incorporated by reference in their entireties), the Lynx massively parallel signature sequencing technology (Brenner et al. (2000). Nat. Biotechnol. 18:630-634; U.S. Pat. No. 5,695,934; U.S. Pat. No. 5,714,330;

herein incorporated by reference in their entireties), and the Adessi PCR colony technology (Adessi et al. (2000). Nucleic Acid Res. 28, E87; WO 00018957; herein incorporated by reference in its entirety).

Next-generation sequencing (NGS) methods share the common feature of massively parallel, high-throughput strategies, with the goal of lower costs in comparison to older sequencing methods (see, e.g., Voelkerding et al, Clinical Chem., 55: 641-658, 2009;

MacLean et al., Nature Rev. Microbiol., 7: 287-296; each herein incorporated by reference in their entirety). NGS methods can be broadly divided into those that typically use template amplification and those that do not. Amplification-requiring methods include pyrosequencing commercialized by Roche as the 454 technology platforms (e.g., GS 20 and GS FLX), the Solexa platform commercialized by Illumina, and the Supported Oligonucleotide Ligation and Detection (SOLiD) platform commercialized by Applied Biosystems. Non-amplification approaches, also known as single-molecule sequencing, are exemplified by the HeliScope platform commercialized by Helicos Biosciences, and emerging platforms commercialized by VisiGen, Oxford Nanopore Technologies Ltd., Life Technologies/Ion Torrent, and Pacific Biosciences, respectively.

In pyrosequencing (Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al, Nature Rev. Microbiol, 7: 287-296; U.S. Pat. No. 6,210,891; U.S. Pat. No. 6,258,568; each herein incorporated by reference in its entirety), template DNA is fragmented, end- repaired, ligated to adaptors, and clonally amplified in-situ by capturing single template molecules with beads bearing oligonucleotides complementary to the adaptors. Each bead bearing a single template type is compartmentalized into a water-in-oil microvesicle, and the template is clonally amplified using a technique referred to as emulsion PCR. The emulsion is disrupted after amplification and beads are deposited into individual wells of a picotitre plate functioning as a flow cell during the sequencing reactions. Ordered, iterative

introduction of each of the four dNTP reagents occurs in the flow cell in the presence of sequencing enzymes and luminescent reporter such as luciferase. In the event that an appropriate dNTP is added to the 3' end of the sequencing primer, the resulting production of ATP causes a burst of luminescence within the well, which is recorded using a CCD camera. It is possible to achieve read lengths greater than or equal to 400 bases, and 106 sequence reads can be achieved, resulting in up to 500 million base pairs (Mb) of sequence.

In the Solexa/Illumina platform (Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. No. 6,833,246; U.S. Pat. No. 7,115,400; U.S. Pat. No. 6,969,488; each herein incorporated by reference in its entirety), sequencing data are produced in the form of shorter-length reads. In this method, single- stranded fragmented DNA is end-repaired to generate 5'-phosphorylated blunt ends, followed by Klenow-mediated addition of a single A base to the 3' end of the fragments. A-addition facilitates addition of T-overhang adaptor oligonucleotides, which are subsequently used to capture the template-adaptor molecules on the surface of a flow cell that is studded with oligonucleotide anchors. The anchor is used as a PCR primer, but because of the length of the template and its proximity to other nearby anchor oligonucleotides, extension by PCR results in the "arching over" of the molecule to hybridize with an adjacent anchor oligonucleotide to form a bridge structure on the surface of the flow cell. These loops of DNA are denatured and cleaved. Forward strands are then sequenced with reversible dye terminators. The sequence of incorporated nucleotides is determined by detection of post-incorporation fluorescence, with each fluor and block removed prior to the next cycle of dNTP addition. Sequence read length ranges from 36 nucleotides to over 50 nucleotides, with overall output exceeding 1 billion nucleotide pairs per analytical run.

Sequencing nucleic acid molecules using SOLiD technology (Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. No. 5,912,148; U.S. Pat. No. 6,130,073; each herein incorporated by reference in their entirety) also involves fragmentation of the template, ligation to oligonucleotide adaptors, attachment to beads, and clonal amplification by emulsion PCR. Following this, beads bearing template are immobilized on a derivatized surface of a glass flow-cell, and a primer complementary to the adaptor oligonucleotide is annealed. However, rather than utilizing this primer for 3' extension, it is instead used to provide a 5' phosphate group for ligation to interrogation probes containing two probe-specific bases followed by 6 degenerate bases and one of four fluorescent labels. In the SOLiD system, interrogation probes have 16 possible combinations of the two bases at the 3' end of each probe, and one of four fluors at the 5' end. Fluor color, and thus identity of each probe, corresponds to specified color-space coding schemes. Multiple rounds (usually 7) of probe annealing, ligation, and fluor detection are followed by denaturation, and then a second round of sequencing using a primer that is offset by one base relative to the initial primer. In this manner, the template sequence can be computationally re-constructed, and template bases are interrogated twice, resulting in increased accuracy. Sequence read length averages 35 nucleotides, and overall output exceeds 4 billion bases per sequencing run.

In certain embodiments, the technology finds use in nanopore sequencing (see, e.g., Astier et al, J. Am. Chem. Soc. 2006 Feb 8; 128(5): 1705-10, herein incorporated by reference). The theory behind nanopore sequencing has to do with what occurs when a nanopore is immersed in a conducting fluid and a potential (voltage) is applied across it. Under these conditions a slight electric current due to conduction of ions through the nanopore can be observed, and the amount of current is exceedingly sensitive to the size of the nanopore. As each base of a nucleic acid passes through the nanopore, this causes a change in the magnitude of the current through the nanopore that is distinct for each of the four bases, thereby allowing the sequence of the DNA molecule to be determined.

In certain embodiments, the technology finds use in HeliScope by Helicos

Biosciences (Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. No. 7,169,560; U.S. Pat. No. 7,282,337; U.S. Pat. No. 7,482,120; U.S. Pat. No. 7,501,245; U.S. Pat. No. 6,818,395; U.S. Pat. No. 6,911,345; U.S. Pat. No. 7,501,245; each herein incorporated by reference in their entirety). Template DNA is fragmented and polyadenylated at the 3' end, with the final adenosine bearing a fluorescent label. Denatured polyadenylated template fragments are ligated to poly(dT) oligonucleotides on the surface of a flow cell. Initial physical locations of captured template molecules are recorded by a CCD camera, and then label is cleaved and washed away. Sequencing is achieved by addition of polymerase and serial addition of fluorescently-labeled dNTP reagents. Incorporation events result in fluor signal corresponding to the dNTP, and signal is captured by a CCD camera before each round of dNTP addition. Sequence read length ranges from 25-50 nucleotides, with overall output exceeding 1 billion nucleotide pairs per analytical run. The Ion Torrent technology is a method of DNA sequencing based on the detection of hydrogen ions that are released during the polymerization of DNA (see, e.g., Science

327(5970): 1190 (2010); U.S. Pat. Appl. Pub. Nos. 20090026082, 20090127589,

20100301398, 20100197507, 20100188073, and 20100137143, incorporated by reference in their entireties for all purposes). A microwell contains a template DNA strand to be sequenced. Beneath the layer of microwells is a hypersensitive ISFET ion sensor. All layers are contained within a CMOS semiconductor chip, similar to that used in the electronics industry. When a dNTP is incorporated into the growing complementary strand a hydrogen ion is released, which triggers a hypersensitive ion sensor. If homopolymer repeats are present in the template sequence, multiple dNTP molecules will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal. This technology differs from other sequencing technologies in that no modified nucleotides or optics are used. The per-base accuracy of the Ion Torrent sequencer is -99.6% for 50 base reads, with -100 Mb generated per run. The read-length is 100 base pairs. The accuracy for homopolymer repeats of 5 repeats in length is -98%. The benefits of ion semiconductor sequencing are rapid sequencing speed and low upfront and operating costs.

The technology finds use in another nucleic acid sequencing approach developed by Stratos Genomics, Inc. and involves the use of Xpandomers. This sequencing process typically includes providing a daughter strand produced by a template-directed synthesis. The daughter strand generally includes a plurality of subunits coupled in a sequence

corresponding to a contiguous nucleotide sequence of all or a portion of a target nucleic acid in which the individual subunits comprise a tether, at least one probe or nucleobase residue, and at least one selectively cleavable bond. The selectively cleavable bond(s) is/are cleaved to yield an Xpandomer of a length longer than the plurality of the subunits of the daughter strand. The Xpandomer typically includes the tethers and reporter elements for parsing genetic information in a sequence corresponding to the contiguous nucleotide sequence of all or a portion of the target nucleic acid. Reporter elements of the Xpandomer are then detected. Additional details relating to Xpandomer-based approaches are described in, for example, U.S. Pat. Pub No. 20090035777, entitled "High Throughput Nucleic Acid Sequencing by Expansion," filed June 19, 2008, which is incorporated herein in its entirety.

Other single molecule sequencing methods include real-time sequencing by synthesis using a VisiGen platform (Voelkerding et al., Clinical Chem., 55: 641-58, 2009; U.S. Pat. No. 7,329,492; U.S. Pat. App. Ser. No. 11/671956; U.S. Pat. App. Ser. No. 11/781166; each herein incorporated by reference in their entirety) in which immobilized, primed DNA template is subjected to strand extension using a fluorescently-modified polymerase and florescent acceptor molecules, resulting in detectible fluorescence resonance energy transfer (FRET) upon nucleotide addition.

In some embodiments, capillary electrophoresis (CE) is utilized to analyze amplification fragments. During capillary electrophoresis, nucleic acids (e.g., the products of a PCR reaction) are injected electrokinetically into capillaries filled with polymer. High voltage is applied so that the fluorescent DNA fragments are separated by size and are detected by a laser/camera system. In some embodiments, CE systems from Life Technogies (Grand Island, NY) are utilized for fragment sizing (See e.g., US 6706162, US8043493, each of which is herein incorporated by reference in its entirety).

2. Hybridization

Illustrative non-limiting examples of nucleic acid hybridization techniques include, but are not limited to, in situ hybridization (ISH), microarray, and Southern or Northern blot. In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA or RNA strand as a probe to localize a specific DNA or RNA sequence in a portion or section of tissue (in situ), or, if the tissue is small enough, the entire tissue (whole mount ISH). DNA ISH can be used to determine the structure of chromosomes. RNA ISH is used to measure and localize mRNAs and other transcripts within tissue sections or whole mounts. Sample cells and tissues are usually treated to fix the target transcripts in place and to increase access of the probe. The probe hybridizes to the target sequence at elevated temperature, and then the excess probe is washed away. The probe that was labeled with either radio-, fluorescent- or antigen-labeled bases is localized and quantitated in the tissue using either

autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes, labeled with radioactivity or the other non-radioactive labels, to simultaneously detect two or more transcripts.

3. Microarrays

In some embodiments, microarrays are utilized for detection of variant nucleic acid sequences. Examples of microarrays include, but not limited to: DNA microarrays (e.g., cDNA microarrays and oligonucleotide microarrays); protein microarrays; tissue

microarrays; transfection or cell microarrays; chemical compound microarrays; and, antibody microarrays. A DNA microarray, commonly known as gene chip, DNA chip, or biochip, is a collection of microscopic DNA spots attached to a solid surface (e.g., glass, plastic or silicon chip) forming an array for the purpose of expression profiling or monitoring expression levels for thousands of genes simultaneously. The affixed DNA segments are known as probes, thousands of which can be used in a single DNA microarray. Microarrays can be used to identify disease genes by comparing gene expression in disease and normal cells.

Microarrays can be fabricated using a variety of technologies, including but not limiting: printing with fine -pointed pins onto glass slides; photolithography using pre-made masks; photolithography using dynamic micromirror devices; ink-jet printing; or, electrochemistry on microelectrode arrays.

Arrays can also be used to detect copy number variations at al specific locus. These genomic micorarrys detect microscopic deletions or other variants that lead to disease causing alleles.

Southern and Northern blotting is used to detect specific DNA or RNA sequences, respectively. DNA or RNA extracted from a sample is fragmented, electrophoretically separated on a matrix gel, and transferred to a membrane filter. The filter bound DNA or RNA is subject to hybridization with a labeled probe complementary to the sequence of interest. Hybridized probe bound to the filter is detected. A variant of the procedure is the reverse Northern blot, in which the substrate nucleic acid that is affixed to the membrane is a collection of isolated DNA fragments and the probe is RNA extracted from a tissue and labeled.

4. Amplification

Variant nucleic acid may be amplified prior to or simultaneous with detection.

Illustrative non-limiting examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), transcription-mediated amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence based amplification (NASBA). Those of ordinary skill in the art will recognize that certain amplification techniques (e.g., PCR) require that RNA be reversed transcribed to DNA prior to amplification (e.g., RT-PCR), whereas other amplification techniques directly amplify RNA (e.g., TMA and NASBA).

The polymerase chain reaction (U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159 and 4,965,188, each of which is herein incorporated by reference in its entirety), commonly referred to as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase copy numbers of a target nucleic acid sequence. In a variation called RT-PCR, reverse transcriptase (RT) is used to make a complementary DNA (cDNA) from mRNA, and the cDNA is then amplified by PCR to produce multiple copies of DNA. For other various permutations of PCR see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159; Mullis et al, Meth. Enzymol. 155: 335 (1987); and, Murakawa et al., DNA 7: 287 (1988), each of which is herein incorporated by reference in its entirety.

Transcription mediated amplification (U.S. Pat. Nos. 5,480,784 and 5,399,491, each of which is herein incorporated by reference in its entirety), commonly referred to as TMA, synthesizes multiple copies of a target nucleic acid sequence autocatalytically under conditions of substantially constant temperature, ionic strength, and pH in which multiple RNA copies of the target sequence autocatalytically generate additional copies. See, e.g., U.S. Pat. Nos. 5,399,491 and 5,824,518, each of which is herein incorporated by reference in its entirety. In a variation described in U.S. Publ. No. 20060046265 (herein incorporated by reference in its entirety), TMA optionally incorporates the use of blocking moieties, terminating moieties, and other modifying moieties to improve TMA process sensitivity and accuracy.

The ligase chain reaction (Weiss, R., Science 254: 1292 (1991), herein incorporated by reference in its entirety), commonly referred to as LCR, uses two sets of complementary DNA oligonucleotides that hybridize to adjacent regions of the target nucleic acid. The DNA oligonucleotides are covalently linked by a DNA ligase in repeated cycles of thermal denaturation, hybridization and ligation to produce a detectable double-stranded ligated oligonucleotide product.

Strand displacement amplification (Walker, G. et al., Proc. Natl. Acad. Sci. USA 89: 392-396 (1992); U.S. Pat. Nos. 5,270,184 and 5,455,166, each of which is herein

incorporated by reference in its entirety), commonly referred to as SDA, uses cycles of annealing pairs of primer sequences to opposite strands of a target sequence, primer extension in the presence of a dNTPaS to produce a duplex hemiphosphorothioated primer extension product, endonuclease-mediated nicking of a hemimodified restriction endonuclease recognition site, and polymerase-mediated primer extension from the 3' end of the nick to displace an existing strand and produce a strand for the next round of primer annealing, nicking and strand displacement, resulting in geometric amplification of product.

Thermophilic SDA (tSDA) uses thermophilic endonucleases and polymerases at higher temperatures in essentially the same method (EP Pat. No. 0 684 315). Other amplification methods include, for example: nucleic acid sequence based amplification (U.S. Pat. No. 5,130,238, herein incorporated by reference in its entirety), commonly referred to as NASBA; one that uses an RNA replicase to amplify the probe molecule itself (Lizardi et al., BioTechnol. 6: 1197 (1988), herein incorporated by reference in its entirety), commonly referred to as Qβ replicase; a transcription based amplification method (Kwoh et al, Proc. Natl. Acad. Sci. USA 86: 1173 (1989)); and, self-sustained sequence replication (Guatelli et al, Proc. Natl. Acad. Sci. USA 87: 1874 (1990), each of which is herein incorporated by reference in its entirety). For further discussion see Persing, David H., "In Vitro Nucleic Acid Amplification Techniques" in Diagnostic Medical

Microbiology: Principles and Applications (Persing et al, Eds.), pp. 51-87 (American Society for Microbiology, Washington, DC (1993)).

5. Detection Methods

Non-amplified or amplified nucleic acids can be detected by a variety of techniques. For example, nucleic acid can be detected by hybridization with a detectably labeled probe and measurement of the resulting hybrids. Illustrative non-limiting examples of detection methods are described below.

One illustrative detection method, the Hybridization Protection Assay (HP A) involves hybridizing a chemiluminescent oligonucleotide probe (e.g., an acridinium ester-labeled (AE) probe) to the target sequence, selectively hydrolyzing the chemiluminescent label present on unhybridized probe, and measuring the chemiluminescence produced from the remaining probe in a luminometer. See, e.g., U.S. Pat. No. 5,283,174 and Norman C. Nelson et al, Nonisotopic Probing, Blotting, and Sequencing, ch. 17 (Larry J. Kricka ed., 2d ed. 1995, each of which is herein incorporated by reference in its entirety).

Another illustrative detection method provides for quantitative evaluation of the amplification process in real-time. Evaluation of an amplification process in "real-time" involves determining the amount of amplicon in the reaction mixture either continuously or periodically during the amplification reaction, and using the determined values to calculate the amount of target sequence initially present in the sample. The amount of initial target sequence present in a sample based on real-time amplification may be determined using methods including those disclosed in U.S. Pat. Nos. 6,303,305 and 6,541,205, each of which is herein incorporated by reference in its entirety. Another method for determining the quantity of target sequence initially present in a sample, but which is not based on a real-time amplification, is disclosed in U.S. Pat. No. 5,710,029, herein incorporated by reference in its entirety.

Amplification products may be detected in real-time through the use of various self- hybridizing probes, most of which have a stem-loop structure. Such self-hybridizing probes are labeled so that they emit differently detectable signals, depending on whether the probes are in a self-hybridized state or an altered state through hybridization to a target sequence. By way of non-limiting example, "molecular torches" are a type of self-hybridizing probe that includes distinct regions of self-complementarity (referred to as "the target binding domain" and "the target closing domain") which are connected by a joining region (e.g., non- nucleotide linker) and which hybridize to each other under predetermined hybridization assay conditions. In a preferred embodiment, molecular torches contain single-stranded base regions in the target binding domain that are from 1 to about 20 bases in length and are accessible for hybridization to a target sequence present in an amplification reaction under strand displacement conditions. Under strand displacement conditions, hybridization of the two complementary regions, which may be fully or partially complementary, of the molecular torch is favored, except in the presence of the target sequence, which will bind to the single- stranded region present in the target binding domain and displace all or a portion of the target closing domain. The target binding domain and the target closing domain of a molecular torch include a detectable label or a pair of interacting labels (e.g., luminescent/quencher) positioned so that a different signal is produced when the molecular torch is self-hybridized than when the molecular torch is hybridized to the target sequence, thereby permitting detection of probe :target duplexes in a test sample in the presence of unhybridized molecular torches. Molecular torches and a variety of types of interacting label pairs are disclosed in U.S. Pat. No. 6,534,274, herein incorporated by reference in its entirety.

Another example of a detection probe having self-complementarity is a "molecular beacon." Molecular beacons include nucleic acid molecules having a target complementary sequence, an affinity pair (or nucleic acid arms) holding the probe in a closed conformation in the absence of a target sequence present in an amplification reaction, and a label pair that interacts when the probe is in a closed conformation. Hybridization of the target sequence and the target complementary sequence separates the members of the affinity pair, thereby shifting the probe to an open conformation. The shift to the open conformation is detectable due to reduced interaction of the label pair, which may be, for example, a fluorophore and a quencher (e.g., DABCYL and EDANS). Molecular beacons are disclosed in U.S. Pat. Nos. 5,925,517 and 6,150,097, herein incorporated by reference in its entirety. Other self-hybridizing probes include probe binding pairs having interacting labels, such as those disclosed in U.S. Pat. No. 5,928,862 (herein incorporated by reference in its entirety) might be adapted for use in the present invention. Probe systems used to detect single nucleotide polymorphisms (SNPs) might also be utilized in the present invention. Additional detection systems include "molecular switches," as disclosed in U.S. Publ. No. 20050042638, herein incorporated by reference in its entirety. Other probes, such as those comprising intercalating dyes and/or fluorochromes, are also useful for detection of amplification products in the present invention. See, e.g., U.S. Pat. No. 5,814,447 (herein incorporated by reference in its entirety). ii. Detection of Variant Proteins

In other embodiments, variant polypeptides are detected. Any suitable method may be used to detect truncated or mutant polypeptides including, but not limited to, those described below.

1. Antibody Binding

In some embodiments, antibodies (See below for antibody production) are used to determine if an individual contains an allele encoding a variant polypeptides. In preferred embodiments, antibodies are utilized that discriminate between variant (i.e., truncated proteins); and wild-type proteins. In some embodiments, the antibodies are directed to the C- terminus of proteins. Proteins that are recognized by the N-terminal, but not the C-terminal antibody are truncated. In some embodiments, quantitative immunoassays are used to determine the ratios of C-terminal to N-terminal antibody binding. In other embodiments, identification of variants is accomplished through the use of antibodies that differentially bind to wild type or variant forms of the polypeptides described herein.

Antibody binding is detected by techniques such as radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels, for example), Western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immunofiuorescence assays, protein A assays, and Immunoelectrophoresis assays, etc.

In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled.

In some embodiments, an automated detection assay is utilized. Methods for the automation of immunoassays include those described in U.S. Patents 5,885,530, 4,981,785, 6,159,750, and 5,358,691, each of which is herein incorporated by reference. In some embodiments, the analysis and presentation of results is also automated. For example, in some embodiments, software that generates a prognosis based on the result of the

immunoassay is utilized. In other embodiments, the immunoassay described in U.S. Patents 5,599,677 and 5,672,480; each of which is herein incorporated by reference.

C. Kits for Detecting Mutant or Variant Alleles

The present invention also provides kits for determining whether an individual contains a wild-type or variant (e.g. , mutant or polymorphic) allele of one or more of the genes described herein as being associated with one or more of NPHP-RC, KIN, and

CAKUT. In some embodiments, the kits are useful for determining whether the subject has one or more of NPHP-RC, KIN, and CAKUT or to provide a prognosis to an individual diagnosed with one or more of NPHP-RC, KIN, and CAKUT. The diagnostic kits are produced in a variety of ways. In some embodiments, the kits contain at least two reagents (e.g., one or more of NPHP-RC, KIN, and CAKUT informative reagent) useful, necessary, or sufficient for specifically detecting two or more distinct mutant or variant allele or protein. In some embodiments, the kits contain reagents for detecting a truncation in the polypeptide encoded by the variant nucleic acid. In preferred embodiments, the reagent is a nucleic acid that hybridizes to nucleic acids containing the mutation and that does not bind to nucleic acids that do not contain the mutation. In other embodiments, the reagent is a pair of primers for amplifying the region of DNA containing the mutation. In still other embodiments, the reagents are antibodies that preferentially bind either the wild-type or truncated or variant proteins. In some embodiments, reagents are one or more sequencing primers.

In some embodiments, the kits include ancillary reagents such as buffering agents, nucleic acid stabilizing reagents, protein stabilizing reagents, and signal producing systems (e.g., florescence generating systems as Fret systems), and software (e.g., data analysis software). The test kit may be packages in any suitable manner, typically with the elements in a single container or various containers as necessary along with a sheet of instructions for carrying out the test. In some embodiments, the kits also preferably include a positive control sample. In some embodiments, markers (e.g., those described herein) are detected alone or in combination with other markers in a panel or multiplex format. For example, in some embodiments, a plurality of markers are simultaneously detected in an array or multiplex format (e.g., using the detection methods described herein).

D. Bioinformatics

For example, in some embodiments, a computer-based analysis program is used to translate the raw data generated by the detection assay (e.g. , the presence, absence, or amount of a given allele or polypeptide) into data of predictive value for a clinician. The clinician can access the predictive data using any suitable means. Thus, in some preferred

embodiments, the present invention provides the further benefit that the clinician, who may not be trained in genetics or molecular biology, need not understand the raw data. The data is presented directly to the clinician in its most useful form. The clinician is then able to immediately utilize the information in order to optimize the care of the subject.

The present invention contemplates any method capable of receiving, processing, and transmitting the information to and from laboratories conducting the assays, information providers, medical personal, and subjects. For example, in some embodiments of the present invention, a sample (e.g., a biopsy or a blood or serum sample) is obtained from a subject and submitted to a profiling service (e.g., clinical lab at a medical facility, genomic profiling business, etc.), located in any part of the world (e.g. , in a country different than the country where the subject resides or where the information is ultimately used) to generate raw data. Where the sample comprises a tissue or other biological sample, the subject may visit a medical center to have the sample obtained and sent to the profiling center, or subjects may collect the sample themselves (e.g., a urine sample) and directly send it to a profiling center. Where the sample comprises previously determined biological information, the information may be directly sent to the profiling service by the subject (e.g., an information card containing the information may be scanned by a computer and the data transmitted to a computer of the profiling center using an electronic communication systems). Once received by the profiling service, the sample is processed and a profile is produced (i.e., presence of wild type or mutant allele), specific for the screening, diagnostic or prognostic information desired for the subject.

The profile data is then prepared in a format suitable for interpretation by a treating clinician. For example, rather than providing raw data, the prepared format may represent a diagnosis or risk assessment (e.g., diagnosis or prognosis of NPHP-RC and/or CAKUT) for the subject, along with recommendations for particular treatment options. The data may be displayed to the clinician by any suitable method. For example, in some embodiments, the profiling service generates a report that can be printed for the clinician (e.g., at the point of care) or displayed to the clinician on a computer monitor.

In some embodiments, the information is first analyzed at the point of care or at a regional facility. The raw data is then sent to a central processing facility for further analysis and/or to convert the raw data to information useful for a clinician or patient. The central processing facility provides the advantage of privacy (all data is stored in a central facility with uniform security protocols), speed, and uniformity of data analysis. The central processing facility can then control the fate of the data following treatment of the subject.

For example, using an electronic communication system, the central facility can provide data to the clinician, the subject, or researchers.

In some embodiments, the subject is able to directly access the data using the electronic communication system. The subject may chose further intervention or counseling based on the results. In some embodiments, the data is used for research use. For example, the data may be used to further optimize the inclusion or elimination of markers as useful indicators of a particular condition or stage of disease.

In some embodiments, the methods disclosed herein are useful in monitoring the treatment of one or more of NPHP-RC, KIN, and CAKUT. For example, in some embodiments, the methods may be performed immediately before, during and/or after a treatment to monitor treatment success. In some embodiments, the methods are performed at intervals on disease free patients to ensure treatment success.

The present invention also provides a variety of computer-related embodiments. Specifically, in some embodiments the invention provides computer programming for analyzing and comparing a pattern of one or more of NPHP-RC, KIN, and CAKUT -specific marker detection results in a sample obtained from a subject to, for example, a library of such marker patterns known to be indicative of the presence or absence of one or more of NPHP- RC, KIN, and CAKUT, or a particular stage or prognosis of one or more of NPHP-RC, KIN, and CAKUT.

In some embodiments, the present invention provides computer programming for analyzing and comparing a first and a second pattern of one or more of NPHP-RC, KIN, and CAKUT -specific marker detection results from a sample taken at least two different time points. In yet another embodiment, the invention provides computer programming for analyzing and comparing a pattern of one or more of NPHP-RC, KIN, and CAKUT -specific marker detection results from a sample to a library of one or more of NPHP-RC, KIN, and CAKUT -specific marker patterns known to be indicative of the presence or absence of one or more of NPHP-RC, KIN, and CAKUT, wherein the comparing provides, for example, a differential diagnosis between an aggressive and a less aggressive one or more of NPHP-RC, KIN, and CAKUT.

The methods and systems described herein can be implemented in numerous ways. In one embodiment, the methods involve use of a communications infrastructure, for example the internet. Several embodiments of the invention are discussed below. It is also to be understood that the present invention may be implemented in various forms of hardware, software, firmware, processors, distributed servers (e.g., as used in cloud computing) or a combination thereof. The methods and systems described herein can be implemented as a combination of hardware and software. The software can be implemented as an application program tangibly embodied on a program storage device, or different portions of the software implemented in the user's computing environment (e.g., as an applet) and on the reviewer's computing environment, where the reviewer may be located at a remote site (e.g., at a service provider's facility).

For example, during or after data input by the user, portions of the data processing can be performed in the user-side computing environment. For example, the user-side computing environment can be programmed to provide for defined test codes to denote platform, carrier/diagnostic test, or both; processing of data using defined flags, and/or generation of flag configurations, where the responses are transmitted as processed or partially processed responses to the reviewer's computing environment in the form of test code and flag configurations for subsequent execution of one or more algorithms to provide a results and/or generate a report in the reviewer's computing environment.

The application program for executing the algorithms described herein may be uploaded to, and executed by, a machine comprising any suitable architecture. In general, the machine involves a computer platform having hardware such as one or more central processing units (CPU), a random access memory (RAM), and input/output (I/O) interface(s). The computer platform also includes an operating system and microinstruction code. The various processes and functions described herein may either be part of the microinstruction code or part of the application program (or a combination thereof) which is executed via the operating system. In addition, various other peripheral devices may be connected to the computer platform such as an additional data storage device and a printing device.

As a computer system, the system generally includes a processor unit. The processor unit operates to receive information, which generally includes test data (e.g., specific gene products assayed), and test result data (e.g., the pattern of gastrointestinal neoplasm-specific marker detection results from a sample). This information received can be stored at least temporarily in a database, and data analyzed in comparison to a library of marker patterns known to be indicative of the presence or absence of one or more of NPHP-RC, KIN, and CAKUT.

Part or all of the input and output data can also be sent electronically; certain output data (e.g., reports) can be sent electronically or telephonically (e.g., by facsimile, e.g., using devices such as fax back). Exemplary output receiving devices can include a display element, a printer, a facsimile device and the like. Electronic forms of transmission and/or display can include email, interactive television, and the like. In some embodiments, all or a portion of the input data and/or all or a portion of the output data (e.g., usually at least the library of the pattern of gastrointestinal neoplasm-specific marker detection results known to be indicative of the presence or absence of one or more of NPHP-RC, KIN, and CAKUT) are maintained on a server for access, e.g., confidential access. The results may be accessed or sent to professionals as desired.

A system for use in the methods described herein generally includes at least one computer processor (e.g., where the method is carried out in its entirety at a single site) or at least two networked computer processors (e.g., where detected marker data for a sample obtained from a subject is to be input by a user (e.g., a technician or someone performing the assays)) and transmitted to a remote site to a second computer processor for analysis (e.g., where the pattern of one or more of NPHP-RC, KIN, and CAKUT -specific marker) detection results is compared to a library of patterns known to be indicative of the presence or absence of one or more of NPHP-RC, KIN, and CAKUT), where the first and second computer processors are connected by a network, e.g., via an intranet or internet). The system can also include a user component(s) for input; and a reviewer component(s) for review of data, and generation of reports, including detection of a one or more of NPHP-RC, KIN, and CAKUT. Additional components of the system can include a server component(s); and a database(s) for storing data (e.g., as in a database of report elements, e.g., a library of marker patterns known to be indicative of the presence or absence of one or more of NPHP-RC, KIN, and CAKUT and/or known to be indicative of a grade and/or a stage of one or more of NPHP- RC, KIN, and CAKUT, or a relational database (RDB) which can include data input by the user and data output. The computer processors can be processors that are typically found in personal desktop computers (e.g., IBM, Dell, Macintosh), portable computers, mainframes, minicomputers, tablet computer, smart phone, or other computing devices.

The input components can be complete, stand-alone personal computers offering a full range of power and features to run applications. The user component usually operates under any desired operating system and includes a communication element (e.g., a modem or other hardware for connecting to a network using a cellular phone network, Wi-Fi, Bluetooth, Ethernet, etc.), one or more input devices (e.g., a keyboard, mouse, keypad, or other device used to transfer information or commands), a storage element (e.g., a hard drive or other computer-readable, computer-writable storage medium), and a display element (e.g., a monitor, television, LCD, LED, or other display device that conveys information to the user). The user enters input commands into the computer processor through an input device.

Generally, the user interface is a graphical user interface (GUI) written for web browser applications.

The server component(s) can be a personal computer, a minicomputer, or a mainframe, or distributed across multiple servers (e.g., as in cloud computing applications) and offers data management, information sharing between clients, network administration and security. The application and any databases used can be on the same or different servers. Other computing arrangements for the user and server(s), including processing on a single machine such as a mainframe, a collection of machines, or other suitable configuration are contemplated. In general, the user and server machines work together to accomplish the processing of the present invention.

Where used, the database(s) is usually connected to the database server component and can be any device which will hold data. For example, the database can be any magnetic or optical storing device for a computer (e.g., CDROM, internal hard drive, tape drive). The database can be located remote to the server component (with access via a network, modem, etc.) or locally to the server component.

Where used in the system and methods, the database can be a relational database that is organized and accessed according to relationships between data items. The relational database is generally composed of a plurality of tables (entities). The rows of a table represent records (collections of information about separate items) and the columns represent fields (particular attributes of a record). In its simplest conception, the relational database is a collection of data entries that "relate" to each other through at least one common field. Additional workstations equipped with computers and printers may be used at point of service to enter data and, in some embodiments, generate appropriate reports, if desired. The computer(s) can have a shortcut (e.g., on the desktop) to launch the application to facilitate initiation of data entry, transmission, analysis, report receipt, etc. as desired.

In certain embodiments, the present invention provides methods for obtaining a subject's risk profile for developing one or more of NPHP-RC, KIN, and CAKUT or having aggressive one or more of NPHP-RC, KIN, and CAKUT. In some embodiments, such methods involve obtaining a blood or blood product sample from a subject (e.g., a human at risk for developing one or more of NPHP-RC, KIN, and CAKUT; a human undergoing a routine physical examination, or a human diagnosed with one or more of NPHP-RC, KIN, and CAKUT), detecting the presence or absence of the variants described herein associated with one or more of NPHP-RC, KIN, and CAKUT in the sample, and generating a risk profile for developing one or more of NPHP-RC, KIN, and CAKUT. For example, in some embodiments, a generated profile will change depending upon specific markers and detected as present or absent or at defined threshold levels. The present invention is not limited to a particular manner of generating the risk profile. In some embodiments, a processor (e.g., computer) is used to generate such a risk profile. In some embodiments, the processor uses an algorithm (e.g., software) specific for interpreting the presence and absence of specific exfoliated epithelial markers as determined with the methods of the present invention. In some embodiments, the presence and absence of specific variants as determined with the methods of the present invention are imputed into such an algorithm, and the risk profile is reported based upon a comparison of such input with established norms (e.g., established norm for various risk levels for developing one or more of NPHP-RC, KIN, and CAKUT, established norm for subjects diagnosed with various variations of one or more of NPHP-RC, KIN, and CAKUT). In some embodiments, the risk profile indicates a subject's risk for developing one or more of NPHP-RC, KIN, and CAKUT. In some embodiments, the risk profile indicates a subject to be, for example, a very low, a low, a moderate, a high, and a very high chance of developing one or more of NPHP-RC, KIN, and CAKUT or having a poor prognosis (e.g., likelihood of long term survival) from one or more of NPHP-RC, KIN, and CAKUT. In some embodiments, a health care provider will use such a risk profile in determining a course of treatment or intervention.

EXPERIMENTAL The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof. Example 1

EXPERIMENTAL PROCEDURES

Research subjects. Blood samples and pedigrees were obtained following informed consent from individuals with NPHP-RC and/or their parents. The diagnosis of NPHP-RC was based on published clinical criteria (Chaki et al., Kidney Int 2011). Rat studies were performed according to Dutch Animal Welfare laws and were locally reviewed by the University of Utrecht Animal Ethical Committee (DEC). Human subjects provided informed consent to the use of their tissue for research purposes. Linkage analysis. For genome-wide homozygosity mapping the GeneChip® Human

Mapping 250k Styl Array from Affymetrix was used. Non-parametric LOD scores were calculated using a modified version of the program GENEHUNTER 2.1 (Kruglyak et al., Am J Hum Genet 58, 1347-1363 1996; Strauch et al, Am J Hum Genet 66, 1945-1957 2000) through stepwise use of a sliding window with sets of 110 SNPs and the program ALLEGRO (Gudbjartsson et al, Nat Genet 25, 12-13 2000) in order to identify regions of homozygosity as described (Hildebrandt et al, PloS Genetics 5, 31000353 2009c; Sayer et al, Nat Genet 38, 674-681 2006) using a disease allele frequency of 0.0001 and Caucasian marker allele frequencies. Bioinformatics. Genetic location is according to the February 2009 Human Genome

Browser data. Statistical analysis. Student"s two-tailed non-paired t-tests and normal distribution two-tailed z-tests were carried out using pooled standard error and s.d. values to determine the statistical significance of different cohorts. Whole-Exome Sequencing. Whole exome capture. Exome enrichment was conducted largely following the manufacturers protocol for the NimbleGen SeqCap EZ Exome v2 (Roche NimbleGen, version 2). Briefly, three micrograms of genomic DNA was fragmented by sonication using the Covaris S2 system to achieve a uniform distribution of fragments with a mean size of 300 bp. The sonicated DNA was purified using AMPure XP Solid Phase Reversible Immobilization paramagnetic (SPRI) beads (Agencourt) followed by polishing of the DNA ends by removing the 3" overhangs and filling in the 5" overhangs resulting from sonication using T4 DNA polymerase and Klenow fragment (New England Biolabs).

Following end polishing, a single "A" -base was added to the 3" end of the DNA fragments using Klenow fragment (3" to 5" exo minus). This prepares the DNA fragments for ligation to specialized adaptors that have a "T"-base overhang at their 3"ends. The end-repaired DNA with a single "A"-base overhang was ligated to paired-end adaptors (Illumina) in a standard ligation reaction using T4 DNA ligase and 2 μΜ - 4 μΜ final adaptor concentration, depending on the DNA yield following purification after the addition of the "A"-base (a 10- fold molar excess of adaptors is used in each reaction). Following ligation, the samples were purified using SPRI beads, amplified by six cycles of PCR to maintain complexity and avoid bias due to amplification and quality controlled by library size assessment on the Agilent Bioanalyzer and quantitation using PicoGreen reagent (Invitrogen).

One microgram of amplified, purified DNA (DNA library) was prepared for hybridization by adding COT1 DNA and blocking oligonucleotides to the DNA library, desiccating the DNA completely and resuspending the material in NimbleGen hybridization buffer. The resuspended material was denatured at 95°C prior to addition of the exome capture library bait material. The DNA library and biotin-labeled capture library were hybridized by incubation at 47°C for 68 h. Following hybridization, streptavidin coated magnetic beads were used to purify the DNA:DNA hybrids formed between the capture library and sequencing library during hybridization. The purified sequencing library was amplified directly from the purification beads using 8 cycles of PCR using Pfx DNA polymerase (Invitrogen). The libraries were purified following amplification and the library size was assessed using the Agilent Bioanalyzer. A single peak between 350-400 bp indicates a properly constructed and amplified library ready for sequencing. Final quantitation of the library was performed using the Kapa Biosciences Real-time PCR assay and appropriate amounts loaded onto the Illumina flowcell for sequencing by paired-end 50 nt sequencing on the Illumina HiSeq2000 sequencer. Sequencing. Sequencing was performed largely as described in (Bentley et al, Nature

456, 53-59 2008). Following dilution to 10 nM final concentration based on the real-time PCR and bioanalyzer results, the final library stock is then used in paired-end (PE) cluster generation at a final concentration of 6-8 pM to achieve a cluster density of 600,000/mm2 (on the Illumina HiSeq2000 instrument, v2.5 reagents). Following cluster generation, lOOnt paired-end sequencing was performed using the standard Illumina protocols. Sequencing each sample in a single lane at paired-end 100 nt conditions on the Illumina HiSeq (v2.5 reagents) generated an average of 19.1Gb of pass-filter sequence data with 94.96% aligning to the genome. This amount of sequence corresponded to an average coverage of 181x over the 44 Mb of capture region. 80.41% of sequencing reads fell with 200 bp of the target region. 98.39%) of the capture region was sequenced to a depth of at least lx, 95.6%> to at least 8x, 92.17% to at least 20x, and 89.42% to at least 30x.

Data Analysis. Raw sequencing data for each individual were mapped to the human reference genome (build hg 19) using the Burrows- Wheeler Aligner (B WA v0.5.81536) 14. The BWA aligned sequencing reads were processed by Picard to label the PCR duplicates. The Genome Analysis Toolkit (GATK, version 5091) was then used to remove duplicates, perform local realignment and map quality score recalibration to produce a "cleaned" BAM file for each individual. SNP calls were made by the Unified Genotyper module in GATK using the "cleaned" BAM files in batch fashion (90 samples per batch). The resulting Variant Call Format (VCF, version 4.0) files were annotated using the GenomicAnnotator module in GATK to identify and label the called variants that are within the targeted coding regions and overlap with known and likely benign SNPs reported in dbSNP vl32 with allele frequencies >5%. The annotated VCF files were then filtered using the GATK variant filter module with a hard filter setting and a custom script for initial filtering. Variant calls that failed to pass the following filters were eliminated from the call set: i) MQ0 >= 4 && ((MQ0 / (1.0 *

DP)) > 0.1); ii) QUAL < 30.0 || QD < 5.0 || HRun > 5 || SB > 0.00; iii) Cluster size 10; iv) not contain dbSNP id; v) inside the targeted regions. Combined VCF files were then split into individual files and remaining variants were tested for recessive segregation in the respective families. For each of the three families with mutation of MRE11, ZNF423 or CEP164 there was only one gene in each family in any of the homozygous regions for which the homozygous variant passed Polyphen 1 and 2 scores, segregated within the family, and was absent from >270 controls, thus validating the pathogenic relevance of the mutation. CEP 164 mutation analysis. Exon-PCR and Sanger sequencing of all 31 coding exons for one affected individual in each of 856 different NPHP-RC families was performed. The number of families examined (by increasing number of organs involved) was: isolated nephronophthisis (5), isolated retinal degeneration (100), Senior-Loken syndrome (168), Joubert syndrome (240), Bardet-Biedl syndrome (195), Meckel syndrome (52), and other severe unsolved NPHP-RC cases (96). 480 individuals from additional NPHP-RC families were also examined using massively-parallel sequencing of exon-PCR from pooled DNA samples (Otto et al, Nat Genet 34, 413-420 2010). Yeast-two-hybrid screening with ZNF423. A subclone of human CEP290 was prepared using high fidelity Taq polymerase, spanning 1770 bp, (562 amino acids, 1917- 2479) and including wild-type stop codon and cloned into pENTR-TOPO as previously described in (Sayer et al, 2006, supra) (clone named "JAS2"). The CEP290 subclone insert was switched to destination vector pDEST32 (binding domain containing yeast-2-hydrid vector, "bait") (Invitrogen). CEP290 (JAS2) was used as bait, fused to the GAL4 DNA binding domain in the pDEST32 vector, and a human fetal brain expression library was screened and cloned into pEXPAD22 GAL4 activation domain fusion vector (Invitrogen). Approximately 1 x 106 clones were screened after cotransforming plasmids into competent MaV203 yeast cells (lithium acetate method) and plating onto His, Leu and Trp deficient medium containing mM 3-aminotriazole. Colonies were replica plated on restrictive media and surviving colonies were used for cDNA extraction. Five ml cultures were grown at 30°C overnight. cDNA was extracted using RPM yeast plasmid isolation kitTM (Bio 101 systems). cDNA was transformed into E. coli, purified and directly sequenced using vector specific primers. Sequence analysis allowed prediction of amino acid sequences (ORFinder), which were then identified by BLAT analysis. Direct yeast-2-hybrid interaction experiments allowed colony growth to be compared to 2 negative controls (respective plasmids without insert) and 5 positive control yeast strains for different interaction strength.

Coimmunoprecipitation with ZNF423. Full length human ZNF423 was subcloned into pcDNA3.1 -NT-GFP using long range PCR (Expand, Roche) and IMAGE clone 100072717 as template. Clones were sequenced to confirm orientation and fidelity. A partial length human CEP290 cDNA spanning 1770 bp as described above (JAS2) was subcloned into a DEST-V5 vector. HEK293 cells were transiently co-transfected with full length GFP- ZNF423 and partial length CEP290-V5 using Lipofectamine 2000. After 24 h cells were washed in phosphate-buffered saline (PBS), pelleted, and lysed in NP-40 buffer (150 mM sodium chloride, 0.5% NP-40, 50 mM Tris pH 7.4, phenylmethanesulphonylfluoride, and protease inhibitors). The lysate was centrifuged for 20 min at 10,000 g at 4°C and the supernatant was precleared with protein G sepharose beads (GE Healthcare, Giles,

Buckinghamshire, UK). After removal of protein G the supernatant was then incubated overnight with protein G and 1 μg of either anti-V5 (Invitrogen) or mouse IgG (Sigma) at 4°C. The beads were washed extensively in lysis buffer and bound proteins resolved by 10% SDS polyacrylamide gel electrophoresis as described above. GFP -tagged proteins were detected with anti-GFP-conjugated to HRP (Santa Cruz) 1 :5,000 and V5-tagged proteins were detected with anti-V5 conjugated to HRP (Invitrogen) 1 :5,000.

Spheroid assays.

mIMCD3 cell culture. mIMCD3 is a mouse inner medullary collecting duct cell line. The cells were cultured in Dulbecco"s Modified Eagle"s Medium (DMEM):F12 (1 : 1) (GlutaMAX, Gibco), supplemented with 10% Fetal Calf Serum (FCS) and penicillin and streptomycin (1% P/S). Cells were incubated at 37°C in 5% carbon dioxide (C02) to approximately 90% confluence.

Transfections. Before exposing the cells to different experimental conditions, cells were seeded in 6-, 24- or 48-well plates, depending on the experimental setup. After at least 6 h, the cells were transfected with Lipofectamine RNAimax (Invitrogen, 13778-075), according to the supplier's protocol. Opti-MEM (Invitrogen, 31985-062) was used to dilute the ON-TARGETplus siRNA SMARTpools (Thermo Scientific Dharmacon) for Non- targeting pool (D-001810-10) or Cepl64 (L-057068-01).

Antibodies. The following primary antibodies were used: mouse anti-acetylated- tubulin (Sigma, T7451), 1 :20000; human anti-Nuclear ANA-Centromere Autoantibody (CREST) (Cortex Biochem, CS1058) 1 : 1000; mouse γΗ2ΑΧ anti-phospho-histone H2A.X (serl39) (Millipore, DAM 1493341. Alexa-488, Alexa-555, Alexa-564 and Alexa-633 and 647 conjugated secondary antibodies were obtained from Invitrogen. mIMCD3 spheroid growth assay. Cells were trypsinized 24 hour post-transfection and resuspended cells were then mixed 1 : 1 with growth factor-depleted matrigel (BD

Bioscience). After the matrigel polymerized for 20 minutes at 37°C, warm medium was dropped over the matrix until just covered. The IMCD3 cells formed spheroids with cleared lumens 3 days later. Medium was removed by pipetting and the gels were washed three times for 10 minutes with warm PBS supplemented with calcium and magnesium. The gels were then fixed in fresh 4% PFA for 30 minutes RT. After washing three times in PBS after fixation, the cells were permeabilized for 15 minutes in gelatin dissolved in warm PBS (350 mg/50 mL) and 0.5% triton-XlOO. Primary antibody (mouse anti-acetylated tubulin, Sigma, at 1 :20,000) was diluted in the permeabilization gelatin buffer and incubated at 4°C overnight. After washing the spheroids 3 times for 30 minutes in permeabilization buffer, goat anti-mouse Alexa-555 secondary antibody (Invitrogen) was diluted 1 :500 in

permeabilization buffer and incubated overnight at 4°C. The next day, spheroids were washed 3 times in permeabilization buffer and mounted with Dapi (1 :2,000) in Fluoroumount-G (Cell Lab, Beckman Coulter). Images were taken with a Zeiss LSM510 confocal microscope and 50 spheroids per condition were scored. GraphPad Prism 5.0 was used to perform two-tailed student t-tests.

RT-qPCR for Cepl64. IMCD3 cells were transfected with non-targeting siControl or siCepl64 oligonucleotides (Dharmacon, ON-TARGETplus SMARTpool Cepl64) using Lipofectamine RNAimax (Invitrogen) in 6 well plates seeded with cells at 50% confluency. Cells were lysed 24, 48 and 72 h after transfection and total RNA was isolated (RNeasy Mini Kit, Qiagen, 74106) and measured (NanoDrop spectrophotometer ND-1000, Thermo Fischer Scientific Inc.). cDNA was synthesized from 500 ng RNA template using the iScript cDNA Synthesis Kit (Bio-Rad, 170-8891) according to the supplier's protocol. Dilutions were made for RT-QPCR analysis to determine the expression of Cepl64, normalized against reference gene RPL27. The primers (Sigma) used are mCepl64 forward 5"- AGAGTGACAACCAGAGTGTCC, mCepl64 reverse 5"- GGAGACTCCTCGTACTCAAAGTT, mRPL27 forward 5"- CGCCCTCCTTTCCTTTCTGC and mRPL27 reverse 5"-

GGTGCCATCGTCAATGTTCTTC. The iQ SYBR Green Supermix (Bio-Rad, 170-8880) was used to multiply and measure the cDNA with a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). All samples were run in triplicate in 20 μΐ reactions. The following PCR program was used: 95°C for 3 min, followed by 40 cycles of 10 s at 95°C, 30s at 51.5/61ºC and 30 s at 72°C, then 10 s at 95°C followed by a melt of the product from 65°C-95°C. The AACT method was used for statistical analysis to determine gene expression levels.

Immunofluorescence and confocal microscopy. For immunostaining mIMCD3 cells, cells were grown on covers lips and fixed for 30 minutes in 4%>PFA followed by a 15 minutes permeabilization step in 0.5%Triton-X100/l%BSA/PBS. Primary antibody incubations (human anti-CREST at 1 : 1,000, mouse anti-acetylated tubulin at 1 :20,000) were performed overnight in 1% BSA/PBS. Secondary antibody incubations were performed for 1 hour at RT. DAPI incubations were performed for 10' at RT. Coverslips were mounted in Fluormount G (Cell Lab, Beckman Coulter). Confocal imaging was performed using Zeiss LSM510 Confocal laser microscope and images were processed with the LSM software. Approximately 250 events per condition were scored. GraphPad Prism 5.0 was used to perform two-tailed student t-tests.

Cell cycle studies.

Generation of IMCD3 and hTERT-RPE cell lines that are doxycycline-inducible for N-GFPCEP164 constructs. Both IMCD3 and hTERT-RPE cells were stably transfected with N-terminally GFP tagged human CEP 164 constructs in a retroviral vector for doxycycline (Dox)-inducible expression (pRetroX-Tight-Pur). Following selection on puromycin for two weeks, cells were induced with 10 ng/ml of doxycyclin for 20 h. Based on the GFP expression levels upon immunofluorescence, clonal cells were generated for IMCD3 cells.

Cell cycle studies. IMCD3 cells Dox-inducible for human N-GFP-Cepl64-WT (wild type) or N-GFPCep 164-Q525X (mutant) were transfected with either non- targeting control siRNA (50 nM, Dharmacon D-001206-14-20) or mouse Cepl64 siRNA (50 nM, Smartpool Dharmacon L-057068-01-0020) using Polyplus™ transfection reagents. Cells were then re- plated and treated for double thymidine (2 mM) blocks beginning at 24 h post transfection. At the same time, cells were also induced with doxycycline (10 ng/ml) for N-GFP-Cepl64-WT or N-GFP-Cepl64-Q525X. Cells were released from second thymidine block for 6 h and fixed with 2% PFA and stained with PI/RNase staining solution (BD Biosciences). FACS analysis for cell cycle histograms was performed and data were then analyzed using Modfit™ software. Mean and SD of % DNA amount for different phases (triplicate samples) were calculated and plotted as bar diagram.

Cell Proliferation studies. IMCD3 cells Dox-inducibly expressing human N-GFPCEP 164-WT or NGFP-CEP 164-Q525X were transfected with either non-targeting control siRNA (50 nM, Dharmacon D-001206-14-20) or mouse Cepl64 siRNA (50 nM, Smartpool Dharmacon L-057068-01-0020) using Polyplus™ transfection reagents. Cells were then re- plated in triplicate of 10,000 cells for each group and treated for thymidine (2 mM) blocks beginning at 24 h post transfection. At the same time, cells were also induced with

doxycycline (10 ng/ml) for N-GFP-CEP 164-WT or N-GFP-CEP164-Q525X expression. Cells were counted at 48, 72, and 96 h post transfection. Roscovitine studies.

Cell culture and cell irradiation. Sh-Neg-IMCD3 cells were maintained in

DMEM:F12 supplemented with 10% FCS, 1% Penicillin/Streptomycin and 5 μg/ml

Puromycin (all from Gibco/Invitrogen, Carlsbad, CA) and maintained in a 5% CO2 humidified atmosphere at 37°C. Roscovitine (Chem Partners, China) was incubated at 80 μΜ for 24 h. Ultraviolet (UV) irradiation was performed using a UV StratalinkerTM

(Stratagene/ Agilent, Santa Clara, CA) at 30 J/m2 dose; cells were left to recover for

1h at 37°C before further analyses. Immunob lotting, immunofluorescence and subcellular fractionation. Cells were lysed as previously described (Bukanov et al, Nature 444, 949-952 2006). Subcellular fractionation was performed using Nuclear Extract Kit according to the manufacturer's instructions (Active Motif, Carlsbad, CA). Protein concentration was determined by BCA protein assay (Pierce/Thermofisher, Rockford, IL). Proteins were resolved on SDS PAGE 5-12% gradient and transferred using iBlot system (Invitrogen). The following antibodies were used: CEP 164 (Novus Biologicals, Littleton, CO), Phospho-Chkl (Ser317) and phospho-H2AX (Serl39) (Cell Signaling, Danvers, MA), Chkl (Upstate/Millipore, Billerica, MA), H2AX and 13.3.3 (AbCam, Cambridge, MA) and Sam-68 (Santa Cruz Biotech. Santa Cruz, CA). Primary antibodies were detected with anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (Promega, Madison, WI) and revealed by ECL (Amersham, Little

Chalfont, Buckinghamshire, UK) as previously described (Bukanov et al, 2006, surpa). For immunofluorescence, cells were fixed for 20 min in 4% paraformaldehyde (Electron

Microscopy Science, Hatfield, PA) in PBS (pH 7.2) and were permeabilized with 0.1 % Triton X100 in PBS. Immunofluorescence was then performed as previously described (Smith et al, J Am Soc Nephrol 17, 2821-2831 2006).

Gene expression analysis. Gene expression analysis was performed as described before (Smith et al, JASN, 2006, 17-pp2821-2831) using TAQ-Man gene expression assays (Applied Biosystems/Invitrogen, Carslbad, CA).

Interaction studies of CEP 164 with TTBK2 and CCDC92.

Yeast two-hybrid. A GAL4-based yeast two-hybrid system was used to screen for binary CEP 164 interactors by using methods described by Letteboer and Roepman (Letteboer and Roepman, 2008). Bait constructs expressing full length CEP 164 (CEP164fl) and several fragments thereof (CEP 1641-550, CEP164551-1100 and CEP1641101-1460) were used to screen two retinal cDNA libraries: a bovine library of randomly primed retina cDNA and a human library of oligo-dT primed retina cDNA (Letteboer and Roepman, Methods Mol Biol 484, 145-159 2008).

GST pulldown. Full length 3xFlag-CCDC92 and 3xFlag-TTBK2 were expressed in COS-1 cells. CEP164fi, CEP1641-550, CEP164551-1100 and CEP1641101-1640 were cloned in pDEST15 and the resulting expression constructs were transformed in BL21DE3 to express glutathione S-transerase (GST) fusion proteins. The GST pulldown was performed as described by Coene et al. (Coene et al, Hum Mol Genet 20, 3592-3605 201 1). The results were assessed by immunoblotting, and Flag-tagged proteins were detected using anti-Flag primary antibodies (Sigma- Aldrich) and goat-anti-mouse IRDye800 secondary antibodies (Rockland, Immunochemicals, Gilbertsville, PA, USA). A Li-Cor Odyssey 2.1 scanner was used for imaging.

Co-immunoprecipitation. 3xHA-CEP164fl was expressed in combination with either 3xFlag- CCDC92fl or 3xFlag-TTBK2fl in COS-1 cells (and vice versa with swapped tags). 3xFlag-dNp63 or 3xHA-dNp63 was used as a control for specificity. The co- immunoprecipitation was performed as described by Coene et al. Transfected COS-1 cells were lysed and incubated with a-M2-agarose from mouse (Sigma- Aldrich) or a-HA affinity matrix from rat (Roche) for 2 h at 4°C. After washing, sample buffer was added to the beads and the sample was heated. Beads were then precipitated by centrifugation and the supernatant was analyzed by immunoblotting to assess if TTKB2 and CCDC92 indeed coimmunoprecipitated with CEP 164.

Interaction studies of CEP 164 with DVL3. Immunoprecipitation coupled to mass- spectrometry. Mouse embryonal fibroblasts were grown in DMEM supplemented with 10% fetal bovine serum at 37°C to confluency and cell pellets are frozen at -80°C. Cells were then lysed in lysis buffer (0.5% NP40; 150mM NaCl; 50 mM Tris pH 7.4) and subjected to immunoprecipitation with antibodies against Dvl3 (sc-8027); Dvl2 (sc-8026) or control IgG (sc-2025; Santa Cruz Biotechnology). Proteomics grade trypsin (Sigma) was added to the beads directly (10 ng/μΐ) and left at 37°C for 12 h. Tryptic digested peptides were desalted using ZipTip CI 8 column (Millipore) based on the manufacturer's protocol. LC-MS/MS was performed on a NanoAcquity UPLC (Waters) on-line coupled to an ESI Q-TOF Premier (Waters) mass spectrometer. Trypsin digested peptides eluted from ZipTip column were diluted in autoclaved M.Q. water (Millipore) and loaded onto a 180 μm x 20 mm

nanoAcquity UPLC Symmetry trap column (Waters) packed with 5 μm BEH C-18 beads. After 1 minute of trapping, peptides were eluted through a 75 μm x 150 mm nanoAcquity (Waters) analytical column packed with 1.7 μm BEH C-18 beads at a flow rate of 400 nL/min using a gradient of 3 - 40% acetonitril with 0.1% formic acid for 35 minutes at a temperature of 35°C. Effluent was directly fed into the ESI source of the mass spectrometer. Raw data was acquired in data independent MSE Identity (Waters) mode. Precursor ion spectra were acquired with collision energy 5 V and fragment ion spectra with a collision energy 20-35 V ramp in alternating 1 sec scans. Raw data was then subjected to a database search using species specific Uniprot and NCBI mouse protein database by the PLGS2.3 software (Waters). Acetyl N-terminal, Deamidation N and Q, Carbamidomethyl C and Oxidation M were set as variable modifications. Peptide accuracy and MS/MS fragment mass accuracy was less than 20 ppm.

Western blotting and immunoprecipitation. Immunoblotting and sample preparations were performed as previously described (Bryja et al, J Cell Sci 120, 586-595 2007b).

HEK293 cells were transfected with the indicated constructs and lysed 48 h later. Samples were analyzed using SDS-PAGE and Western blotting, or subjected to immunoprecipitation. The antibodies used include the following: Dvl3 (sc-8027), Dvl2 (sc-8026), mouse IgG (sc- 2025), from Santa Cruz Biotech, CEP164 (4533.00.02) from Sdix, HA.11 (MMS-101P) from Covance, GFP (3H9), RFP (3F5) from Chromotek and FLAG M2 (F1804), GST (Gl 160) from Sigma. Immunoprecipitation was performed as previously described (Bryja et al., Proc Natl Acad Sci USA 104, 6690-6695 2007a). The antibodies used for immunoprecipitation were RFP Trap from Chromotek, HA.11 (MMS-101 P) from Covance, anti FLAG (F 1804; Sigma), anti Dvl3 (sc-8027), anti Dvl2 (sc-8026) (all Santa Cruz Biotechnology).

GST pulldown. Production of recombinant GST-tagged fragments of Cepl64

(Sivasubramaniam et al, Genes Dev 22, 587-600 2008) was induced by adding 0.2 mM IPTG and grown for 4 h at 37°C. The bacteria were spun down at 4000 g/4°C/10 min and the pellet was resuspended in 10 ml of GST lysis buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 5mM MgC12, protease inhibitors (Roche)) and stored at -80°C. Then the solution was thawed, sonicated for 3x30 seconds and spun down 15000 g/4°C/15 min. The supernatant was then used for incubation with GST beads for 2 h at rotator (100 μl of beads per 100 ml of original bacterial culture). After incubation beads were washed 3 times with 1 ml of GST lysis buffer and frozen at -80°C as 25% slurry in GST lysis buffer +20 % glycerol. HEK293T cells were transfected according to the scheme, grown for 24 or 48 h and lysed in 0.5 % NP 40 lysis buffer (0.5 % NP40, 50 mM Tris pH 7.4, 1 mM EDTA, 150 mM NaCl) with added protease inhibitors (Roche) and phosphatase inhibitors (Calbiochem). The lysate was spun down at 16100 g/4°C/10 minutes and the supernatant was used for overnight incubation with 15 μl of solid GST beads containing GST recombinant proteins on the rotator. After the incubation samples were washed with 800 μl 0.5 % NP 40 lysis buffer, the GST beads were collected at 0.1 g/4°C/l min, the supernatant was aspirated out and this washing was repeated 6 times. Proteins were eluted with 45 μΐ of 2x Laemmli buffer.

Immunohistochemistry. HEK-293 cells were seeded at approx. 2x105 cells/well on collagen coated covers lips in 24-well plates. Cells were fixed in fresh 4% paraformaldehyde, permeabilized with 0.05% Triton-X100, blocked with PBTA (3% BSA, 0,25% Triton, 0,01% NaN3) for 1 hour and incubated overnight with primary antibodies (CEP 164 (4533.00.02) from Sdix and Dvl2 (sc-8026 from Santa Cruz Biotechnology). Next day, coverslips were washed in PBS, and incubated with secondary antibodies: Alexa 488, Alexa 568 (Invitrogen), washed with PBS, stained with DAPI (1 :5000) and mounted on coverslips. Cells were visualized using a Leica TCS SP-5 confocal microscope. zebrafish ruvbll morphants. ruvbllhil055b/+ heterozygous fish were acquired from the Zebrafish International Resource Center (ZIRC, Oregon). For histological analyses, embryos were fixed at 72 hpf with 4% PFA/PBS and embedded in JB-4 resin (PolySciences) following the manufacturers protocol. 6-μιη sections were obtained using a Leica R2265 microtome and stained with methylene blue following published procedures (Zhou et al., Am J Physiol Renal Physiol 299, F55-62 2010). For

Figure imgf000047_0001
staining, embryos were fixed at 27 hpf with 4% PFA/PBS +1% DMSO for overnight, permeablized with acetone at -20°C for 7 minutes, and stained with antibody against phosphorylated zebrafish H2AX (1 : 1,000, from Amatruda lab at UT Southwestern). Alex488-anti rabbit IgG was used at 1 : 1,000. The IF procedure followed a standard protocol. After washing off secondary antibody, embryos were mounted in Vector-Shield Mounting Media and imaged with Leica SP5X confocal system. Z- stacks were processed to reduce noise and overlayed to show the representative images of IF staining for γΗ2ΑΧ. Morpho linos were: cepl64 MO: 5"- TATATGCTCTTCTCCATCACCTCAT; p53 MO: 5"- GCGCCATTGCTTTGCAAGAATTG. Coimmunoprecipitation with Ruvbll . HEK293T cells were transiently transfected using the calcium phosphate method and the indicated plasmids and co-immunopresipitation was performed as described previously (Habbig et al, J Cell Biol 193, 633-642 2011).

Enhanced chemiluminescence was detected with a digital imaging system (Fusion, Peqlab). Human Ruvbll and human NPHP5 were cloned from a human cDNA library. Human NPHPl and NPHP4, mouse NPHP2 and human EPS15L1 1-225 have been described previously (Habbig et al, 2011, supra; Liebau et al, J Biol Chem 286, 14237-14245 2011; Otto et al, Nat Genet 34, 413-420 2003).

Multicolor Competition Assay (MCA). MCA was performed as described previously (Smogorzewska et al, Cell 129, 289-301 2007). Briefly, 1.7 x 105 U20SGFP were reverse transfected with 64 pmol siRNA duplex using Lipofectamine RNAiMAX (Invitrogen) as per manufactures instructions. 48 h post transfection siRNA treated U20S-GFP were mixed with anti-Luciferase siRNA treated U20S-RFP cells at a 1 : 1 ratio. 72 h post transfection cells were treated with DNA damaging agents as depicted. Cells were harvested for FACS analysis 7 days post treatment. siRNA duplex sequences were as follows:

Luc: CGTACGCGGAATACTTCGA (siRNA targeting firefly luciferase niRNA), CEP164#1

GGACCATCCATGTGACGAA,

CEP164#2: GGCTGGAACGTGTCAAGAA

CEP164#3: GAGTTGGAGTCTCAACAGA,

ZNF423#1 : GG AG AAC C AC AAG AAC ATT

ZNF423#2: CGAGTGCAGTGTCAAGTTT,

ZNF423#3: GCATCAACCACGAGTGTAA, all from Ambion.

BRCA2: (Stealth siRNA, Invitrogen) used as a combination of three siRNAs:

GGAACCAAATGATACTGATCCATTA, GGAGGACTCCTTATGTCCAAATTTA,

GAGCGCAAATATATCTGAAACTTC;

ATM (Stealth siRNA, Invitrogen) used as a combination of three siRNAs):

GCGCAGTGTAGCTACTTCTTCTATT, GGGCCTTTGTTCTTCGAGACGTTAT, GC AAC ATTTGC CT AT ATC AGC AATT . anti-CEP 164 antibody for immunoblot was from Novus (cat #45330002).

Quantitative RT-PCR. Intron spanning primer pairs were designed for quantitative RT-PCR of ZNF423 and beta-actin as control. RNA was extracted from the siRNA treated U20S cells using QIAGEN RNeasy Plus Mini Kit. First-strand cDNAs were then synthesized using the Invitrogen Superscript III Reverse Transcriptase kit. Quantitative RT- PCR was performed using Platinum SybrGreen Super Mix (Invitrogen) according to the manufacturer's instructions. Sequences of primer pairs were: Beta-actin forward:

GCTACGAGCTGCCTGACG, Beta-actin reverse: GGCTGGAAGAGTGCCTCA, ZNF423 forward: GTCTCTGGCAGACCTGACG, ZNF423 reverse:

AGAGTTGTGGGTCGTCATCA.

ZNF423 DNA damage response. Lentivirus constructs were obtained in a pLKO vector (Sigma) modified to express Emerald GFP in place of the Puromycin marker.

Transduction efficiencies were -70% by GFP fluorescence. For DDR assays, transduced cells were plated at 1x105 per well in 12-well plate containing poly-L-lysine coated

glass cover slides one day before irradiation with a calibrated source (Moores UCSD Cancer Center). Two h after irradiation, cells were fixed with 4% PFA. Mouse

Figure imgf000049_0002
(Upstate, clone JBW30) and rabbit anti-ZNF423 antibodies were added at room temperature for 2 h. Donkey anti-mouse (Alexa fluor 555) and anti-rabbit (Alexa fluor 488) antibodies (Jackson Immunoresearch) were incubated at room temperature for 1 h. Images were captured on an Olympus FV1000 confocal microscope. Signal intensities of
Figure imgf000049_0001
were quantified from image files in ImageJ software. Distribution of foci in Zfp423 -knockdown cells did not meet the conditions of a ttest and were analyzed by the Mann- Whitney U (Wilcoxon rank sum) test implemented in R 2.8.1.

FACS analysis of γΗ2ΑΧ. Both control and Cepl64 stable knockdown IMCD3 cells were irradiated with indicated doses (Fig. 6Q). Cells were then fixed 60 min post-irradiation with 2% paraformaldehyde, permeabilized with 0.1% SDS and stained with rabbit γΗ2ΑΧ (Cell Signaling). Alexa-fluor-488 conjugated secondary antirabbit antibody was used to analyze the mean fluorescent intensity (MFI) of samples in triplicate in the FACS facility (Cancer Center, University of Michigan). Results shown are representation of mean and SD of triplicate samples.

Statistical analysis. Student's two-tailed non-paired t-tests and normal distribution two-tailed z-tests were carried out using pooled standard error and s.d. values to determine the statistical significance of different cohorts. RESULTS

Whole exome resequencing accelerates discovery of NPHP-RC genes. Identification of monogenic causes of ciliopathies is limited by their rarity (Attanasio et al., Nat Genet 39, 1018-1024 2007), necessitating methods to identify ciliopathy-causing genes in single families, which include whole exome resequencing (WER). However, WER typically yields hundreds of variants from normal reference sequence as "candidate mutations" (Ng et al, Nature 461, 272-276 2009), whereas only a single-gene mutation will represent the disease cause. To overcome this limitation, WER was combined with homozygosity mapping (Hildebrandt et al, PloS Genetics 5, 31000353 2009c) in sib pairs affected with NPHP-RC and performed functional analysis of the identified genes (Otto et al., Nat Genet 42, 840-850 2010a).

Homozygosity mapping yielded positional candidate regions of homozygosity by descent (Hildebrandt et al, 2009c) in families A3471 (2 regions), F874 (9 regions), and KKESH001-7 (14 regions) (Figure 1), who had one or more features of NPHP-RC, including NPHP, retinal degeneration, liver fibrosis, or cerebellar degeneration/hypoplasia (Table 1). WER was then performed in one affected individual of each of the three NPHP-RC families (Ng et al, 2009, supra; Otto et al, 2010a, supra). Each of three NPHP-RC genes

consecutively identified by this approach, MREl 1, ZNF423 and CEP 164, indicated a functional connection to the DDR pathway (Figure 1, Table 1).

A mutation of MREl 1 causes progressive cerebellar degeneration. In family F3471 two siblings had cerebellar vermis hypoplasia (CVH), a central feature of NPHPRC (Table 1). Mapping regions of homozygosity by descent yielded 2 candidate loci (Figure 1A). WER detected a homozygous truncation mutation (p.R633X) of MREl 1 (Figure IB; Table 1) previously described for CVH in a Pakistani family (Stewart et al., 1999). Family F3471 is also from Pakistan, indicating a founder effect for this allele. MREl 1 is an essential component of the ATMChk2 pathway of DDR (Figure 7), where it recruits ATM (ataxia telangiectasia-mutated) to sites of DNA double-strand breaks (Figure 7A). Rediscovery of this MREl 1 mutation in family F3471 thus generated an unexpected link between NPHP-RC phenotype and the ATM pathway of DDR signaling (Figure 7A).

Patients with the NPHP-RC Joubert syndrome have defects in ZNF423. Another link of NPHP-RC to the ATM pathway of DDR signaling emerged from homozygosity mapping and WER in two siblings (F874) with infantile onset NPHP, CVH, and situs inversus (Table 1). SNP mapping yielded nine candidate regions of homozygosity by descent (Figure 1C). Both affected individuals had a homozygous missense mutation (p.P913L; conserved in vertebrates) of ZNF423 (Figure ID). In addition, when examining 96 additional Joubert syndrome subjects, two heterozygous-only mutations of ZNF423 were detected: p.P506fsX43 in family A106 and p.H1277Y in individual Al 11-21 (Table 1). Mutations of the mouse ortholog Zfp423 cause reduced proliferation and abnormal development of midline neural progenitors resulting in a loss of the cerebellar vermis (Alcaraz et al., Proc Natl Acad Sci U S A 103, 19424-19429 2006; Cheng et al, Dev Biol 307, 43-52 2007) similar to that seen in Joubert syndrome patients with CVH.

ZNF423 encodes a protein with 30 zinc fingers (Figure 2A). A mouse mutation disrupting only the last zinc finger, similar to p.H1277Y, is sufficient to cause a severe phenotype (Cheng et al, 2007, supra). Mouse models also display phenotypic variability that is subject to modifier genes, environment, and stochastic effects (Alcaraz et al., Hum Mol Genet 2011; Alcaraz et al., 2006, supra), consistent with the variable presentations of NPHP- RC patients. The homozygous mutation p.P913L, located between zinc fingers 21 and 22 (Figure 2A), may exert recessive loss-of-function, analogous to the Zfp423 mouse models. Of the heterozygous-only mutations, one (p.N506fsX43) truncates the protein before the 11th zinc finger, whereas the other mutation (p.H1277Y) abrogates a zinc coordinating histidine that is part of the "knuckle" in the last zinc finger, which is required for interactions with

EBF family transcription factors (Tsai and Reed, Mol Cell Biol 18, 6447-6456 1998), and is important for ZNF423 function (Figure 2A).

It was next examined whether the heterozygous-only mutations (Table 1) lead to loss of function via a dominant mechanism, using a proliferation assay in P19 cells (Figure 2B-D). Mutations were engineered into a FLAG-tagged ZNF423 cDNA and assayed by a S-phase index, defined as the proportion of transfected cells that incorporate BrdU in 1 h, 48 h after transfection. Simple loss of function alleles should not interfere with endogenous Zfp423 activity in this assay. Indeed, overexpression of either wild-type or the homozygous p.P913L allele had no effect (Figure 2D). However, transfection with either the p.P506fsX43 frame- shifting allele, which removes the zinc fingers required for SMAD and EBF interactions, and the H1277Y substitution allele, which destroys the terminal zinc finger required for EBF interaction, reduced the mitotic index to little more than half that of cells transfected with GFP control vector or other alleles of ZNF423 (Figure 2B-D). A dominant mechanism is plausible for the two heterozygous mutations, as each is predicted to interfere selectively with a subset of interaction domains (Figure 2A). Neither subject had siblings, and DNA from parents was not available to determine whether the mutations occurred de novo.

Five additional putative mutations were identified in highly conserved (including histidine knuckle) residues of ZNF423 among Joubert syndrome families (Table 2). While these mutations have not been confirmed functionally, the high incidence of predicted deleterious mutations found in patients but absent from 270 healthy control individuals, dbSNP, and 1,000 Genomes Project data further support identification of ZNF423 as a causal gene in NPHP-RC and JBS.

ZNF423/OAZ was recently shown to interact with the DNA ds-damage sensor PARP1 (poly-ADP ribosyl polymerase 1) (Ku et al, 2003), which recruits MRE11 and ATM to sites of DNA damage (Figure 7 A). This indirectly linked ZNF423 to the ATM pathway of DNA damage signaling (Figure 7A). It was therefore tested whether ZNF423 mutations affect interaction between ZNF423 and PARP1. Coimmunoprecipitation verified the association of ZNF423 and PARP1 in reciprocal assays (Figure 2E). The truncating mutation P506fsX43, which was detected in a JBTS patient (Table 1), abrogates this interaction (Figure 2E), while H1277Y inhibits multimerization of ZNF423 (Figure 2E). In addition, depletion of ZNF423 mRNA caused sensitivity to DNA damaging agents (see below).

Furthermore, ZNF423 was identified as a direct interaction partner of CEP290/NPHP6, which is mutated in NPHP-RC (Sayer et al, Nat Genet 38, 674-681 2006; Valente et al, Nat Genet 38, 623-625 2006). In a yeast two-hybrid screen of human fetal brain library with a CEP290 (JAS2; amino acids 1917-2479) 'bait' 3 in- frame "prey" sequences corresponding to ZNF423 (amino acids 178-406) were found. This interaction was confirmed in a direct yeast two-hybrid assay (Figure 2F) and by co-immunoprecipitation of CEP290/NPHP6 with ZNF423 expressed in HEK293T cells (Figure 2G). CEP290/NPHP6 is known to interact with the NPHP-RC protein NPHP5 (Schafer et al, 2008) and localizes to the ciliary transition zone (Sang et al, Cell 145, 513-528 2011). A nuclear function of CEP290/NPHP6 is likely: it contains a nuclear localization sequence (NLS), binds the transcription factor ATF4, and localizes to the nucleus by cell fractionation (Sayer et al., 2006, supra). Direct binding between ZNF423 and CEP290/NPHP6, whose mutation also causes a CVH phenotype (Sayer et al., 2006; Valente et al., 2006), is consonant with other NPHP-RC protein interactions in dynamic complexes (Sang et al, 2011, supra).

Mutations of CEP 164 cause NPHP-RC. Leber congenital amaurosis (LCA) is an early-onset form of isolated retinal degeneration (RD) that can be allelic with NPHP-RC. For example, null mutations of CEP290/NPHP6 cause severe multiorgan NPHP-RC variants of JBTS and MKS syndromes (Helou et al, J Med Genet 44, 657-663 2007), whereas hypomorphic mutations cause LCA only (Chang et al., Hum Mol Genet 15, 1847-1857 2006; den Hollander et al., Am J Hum Genet 79, 556-561 2006). By homozygosity mapping in a Saudi family (KKESHOOl) of first-cousin parents with a child who had LCA with nystagmus, hyperopic discs, vascular attenuation, diffuse retinal pigment epithelium atrophy, and non- recordable ERG (Table 1), 14 candidate regions (Figure IE) were identified. By whole exome resequencing a homozygous point mutation in CEP 164 (centrosomal protein 164 kDa) was identified that abolished the termination codon, adding 57 amino acid residues to the open reading frame (p.X1460WfsX57) (Figure IF, Table 1). The mutation was absent from 96 Saudi healthy controls and from 224 North American LCA patients who lack mutations in other known LCA genes.

CEP 164 was also considered as a candidate gene for NPHP-RC, because it is part of the human centrosomal proteome (Andersen et al., Nature 426, 570-574 2003). Exon-PCR and Sanger sequencing of all 31 coding exons for one affected individual in each of 856 different NPHP-RC families was performed. Both mutated CEP 164 alleles were detected in each of 3 additional families with NPHP-RC (Table 1; Figure 8). Specifically, i) a

homozygous missense mutation (p.Ql IP; conserved to Chlamydomonas) was detected in two siblings of family F319 with Senior- Loken syndrome (NPHP with retinal degeneration) (Table 1), ii) compound heterozygosity for a missense mutation (p.R93W; conserved to

Chlamydomonas) and a truncating mutation (p.Q525X) in three siblings of family F59 with Senior-Loken syndrome, one of whom also had seizures and intellectual disability and, iii) a homozygous nonsense mutation (p.R576X) in individual NPH-505 with Senior-Loken syndrome, partial vermis aplasia and bronchiectasis (Table 1) were detected. In individuals with LCA heterozygous mutations, for which a second recessive allele was not detected, were identified. These were p.R93W, also present in F59, and p.Q1410X, which was detected in two families with phenotypic features of Joubert syndrome and oral-facial-digital syndrome (OFG6), and heterozygously in two out of 96 healthy control individuals. All other mutations were absent from >96 ethnically matched healthy control individuals and from healthy controls of the 1000 genomes project (Table 1). Recessive mutations of CEP164 were identified as a new cause of NPHP-RC. Because of the significant overlap of phenotypic features with other forms of NPHP-RC, the alias "NPHP 14" was identified for the CEP164 protein. Although the number of families with CEP 164 mutation is small, the findings indicate a genotype phenotype correlation (Table 1). First, the patient with the mildest phenotype had isolated LCA only and a homozygous mutation (p.X1460WfsX57) that removes the termination codon, adding 57 new amino acids to the C-terminus. This may reduce protein function, because overexpression of full length CEP 164 with a C-terminal GFP tag strongly reduced centrosomal labeling compared to an Nterminal GFP tag (Figure 3 A-D). Second, the homozygous missense allele (p.Q11P) caused a multiorgan phenotype of NPHP with retinal degeneration (Senior-Loken syndrome; SLSN). Third, compound heterozygosity for one missense and one truncating allele (p.R93W; p.Q525X) caused SLSN with additional central nervous system involvement. Fourth, a homozygous truncating mutation (p.R576X) caused a severe phenotype that combined Joubert syndrome (NPHP, retinal degeneration and vermis hypoplasia) with bronchiectasis, a classic feature of primary ciliary dyskinesia (PCD), a disease of motile cilia. These data further support the gradient of genotype-phenotype correlations characteristic of NPHP-RC, in which null mutations cause the severe dysplastic phenotypes of Meckel syndrome and Joubert syndrome, whereas hypomorphic alleles cause the milder degenerative phenotypes of NPHP and SLSN (Hildebrandt et al., N Engl J Med 364, 1533-1543 2011).

CEP 164 is transcribed into 3 common iso forms (Figure 8A-C). In addition to the centrosome, CEP 164 is part of the photoreceptor sensory cilium proteome (Liu et al., Mol Cell Proteomics 6, 1299-1317 2007). Homozygous truncating mutations found in exons common to iso forms 1 and 3, but not in iso form 2 support the idea that isoforms 1 and/or 3 are relevant for the NPHP-RC phenotype (Figure 8B). The deduced CEP 164 amino acid sequence has a WW domain (aa 57-89), which interacts with the DDR protein ATRIP (Figure 8D). CEP 164 also contains a segment of 6 coiled-coil domains (Figure 7C), which are frequently found in NPHP-RC genes (Hildebrandt et al, 2009a). CEP 164 is conserved across species including the green alga Chlamydomonas reinhardtii, indicating a conserved function with strong sequence constraints. To study expression and subcellular localization of the CEP 164 protein antibodies against human CEP 164 were utilized by immunob lotting and immunofluorescence (Figure 9).

Mutation of CEP 164 abrogates mother centriole localization. By confocal microscopy of GFP-labeled CEP 164 protein with other labels, it was shown that CEP 164 co localizes in hTERT-RPE cells with the mother centriole, with the mitotic spindle poles, and with the abscission structure in a cell cycle-dependent way (Figure 10), a feature characteristic of proteins involved in single-gene ciliopathies (Otto et al, Nat Genet 42, 840-850 2010a) (Graser et al, J Cell Biol 179, 321-330 2007). Subcellular localization of CEP164 in mouse retina (Figure 9C) as well as in MDCK2 (Madin Darby canine kidney) cell lines, IMCD3 (mouse kidney inner medullary collecting duct) cell lines (Figure 3), and hTERT-RPE (human retinal pigment epithelium) cell lines (Figure 9D-F, Figure 11 A) that stably express GFP-tagged human full length CEP 164 wild type isoform were generated from a doxycyclin- inducible stable expression construct (NGFP-hCEP164-WT). It was found using the a- CEP164-NR antibody in MDCK cells as well as upon induction with doxycyclin (10 ng/ml) of N-terminally GFP tagged NGFP-hCEP164-WT in IMCD3 cells, CEP 164 localizes to mother centrioles of cells (Figure 3A-B). In contrast, the signal at centrosomes was abrogated upon overexpression of an N-terminally GFP-tagged truncated CEP 164 construct

representing the mutation p.Q525X (Figure 3C). Furthermore, the signal occurred at a reduced number of centrosomes upon overexpression of C-terminally GFP-tagged human full-length construct (Figure 3D), which mimics the mutation p.X1460WfsX57 occurring in NPHP-RC family KKESH001 that causes a read-through of the stop-codon X1460, adding 57 aberrant amino acid residues to the Cterminus of CEP 164 (Table 1). Corresponding data were obtained upon CEP 164 expression in hTERT RPE cells (Figure 9D-E). A lack of

centrosomal localization for the truncating mutation p.Q525X and for an equivalent of the p.Q1460WextX57 mutation was demonstrated.

Endogenous Cepl64 levels regulate cilia in 3D epithelial spheroid structures. Loss of function of several genes that cause nephronophthisis in NPHP-RC cause disruption of 3D architecture of renal epithelial cell culture (Otto et al., Nat Genet 42, 840-850 2010a; Sang et al, Cell 145, 513-528 2011). To evaluate CEP164 by this criterion, murine kidney IMCD3 cells were transfected with siRNA oligonucleotides against murine Cepl64, or random sequences (Ctrl) in 3D spheroid growth assays. After 3 days, siCtrl transfected cells formed spheroid structures with a clear lumen, apical cilia, defined tight junctions and clear basolateral structure. Cells transfected with siCepl64 developed spheroids with overall normal architecture and size, but with markedly reduced frequency of cilia (Figure 3E-H). While 49% of the spheroid cells transfected with siCtrl generated detectable cilia, only 33% of the siCepl64 transfected cells grown in 3D cultures were ciliated (p<0.0001). It was concluded that Cepl64 affects ciliogenesis or maintenance, but that the overall architecture of renal 3D growths is not as grossly affected as previously seen for knockdown of other NPHP- RC genes (Sang et al, 2011, supra). To exclude the possibility that the effect observed was due to off-target effects, two IMCD3 clones stably transfected with inducible full-length human CEP 164 (IMCD3-NGFP- CEP164- WT clones 2 and 8) were utilized. Both lines form spheroids in 3D cultures. siRNA of the endogenous murine Cepl64 was performed and no irregularities except for reduced cilia frequency were observed. However, after doxycycline induction of siCepl64-resistant human NGFP-CEP164, ciliary frequencies were restored, with 57% of the siCepl64 transfected cells ciliated (p=<0.0001) (Figure 3G). Rescue was not observed for IMCD3 cells expressing a patient mutation (NGFP-CEP164-Q525X, Figure 3H). Reducing cellular levels of Cepl64 affects renal ciliation frequencies without gross disturbances to tubular

architecture, a phenomenon that can be rescued with WT but not mutant CEP 164.

NPHP-RC proteins colocalize with the DDR protein TIP60 to nuclear foci. A non- centrosomal localization for CEP 164 was recently described by demonstrating its

translocation to nuclear foci in response to DNA damage (Pan and Lee, Cell Cycle 8, 655- 664 2009; Sivasubramaniam et al, Genes Dev 22, 587-600 2008). In this context CEP164 is thought to play a role in DNA damage-response (DDR) signaling where it interacts with the DDR protein ATRIP (Figure S2D), is activated by the DDR proteins ATM and ATR, and is necessary for checkpoint- 1 (Chkl) activation. Abrogation of CEP 164 function leads to loss of G2/M cell cycle checkpoint and aberrant nuclear divisions (Sivasubramaniam et al., 2008, supra). Because the NPHP-RC gene products MREl 1 and CEP 164 are essential components of DDR signaling, and because ZNF423 interacts with the bona fide DDR protein PARP1 (Ku et al., Biochem Biophys Res Commun 311, 702-707 2003), the identification of mutations in these genes support a role of DDR signaling in the pathogenesis of NPHP-RC (Figure 7). Nuclear foci localization for the newly identified NPHP-RC causing gene products ZNF423 and CEP 164 and for previously identified NPHP-RC gene products was examined.

Localization of SDCCAG8 (alias NPHP10), in which NPHP-RC mutations were previously identified (Otto et al, 2010a, supra), shows nuclear foci in hTERT-RPE cells in addition to its centrosomal localization (Figure 4B-D). Transient shRNA knockdown confirmed specificity of the signal (Figure 10B-D). SDCCAG8/NPHP 10 did not colocalize with markers for PLM bodies (Janderova-Rossmeislova et al, J Struct Biol 159, 56-70 2007) or CENP-C (marking chromosomal centromeres) (Figure 11 A). In contrast

SDCC AG8/NPHP 10 fully colocalized with SC35 in hTERT-RPE cells (Figure 4A-C). SC35, also known as serine/arginine-rich splicing factor 2 (SRSF2), is a splicing factor that plays a role in DDR by controlling cell fate decisions in response to DNA damaging agents (Edmond et al, EMBO J. 2010; Reinhardt et al, Cell Cycle 10, 23-27 2011). SC35 marks hubs of enhanced gene expression (Szczerbal and Bridger, Chromosome Res 18, 887-895 2010), is phosphorylated by topoisomerase I (Elias et al, Exp Cell Res 291, 176-188 2003), and is required for genomic stability during mammalian organogenesis (Xiao et al., Mol Cell Biol 27, 5393-5402 2007). Moreover, ZNF423 also fully colocalizes (Figure 4D), and CEP 164 partially colocalizes (Figure 4E) with SC35 in nuclear foci. Consequently, ZNF423 and CEP164 also colocalize with SDCC AG8/NPHP 10 in SC35-positive nuclear foci (Figure 4F,G).

SC35 functions within a TIP60 complex, in which TIP60 acetylates SC35 on lysine

52 (Figure 7B), modifying the role of SC35 in the promotion of apoptosis and inhibition of G2/M arrest (Edmond et al., EMBO J. 2010), which is regulated by the checkpoint proteins Chkl and Chk2 (Figure SID). The TIP60 protein, together with the heterotrimeric MRN complex (of which MRE11 is a component) constitutes the major activator of ATM within the ATM pathway of DDR signaling (Ciccia and Elledge, Mol Cell 40, 179-204 2010)

(Figure 7A). In hTERT-RPE cells the ATM activator TIP60 colocalizes to nuclear foci with SC5/SRSF2 (Figure 4H) and partially with the newly identified NPHP-RC protein CEP 164 (Figure 41). A new group of NPHP-RC proteins that colocalize to nuclear foci with the DDR proteins TIP60 and SC35 is thus identified. These gene products include the newly identified NPHP-RC proteins in ZNF423 and CEP164 as well as SDCCAG8/NPHP10. The protein OFD1, which is mutated in the ciliopathy oral-facial-digital syndrome, is part of the TIP60 complex, which is a major ATM activator within DDR signaling; OFD1 has been identified as a direct interaction partner of SDCCAG8/NPHP 10 (Figure 7B) (Otto et al, 2010a, supra).

CEP164CEP164 colocalizes with the DDR proteins TIP60 and CHK1 in nuclear foci upon DNA damage. Because one of the central mechanisms controlled by DDR signaling is cell cycle regulation through phosphorylation of checkpoint- 1 (Chkl) and checkpoint-2 (Chk2) proteins (Figure 7D), it was tested whether checkpoint proteins are recruited to SC25/SRSF2 -positive nuclear foci. SC35 and p317-Chkl colocalize to nuclear foci in hTERT-RPE cells (Figure 4J). As colocalization conveys only a static image of DDR components, it was then tested whether localization of CEP 164 to nuclear foci was inducible by DNA damage as suggested (Sivasubramaniam et al, 2008, supra). Following irradiation with 20-50 J/m2 of UV light, CEP164-positive nuclear foci condensed to larger size and colocalized with newly appearing TIP60 foci of similar size (Figure 4K-M). Similarly, a pattern of broad CEP 164 speckles, which were CHK1 -negative and locate to DAPI-negative domains in untreated cells (Figure 4N), changed to a pattern of multiple smaller foci that were positive for both CEP 164 and CHK1 (Figure 40). Colocalization of TIP60 and p317- CHK1 in these foci (Figure 4P) demonstrated that the UV-inducible foci observed in HeLa cells are likely equivalent to the foci positive for TIP60 and SC35 that were observed as positive for NPHP-RC proteins in hTERT-RPE cells (Figure 4 A- J). It was thus demonstrated that CEP 164 translocates in response to DNA damage to nuclear foci that contain the DDR proteins TIP60 and CHK1.

Reducing endogenous levels of Cepl64 causes anaphase lagging chromosomes. Lagging chromosomes on anaphase spindles ("anaphase lag") are a hallmark of many mutations that affect mitotic checkpoint integrity. Acute reduction of Cepl64 by siCepl64 knockdown in IMCD3 cells exacerbated the incidence of anaphase lag from 1% in siCtrl controls to 21% in siCepl64-treated cells, in over 250 anaphases scored from unsynchronized cells in a total of five independent experiments (Figure 5A-B, p=0.04). CREST antiserum and DAPI confirmed the presence of incomplete mitotic congression and unattached kinetochores during late anaphase. This phenomenon was specific, since doxycycline-inducible expression of WT-CEP164 during Cepl64 siRNA knockdown reduced the incidence of anaphase lag to just 4% (Figure 5B). Untransfected (non-siRNA treatment) IMCD3 cells had no detectable anaphase lag (0%). These data indicate a requirement for Cepl64 at the G2/M checkpoint.

Human wild type CEP 164 but not its NPHP-RC truncation rescues IMCD3 cell proliferation. In clonally selected IMCD3 cells expressing wild type human CEP 164 cDNA construct N-GFPCEP164-WT under doxycycline (Dox) control, depletion of endogenous mouse Cepl64 retarded proliferation in comparison to either undepleted control cells or undepleted cells that were Doxinduced to overexpress N-GFP-CEP164-WT alone (Figure 5C). Cepl64-depleted growth was rescued by Dox-induced expression of human N-GFP- CEP164-WT (Figure 5C). Cells expressing truncated cDNA construct N-GFP-CEP164- Q525X, modeling the NPHP-RC mutation in family F59, exhibited retarded growth, even when the endogenous Cepl64 was present (Figure 5D), consistent with a dominant negative effect. Further depletion of the endogenous Cepl64 in N-GFP-CEP164-Q525X expressing cells showed an additive effect on growth retardation, confirming the dominant negative effect of N-GFP-CEP164-Q525X in this experimental system (Figure 5D). CDK inhibition translocates Cep164 to the nucleus. CEP 164 is required for DNA damage-induced phosphorylation of Chk1, and down regulation of CEP 164 significantly reduces DDR (Maude and Enders, Cancer Res 65, 780-786 2005; Sivasubramaniam et al, 2008, supra). On the other hand, the DDR pathway can also be activated by the small molecule CDK inhibitor roscovitine, which in addition reduces Chk1 expression (Maude and Enders, 2005, supra). Roscovitine also reduces the development of kidney cysts in the Nphp9 mouse model, Jck (Bukanov et al, Nature 444, 949-952 2006). The influence of roscovitine (targeting CDK2, 5, 7 and 9) on DDR activation in IMCD3 cells was assayed.

Immunofluorescence shows increased uniform distribution of γΗ2ΑΧ (activated H2AX phosphorylated at Serl39) in the nucleus of IMCD3 cells upon roscovitine treatment in irradiated cells, indicating partial DDR activation (Figure 5E). Second, in cells treated with roscovitine, UV irradiation caused enhanced γΗ2ΑΧ staining with a prominent nuclear foci pattern, characteristic of strong DDR activation (Figure 5F). Immunoblotting showed that roscovitine decreased the amount of CEP 164 present in both control and UV-irradiated cells (Figure 5G-H). This was most likely due to translocation of CEP 164 into the nucleus upon roscovitine treatment, as shown by subcellular fractionation (Figure 5H). UV radiation increased phosphorylation of Chkl at Ser317 (p-Chkl) (Figure 5G), and roscovitine decreased Chkl protein expression and abrogated UV-induced p-Chkl in both cytoplasm and nucleus (Figure 5G-H). Together, these data indicate that CDK inhibition by roscovitine causes nuclear translocation of CEP 164 and inhibits Chkl activation. γΗ2ΑΧ activation by roscovitine may restore cell cycle control by Chk2 activation instead (Maude and Enders, Cancer Res 65, 780-786 2005).

CEP 164 directly interacts with CCDC92 and TTBK2. NPHP-RC proteins are known to interact with other NPHP-RC proteins in dynamic complexes that have been termed the "NPHP-JBTS-MKS interaction network" (Sang et al, Cell 145, 513-528 2011). In order to identify novel direct interaction partners of CEP 164 yeast two-hybrid screening of both a random-primed bovine retina and an oligo-dT primed human retina cDNA library was performed. Full length and partial cDNA clones of CEP 164 were used as baits: CEP164fl (encoding the full length protein), CEP 1641 -550 (encoding amino acids 1 -550), CEP 164551 - 1100 (encoding amino acids 551-1100) and CEP 1641101-1460 (encoding amino acids 1101- 1460). CCDC92 (coiled coil domain containing protein 92) and TTBK2 (tau tubulin kinase 2) were identified as direct interactors of CEP 164 (Figure 1 ICJ). CCDC92 was the protein with the most positive clones in the screen with the human oligo-dT retinal cDNA library, while TTBK2 appeared to be the main CEP 164 interactor in the bovine library. Two overlapping CCDC92 clones were found with baits CEP164fi, CEP1641-550 and CEP1641101-1460 in the human library, however, no CCDC92 clones were identified with the bovine library (Figure 11C). TTBK2 was identified in both screens, with 12 hits (4 different clones) in the bovine, and a single clone in the human retina cDNA library (Figure S5C). Interactions between CEP 164 and each of these new partners were validated by GST pulldown (Figure 1 ID) and co-immunoprecipitation (Figure 11E-H). Immunofluorescence showed that CCDC92 fully colocalizes with CEP 164 at the mother centriole (Figure 111). TTBK2 weakly colocalizes with CEP 164 at one of the centrioles, but yields a strong signal at the mid body in dividing hTERT-RPE cells (Figure 11 J). Tau tubulin kinase 2 (TTBK2) is a member of the casein kinase family encoded by the gene mutated in cerebellar ataxia type 11 (Houlden et al., 2007). TTBK2 can phosphorylate tubulin, and its kinase activity is required for the phosphorylation of tau by GSK-3β. Tau is a microtubule associated protein that is also found in the nucleus, where it is a key player in the early stress response/DNA damage protection of neurons (Sultan et al, Biol Chem 286, 4566-4575 2011).

CEP 164 interacts with NPHP3 and DVL3. To determine whether CEP 164 interacts with known NPHP-RC proteins, HEK 293T cells were cotransfected with N-terminally V5- tagged human full-length CEP 164 and the seven different FLAGtagged human full length proteins NPHP1-NPHP5, NPHP8, NPHP9 or the control protein CD2AP. NPHP proteins were precipitated, using anti-Flag M2 beads. Interaction of CEP 164 with NPHP3 and weakly with NPHP4 was detected (Figure 11 A-B), demonstrating that CEP 164 is in a complex with other known NPHP-RC proteins (Figure 7 A-B). The DDR protein DDB1 interacted with NPHP2 (Figure 12C-D).

The dishevelled protein (Dvl) is a central component of the Wnt pathway and it has been shown that NPHP2/inversin interacts with Dvl targeting it for proteasomal degradation, thereby liberating the β-catenin destruction complex and triggering a switch from canonical to non-canonical Wnt signaling (Germino, Nat Genet 37, 455-457 2005; Simons et al, 2005). In loss of function of the NPHP-RC protein NPHP2/INVS, which causes NPHP type 2 in humans (Otto et al, Nat Genet 34, 413-420 2003), this switch is lacking (Simons et al, Nat Genet 37, 537-543 2005). By immunoprecipitation coupled to mass spectrometry using endogenous Dvl3 as bait interaction between Dvl3 and CEP 164 (Figure S6E-H) was identified. Immuno cytochemistry revealed that endogenous Dvl3 and CEP 164 share centrosomal localization in most cells analyzed (Figure 12 A). To further study CEP164-Dvl3 interaction a GST-pull down assay in HEK293T cells, as well as domain mapping, using a set of Dvl3 deletion constructs (Angers et al. Nat Cell Biol 8, 348-357 2006), was performed. It was demonstrated that GST-CEP164 (aa2-195), which was shown to interact with the DDR protein ATRIP (Sivasubramaniam et al, 2008, supra), is sufficient to pull down endogenous Dvl3 from the cellular lysate (Figure 12B). Domain mapping for Dvl3 indicates that CEP 164 interacts with the pro line-rich region of Dvl3, because only mutants containing this sequence efficiently co-immunoprecipitate with CEP164-GFP (Figure 12C). Only wild type CEP 164- mCherryRFP but not the NPHP-RC causing mutant CEP164-Q525X detected in family F59 (Table 1) can be efficiently immunoprecipitated with Dvl3 (Figure 12D), further supporting its pathogenic role. cepl64 loss of function causes NPHP-RC and DDR activation in zebrafish. To test in a vertebrate animal model whether loss of cepl64 function results in both, an NPHP-RC phenotype as well as DDR activation, cepl64 knockdown was performed in zebrafish embryos using a morpholino-oligonucleotide (MO) that targets the exon 7 splice donor site of cepl64 (Fig. 6). A p53 MO was injected to reduce off-target MO effects (Robu et al, PLoS Genet 3, e78 2007). At 28 hours post fertilization (hpf) the ciliopathy phenotypes of ventral body axis curvature and cell death were observed (Fig. 6AC). Embryos showed increased expression of phosphorylated γΗ2ΑΧ (Fig. 6D-E). At 48 hpf, cepl64 morphants displayed the typical ciliopathy phenotype of abnormal heart looping as a laterality defect (Fig. 6F-I). Furthermore, at 72 hpf, embryos developed further NPHP-RC phenotypes, including pronephric tubule cysts (Fig. 6J-K), as well as hydrocephalus and retinal dysplasia (Fig. 6L- O). To test in a vertebrate animal model whether loss of cepl64 function results in both, an NPHP-RC phenotype as well as DDR activation, cepl64 knockdown was performed in zebrafish embryos using a morpholino-oligonucleotide (MO) that targets the exon 7 splice donor site of cepl64 (Fig. 6). A p53 MO was injected to reduce off-target MO effects (Robu et al., 2007,supra). At 28 hours post fertilization (hpf) the ciliopathy phenotypes of ventral body axis curvature and cell death were observed (Fig. 6AC). Embryos showed increased expression of phosphorylated γΗ2ΑΧ (Fig. 6D-E). At 48 hpf, cepl64 morphants displayed the typical ciliopathy phenotype of abnormal heart looping as a laterality defect (Fig. 6F-I). Furthermore, at 72 hpf, embryos developed further NPHP-RC phenotypes, including pronephric tubule cysts (Fig. 6J-K), as well as hydrocephalus and retinal dysplasia (Fig. 6L- O). Depletion of CEP 164 or ZNF423(Zfp423) causes sensitivity to DNA damaging agents. To assess whether depletion of CEP 164 causes sensitivity to DNA damage, Cepl64 expression was stably suppressed in the mouse renal cell line IMCD3 (Fig. 6P-Q). Cepl64 knockdown resulted in a dose-dependent increase of γΗ2ΑΧ intensity levels in a FACS analysis, signifying increased radiation sensitivity to IR and perturbed DDR. Cellular sensitivity to IR was also seen in cells depleted of CEP 164 using a multicolor competition assay (MCA) (Smogorzewska et al, 2007) (Figure S7A-B).

To test whether ZNF423(Zfp423) affects DDR, P19 cells, which express high levels of endogenous Zfp423 (Fig. 6R-T) were examined. Replicate cultures infected with lentivirus expressing either scrambled control or Zfp423 -targeted shRNA were exposed to 0-10 Gy of X-irradiation and imaged for Zfp423 and nuclear γΗ2ΑΧ foci (Fig. 6R). Quantification showed significantly increased γΗ2ΑΧ intensities in Zfp423 -depleted cells at lower (0.5 and 1.0 Gy) exposures (Fig. 12), but the effect was non-significant when corrected for number of exposures. To determine whether sensitivity to lower dose is reproducible, 32 additional cultures were exposed at 1.0 Gy (Fig. 6T). Normalized γΗ2ΑΧ fluorescence in Zfp423 knockdown had both higher mean (9.6 vs. 4.7) and median (6.6 vs. 5.2) values than control (Fig. 6T). These data replicate the radiation sensitivity with high significance (p=0.018, Mann- Whitney U test, 2 tails), indicating that P19 cells require Zfp423 for quantitatively normal DDR.

Figure imgf000063_0001

Example 2

MATERIALS AND METHODS

Human subjects. Blood or DNA samples were obtained from 40 individuals diagnosed with CAKUT from 38 different families. The diagnosis was made by pediatric nephrologists on the basis of imaging studies. After informed consent was obtained, detailed clinical data and pedigree information was referred to us by the specialists through a standardized clinical questionnaire. The cohort of 40 individuals included 29 (72.5%) individuals diagnosed with unilateral renal agenesis, 3 (7.5%) individuals with renal hypodysplasia, 7 (17.5%) individuals with vesicoureteral reflux, and 1 (2.5%) individual with bilateral duplex systems.

Whole-genome amplification and DNA pooling. To normalize various DNA samples, whole-genome amplification was performed on DNA of 40 different individuals and 96 healthy control samples using Phi29-based DNA polymerase strand displacement amplification (GenomiPhi DNA amplification kit, GE Healthcare, Piscataway, NJ).

Subsequently, whole-genome amplified DNA was purified using 96-well spin columns (Rapid 96TM PCR purification system, Marligen Biosciences, Reutlingen, Germany). DNA of 20 individuals were pooled at 2 mg each and diluted to 60 ng/ml. Pooling was repeated to represent 40 individuals. In addition, an equimolar DNA pool was generated by pooling 96 DNA samples derived from healthy individuals of Caucasian origin (Human Random Control DNA Panel- 1 (HRC-1); European Collection of Cell Cultures, Salisbury, UK).

PCR amplification and massively parallel exon resequencing. All 313 exons (342 amplicons) of 30 candidate genes, ATGR2, BMP4, CDC5L, EMX2, EYAl, FOXCl, FRASl, FREM2, GATA1, GDF1 1, GDNF, GFRA1, GREMl, HOX11, HOXAl l, KALI , PAX2, RET, ROB02, SALLl, SIX1, SIX2, SIX4, SIX5, SLIT2, SPRYl, TCF2 (HNFlb), UPK3A, and USF2, were individually amplified using exon-flanking primers on each of the DNA pools. PCR products from the same pool were combined and enzymatically modified. The modified PCR fragment mixture was then used to construct an Illumina sequencing library using 'Genomic DNA Sample Prep Kit' as previously described (Otto et al, J Med Genet 2010). Each library was run on a single lane of a Solexa/Illumina Genome Analyzer GAII platform, generating about 15-20 million single-end sequence reads of 39 bases each. Sequence analysis and mutation carrier identification. Sequence alignment was performed with CLC Genomics Workbench software (CLC-bio, Aarhus, Denmark) using imported and annotated human reference genome assembly NCBI36/hg 18 as a reference. Sequence reads were mapped to exonic coding regions plus adjacent 100 bp intronic sequence of all 313 exons. Variant calls were obtained using the following filter parameters: Coverage >400-fold, variant frequency >1%, and a minimum variant count of five reads. The variant analysis included coordinates of obligatory splice sites, and all variants predicted to change the amino-acid sequence (missense, nonsense, and coding indels). Variants present in dbSNP130, the Ί000 Genomes Project' (270 control individuals), or in the healthy control pool of 96 individuals (HRC-1) were excluded from further analysis. To prioritize for downstream analysis, missense variants were scored according to the information of evolutionary conservation and the likelihood of a potential protein-damaging effect using PolyPhen software predictions (Ramensky et al, Nucleic Acids Res 2002; 30: 3894-3900). All variants with a predicted 'probably damaging' or 'possibly damaging' effect and a score above 1.4 were further analyzed. The selected variants were amplified by PCR for each individual in the corresponding DNA pool with subsequent Sanger sequencing to identify the mutation carrier.

RESULTS

Two times 20 DNA samples of individuals with CAKUT (29 with unilateral renal agenesis and 11 with other forms of CAKUT) were pooled and exon PCR was performed in 30 candidate genes. Massively parallel exon resequencing of 342 different exon PCR products generated was carried out on an Illumina/Solexa GAII platform (one pool per lane of a flow cell) delivering about 5.3 million short reads of 39 bases (5.22 million in pool no. 1 and 5.37 million in pool no. 2). Following gapped alignment to the human normal reference sequence, 75% of all reads mapped back to one of the 342 target sequences that contained the exons plus 100-bp adjacent intronic sequence. The sequence concatenation of all 342 amplicons amounts to a total length of 175.7, 68.2 kb of which were exonic coding regions. The median coverage depth for the coding regions was 1228-fold (mean 1402-fold). On average, about 95% of nucleotides in targeted coding regions had at least 400-fold coverage depth. This translates into a depth of at least 20-fold per patient, which is sufficient to cause a heterozygous change.

Mutation carrier identification by Sanger sequencing. Massively parallel exon resequencing of all PCR products of 40 individuals revealed initially a total of 114 variants from normal reference sequence within the coding regions and splice sites of the 30 candidate genes analyzed, 47 of which were known single-nucleotide polymorphisms (SNPs). Eight additional variants were present in a cohort of 96 Caucasian healthy control individuals and are thought to be either as yet unannotated SNPs or false calls due to software base calling or alignment artifacts. Of the remaining 59 variants, 11 were predicted to truncate the protein products and 16 others had a PolyPhen score higher than 1.4 (predicted to be 'possibly damaging'). These 27 variants were assumed to affect the function of encoded proteins, and thus were followed by direct Sanger sequencing to identify the mutation carrier(s) out of the respective two pools of 20 individuals each. This carrier analysis led to the confirmation of 10 of the 27 variants ('true positives') in 11 individuals from 11 different families, whereas 17 of the 27 variants could not be confirmed ('false positives'). Segregation study was conducted in all confirmed cases where DNA of relatives was available and 3 out of 10 variants were not segregating. The remaining seven variants (Table 2, Figure 13) were heterozygous missense mutations in four genes. Four mutations were in FRASl (p.L259R, p.D998Y, p.R3273H, and p.H3757Q) and one in FREM2 (p.T2338I). Mutations in FRASl and FREM2 have never been reported in nonsyndromic CAKUT in humans. All five mutations were absent from 96 healthy control individuals of Caucasian origin and 270 control individuals from the Ί000 Genomes project'. Two mutations in FRASl (p.D998Y and p.H3757Q) and one mutation in FREM2 (p.T2338I), which were detected in individuals of Arabic and Indian origin, respectively, were additionally screened and were absent in 141 ethnically matched healthy controls. As FRASl and FREM2 were known to inherit recessively, all other coding exons in respective mutation carriers were amplified and sequenced to exclude the presence of the second mutation. No further mutations were found except in A2381 II-2 (FRASl; R3273H), where an additional nonsense mutation was detected (R2621X). The R2621X change was 2349-fold covered with frequency of 4.5% but had been systematically masked out. The two mutations (R3273H and R2621X) were in trans as R3273H as heterozygously present only in the mother, whereas R2621X was heterozygously present only in the father. The elder female sibling of A2381 II-2 had died in utero of bilateral renal agenesis and absent bladder;

however, the DNA was not available for study.

Two other variants were novel mutations in known CAKUT genes: RET (p.V704F) and BMP4 (p.H121R; Table 2, Figure 13). Family Al 143 and Al 184 (both Macedonian) shared the same novel change in BMP4 (p.H121R). Both mutations were absent from 96 healthy control individuals and 270 control individuals from the Ί000 Genomes project'.

Figure imgf000066_0001
Example 3

Methods

Research subjects. Blood samples and pedigrees were obtained after obtaining informed consent, from individuals with KIN. Approval for research on human subjects was obtained from the University of Michigan Institutional Review Board and the other institutions involved. Diagnosis with NPHP -related ciliopathy was based on published clinical criteria. The clinical features of many individuals with KIN have been reported (Table 3) (Palmer et al, Diagn. Cytopathol. 35, 179-182 (2007); Spoendlin, et al. Am. J. Kidney Dis. 25, 242-252 (1995); Baba et al, Pathol. Res. Pract. 202, 555-559 (2006); Godin, et al, Verine et al, Ann. Pathol. 30, 240-242 (2010); Moch et al, Pathologe 15, 44-48 (1994)).

Homozygosity mapping. For genome-wide homozygosity mapping (Hildebrandt, F. et al. PLoS Genet. 5, e1000353 (2009)), the Human Mapping 250k Styl array and the Genome- wide Human SNP 6.0 Array from Affymetrix were used. Genomic DNA samples were hybridized and scanned using the manufacturer's standard protocol at the University of Michigan Core Facility. Non-parametric logarithm of odds (LOD) scores were calculated using a modified version of the GENEHUNTER 2.1 program (Kruglyak, et al., Am. J. Hum. Genet. 58, 1347-1363 (1996); Strauch, K. et al. Am. J. Hum. Genet. 66, 1945-1957 (2000)) through stepwise use of a sliding window with sets of 110 SNPs using ALLEGRO35.

Genetic regions of homozygosity by descent (homozygosity peaks) were plotted across the genome as candidate regions for recessive disease-causing genes (Fig. 14a) as described (Hildebrandt, F. et al. PLoS Genet. 5, e1000353 (2009); Otto, E.A. et al. Nat. Genet. 42, 840- 850 (2010)). Disease allele frequency was set at 0.0001, and European ancestry marker allele frequencies were used.

Whole-exome sequencing. Exome enrichment was conducted following the manufacturer's protocol for NimbleGen SeqCap EZ Exome v2' beads (Roche NimbleGen). The kit interrogates a total of approximately 30,000 genes (-330,000 coding sequence (CCDS) exons). Massively parallel sequencing was performed as described (Bentley, D.R. et al. Nature 456, 53-59 (2008)).

Mutation calling. Following exome sequencing, mutation calling was performed using CLC Genomics Workbench software. The minimum length fraction with which a read had to match the reference sequence was set to 90%. For SNP detection, the minimum quality score of the central base and the minimum average quality score of surrounding bases were kept at default values (20 and 15, respectively). Quality assessment was performed within a window of 11 bases. Only reads that uniquely aligned to the reference genome were used for variant SNP or deletion/insertion polymorphism (DIP) calling. In individuals with evidence of homozygosity by descent, the threshold for the number of reads (minor allele frequency) was set to >55%.

Filtering of variants from normal reference sequence (VRS). For DIPs and SNPs, we used the following a priori criteria to restrict the high number of VRS (average of 53,272 for DIPs and 315,372 for SNPs). (i) We retained exonic variants (missense, nonsense and indels) and obligatory splice-site variants only, (ii) We included only VRSs that were not listed in the SNP129 database of innocuous polymorphisms, (iii) We evaluated exonic changes only within genomic regions in which homozygosity mapping showed linkage for both affected siblings (retaining on average 38 for DIPs and 169 for SNPs). (iv) Variants were analyzed using the BLAT program at the UCSC human genome Bioinformatics Browser for the presence of paralogous genes, pseudogenes, misalignments at ends of sequence reads and for whether the variant was a known variant in dbSNP132 with an allele frequency of >1% in populations of European ancestry. In families in whom mapping showed homozygosity by descent, we retained only homozygous variants and examined all of them in the sequence alignments within CLC Genomics Workbench software for the presence of mismatches indicating potential false alignments or poor sequence quality, (v) Sanger sequencing was performed to confirm the remaining variants in original DNA samples and to test for intrafamily segregation in a recessive mode, (vi) Finally, remaining variants were ranked by whether mutations truncated the conceptual reading frame (nonsense, frameshift and obligatory splice variants), by analysis of evolutionary conservation of missense variants, by using web-based programs predicting the impact of disease candidate variants on the encoded protein and by whether variants were known disease-causing mutations.

Segregation analysis by Sanger sequencing. Sanger dideoxy- terminator sequencing was applied for confirmation and segregation of potential disease-causing variants in the respective affected subjects, their affected siblings and their parents. In affected subjects in whom only one heterozygous mutation was detected by exome capture and massively parallel sequencing, all exons and flanking intronic sequences of the respective gene(s) were analyzed by Sanger sequencing. PCR was performed using a touchdown protocol described previously (Otto, E.A. et al. Hum. Mutat. 29, 418-426 (2008)). Sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit on an ABI 3730 XL sequencer (Applied Biosystems). Sequence traces were analyzed using Sequencher (version 4.8) software (Gene

Codes Corporation).

Web-based variant analysis. Predictions of the possible impact of an amino-acid substitution on chemical change, evolutionary conservation and protein function were obtained using the following web-based programs: PolyPhen, PolyPhen-2, SIFT and

MutationT aster. GERP calculation was performed at the SeattleSeq website.

Cell culture. Human fibroblasts were grown in 3% oxygen in DMEM supplemented with 15% FBS, 100 U/ml of penicillin, 0.1 mg/ml streptomycin and nonessential amino acids

(all from Invitrogen). BJ cells are normal foreskin fibroblasts and were obtained from ATCC. Fibroblasts were immortalized using pWZLhTERT and/or pMSCVNeo HPV16E6E7 plasmids. LCLs were immortalized using Epstein-Barr virus (EBV) and were grown in RPMI supplemented as above, except with 20% FBS.

DNA damage sensitivity assay. Cells were plated in a 6-well plate in triplicate at a density of 5 x 104 cells per well for primary fibroblasts and LCLs or 2.5 x 104 cells per well for transformed fibroblasts. Immediately after plating for LCLs or 24 h later for fibroblasts,

MMC or DEB was added at a final concentration of 0-100 nM for MMC or 0.75-1 μg/ml for

DEB. After 6-8 d of culture, cell numbers were determined using a Z2 Coulter Counter

(Beckman Coulter). Cell number after MMC or DEB treatment was normalized to cell number in the untreated sample to give the percentage of survival.

RNA interference (RNAi). For siRNA experiments, Al 170-22 E6E7/hTERT cells were transfected with a pool of three siRNAs using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instruction, with the final concentration of total siRNA at 25 nM.

Mutagenesis. Mutagenesis of FANl was performed on a pDONR223 FANl cDNA construct (Smogorzewska, A. et al. Mol. Cell 39, 36-47 (2010)) using a multisite

mutagenesis kit (Agilent). Other FANl mutants were previously described (Smogorzewska et al, supra).

Antibodies. Antibody to FANl (RC394) was raised in a rabbit using GST-FANlaal- 90 as an antigen and was affinity purified against HIS-FANlaal-90. Commercial antibodies were purchased to HA (Covance, MMS-101R), XPF/ERCC4 (Bethyl Laboratory, A301- 315A), MUS81 (Sigma, M1445) and FANCD2 (Novus, NB100-182).

Breakage analysis. Cells were exposed to 0.01 or 0.1 μg/ml DEB for 72 h or with 50 nM MMC for 24 h, arrested with colcemid (0.167 μg/ml) for 2 h, harvested, incubated for 10 min at 37 °C in 0.075 M KC1 and fixed in freshly prepared methanol: glacial acidic acid (3: 1). Cells were stored at 4 °C and, when needed, were dropped onto wet slides and air dried at 40 °C for 1 h before staining with Karyomax Giemsa (Invitrogen) Gurr Buffer for 3 min. After rinsing with fresh Gurr Buffer and then with distilled water, slides were fully dried at 40 °C for 1 h and were scanned using the Metasystems Metafer application.

Cell cycle analysis. Cells were left untreated, were treated with 100 nM MMC and grown for 48 h, or were treated with 0.1 μg/ml of DEB and grown for 72 h. Collected cells were resuspended in 300 μl of PBS. While vortexing, 700 μΐ of ice-cold 100% ethanol was added dropwise, and suspensions were stored at -20 °C at least overnight. Thirty minutes before fluorescence-activated cell sorting (FACS), cells were spun down, resuspended in propidium iodine mix (1 ml of PBS, 10 μl of RNase (from a stock solution of 20 mg/ml) and 10 ml of propidium iodine (from a stock solution of 1 mg/ml)) and analyzed using a

FACSCalibur instrument (Becton Dickinson). Cell cycle analysis was performed using Flow Jo software (Tree Star).

Morpho lino-mediated knockdown of fanl in zebrafish. Morpholino oligonucleotides were obtained from Gene Tools. Morpholinos (fanlD7 at 0.1 mM, standard control morpholino at 0.2 mM and p53 morpholino at 0.2 mM) were injected into zebrafish embryos at the 1-4 cell stages. Embryos were then fixed at 27 h.p.f. with 4% paraformaldehyde in PBS with 1% DMSO overnight, were permeabilized with acetone at -20 °C for 7 min and were stained with antibody against phosphorylated zebrafish γΗ2ΑΧ (1 : 1 ,000 dilution; a gift from Amatruda (UT Southwestern), or antibody against cleaved Caspase-3 (1 :200 dilution; BD Biosciences). Alex568-conjugated secondary antibody to rabbit IgG was used at a 1 :2,000 and a 1 : 1,000 dilution, respectively, for each primary antibody. The

immunofluorescence procedure followed a standard protocol. After washing off secondary antibody, embryos were mounted in Vector-Shield Mounting Media and were imaged with a Leica SP5X confocal system. Z stacks were processed to reduce noise and were overlayed to obtain representative images of immunofluorescence for

Figure imgf000070_0001
and cleaved caspase. DAPI was used to stain nuclei.

Quantitative RT-PCR. cDNA from 48 human tissues was purchased from OriGene (Tissue SCANTM Normal Tissue qPCR Arrays, HMRT502). Quantitative RT-PCR was performed using the TaqMan Gene Expression Assay kit (Applied Biosystems) according to the manufacturer's instructions. Briefly, 1 μl of cDNA was mixed with 10 μl of 2x TaqMan Universal Master Mix and 1 μl of 20x TaqMan Gene Expression Assay, bringing the total volume to 20 μl with RNase-free water. Target amplification was performed in 96-well plates using the StepOnePlus Real-Time PCR System (Applied Biosystems). TaqMan probes for FAN1 (Hs00429686_ml), FANCD2 (Hs00276992_ml) and GAPDH (Hs02758991_gl) were purchased from Applied Biosystems. PCR thermal cycling conditions included an initial 10- min hold at 95 °C to activate the AmpliTaq Gold DNA polymerase followed by 40 cycles of denaturation (15 s at 95°) and annealing and primer extension (15 s at 60 °C). More than three RT-PCR analyses were executed for each sample, and the obtained threshold cycle values were averaged. Relative RNA expression levels were calculated via a comparative threshold cycle (CT) method using GAPDH as control, where ACT = CT(GAPDH) - CT(FAN1 or FANCD2). Fold change in gene expression, normalized to GAPDH and relative to the control sample (FAN1 expression in the kidney), was calculated as 2-AACTD

Immunocytochemistry in the FHH rat and in humans with kidney disease. Kidney tissue coupes from FHH rats30,38 (n = 10) embedded in paraffin were deparafmized, treated with PO block for 15 min and incubated at 100 °C in citrate-HCl buffer for 20 min. The coupes were stained with mouse antibody to γΗ2ΑΧ (1 :200 dilution) overnight at 4 °C.

Samples were incubated with horseradish peroxidase (HRP)-conjugated secondary polyclonal rabbit antibody to mouse (1 : 100 diltuion; Dako, P0260) for 30 min at room temperature. Finally, samples were incubated with Bright Vision Poly HRP-Anti Rabbit IgG

(Immunologic, DPVR55HRP) for 1 h at room temperature. The Nova RED substrate kit for Peroxidase (Vector, SK-4800) was used, and samples were counterstained with hematoxylin. Analysis was performed using Aperio ImageScope software. Ten random tubular fields in the cortex (approximately 270 cells per field) were analyzed for positively stained nuclei using an in-house algorithm macro. GraphPad Prism 5.0 was used to calculate the R2 and P values.

Statistical analysis. Student's two-tailed nonpaired t tests and normal distribution two- tailed z tests were carried out using pooled standard error and s.d. values to determine the statistical significance of differences between cohorts.

Bioinformatics. Genetic locations are annotated according to March 2006 Human

Genome Browser data.

Results

By homozygosity mapping in family A1170, we defined seven candidate regions of homozygosity by descent. Exome sequencing identified a homozygous nonsense mutation (coding for p.Trp707*) in FAN1 (which encodes the Fanconi anemia-associated nuclease 1 protein) in both affected siblings (Table 3). No additional homozygous truncating mutations were detected in any other genes within the mapped candidate regions in family Al 170. DNA samples were obtained from five published families with KIN and five unpublished cases (Table 3). Clinical phenotypes have been published for families A4385 (Godin, M. et al. Am. J. Kidney Dis. 27, 166 (1996)), A4393 (Verine, et al, Ann. Pathol. 30, 240-242 (2010)), Al 170 (Auerbach, A.D. & Wolman, Nature 261, 494-496 (1976)), A4433 (Spoendlin, M. et al. Am. J. Kidney Dis. 25, 242-252 (1995)) and A4333 (Baba, et al. Pathol. Res. Pract. 202, 555-559 (2006)). With Sanger sequencing of all FANl exons, we found 12 different mutations of FANl in 9 of the 10 families with KIN (Table 3), detecting both mutated alleles in 9 families (Table 3). Eight of the 12 mutations truncated the conceptual reading frame (Table 3). Three missense mutations (encoding p.Gln929Pro, p.Gly937Asp and

p.Asp960Asn) altered amino-acid residues that have been conserved throughout evolution and are located in the FANl nuclease domain (VRR-NUC) (Smogorzewska, et al., supra). All mutations were absent from 96 healthy controls. Recessive mutations of FANl were identified as a major cause of KIN, an NPHP-like fibrotic kidney disease.

FANl is considered to be an effector of the Fanconi anemia pathway, a DDR signaling pathway involved in the repair of ICL damage (Knipscheer, P. et al. Science 326, 1698-1701 (2009)). Individuals with Fanconi anemia are characterized by developmental abnormalities, bone marrow failure and predisposition to cancer (Auerbach, A.D. Mutat. Res. 668, 4-10 (2009)). However, no FANl mutations have been detected in individuals with Fanconi anemia of unassigned complementation groups. The FANl protein is recruited to sites of ICL damage by interacting with a monoubiquitinated FANCI-FANCD2 complex through its UBZ domain (Kratz, K. et al. Cell 142, 77-88 (2010); Liu et al, Science 329, 693-696 (2010); MacKay, C. et al. Cell 142, 65-76 (2010); Smogorzewska, A. et al. Mol. Cell 39, 36-47 (2010)). In vitro, FANl has nuclease activity.

FANl expression was examined in fibroblasts and lymphoblastoid cell lines (LCLs) from individuals with KIN (Fig. 15a). No FANl protein was detected in the three individuals (Al 170-22, A4385-22 and A4466-21) who had two truncating mutations in FANl (Fig. 15a). Conversely, the protein was detected in the cell line from individual A4486-23 with a missense mutation (encoding p.Asp960Asn) in the nuclease domain of FANl (Fig. 15a).

As depletion of FANl sensitizes human cell lines to ICL-inducing agents (Mihatsch et al, supra; Kratz et al, supra; Liu et al, supra; MacKay et al, supra; Smogorzewska et al, supra), FANl-mutant cells from individuals with KIN were examined for genome instability upon exposure to mitomycin C (MMC) (Fig. 15b). Chromatid breaks and radial

chromosomes were observed on metaphase spreads (Fig. 15b), which is consistent with a role for FANl in genome maintenance and DDR. The levels of genome instability observed in KIN cell lines were not as high as in Fanconi anemia cell lines that lack FANCA gene function (RA3087 and RA3157), but were above background levels seen in wild-type cells. The results of the classic test for Fanconi anemia, diepoxybutane (DEB) breakage (Auerbach, A.D. & Wolman, S.R. Nature 261, 494-496 (1976)), were negative in all FANl -mutant cell lines tested but positive in the control FANCA-mutant cell lines (RA3087 and RA3157).

Despite the differences in chromosomal instability following MMC and DEB exposure, survival of FANl -mutant cell lines from affected individuals was severely compromised after exposure to either ICL-inducing agent (Fig. 15c,d). ICL sensitivity was observed in multiple cell lines from affected individuals (Fig. 15c,d). In contrast, cell cycle arrest in late-S/G2 phase, which is characteristic of Fanconi anemia cells, was seen in FAN1- mutant cells only after MMC and not after DEB exposure (Fig. 15e). These observed differences between FANCA-and FANl -mutant cells can be explained by differential engagement of FANl versus other Fanconi anemia pathway-directed nucleases in the repair of different ICL lesions or by different processing of the same kind of lesion by these distinct nucleases. Unlike the Fanconi anemia pathway defect, the FANl deficiency clearly resulted in high ICL sensitivity in the survival assays but did not lead to the profound genomic instability seen in Fanconi anemia cells. These differences in the cellular phenotype of FANl- and FANCA-deficient cell lines may explain the lack of phenotypic similarity between FANl -deficient individuals, who present with KIN, and individuals with Fanconi anemia, who have bone marrow failure and cancer predisposition.

A different phenotype for KIN is also consistent with the finding that FANl is not necessary for activation of the Fanconi anemia pathway, as judged by the presence of normal FANCD2 ubiquitination in FANl -deficient cells.

To complement the FANl defect, fibroblasts of individual Al 170-22 were transduced with wild-type FANl cDNA or with FANl cDNA carrying KIN-associated mutations or mutations known to inhibit nuclease activity (encoding p.Glu975Ala/Lys977Ala) or interaction with FANCD2 (p.Cys44Ala/Cys47Ala)6 (Fig. 16a,b). No FANl protein was detected in immunoblotting of fibroblasts expressing the two truncating variants (p.Trp707* and p.Arg679Thrfs*5), indicating that they are unstable, whereas p.Leu925Profs*25 yielded a shortened protein product (Fig. 16b). Whereas wild-type FANl did complement the MMC sensitivity defect, none of the cDNAs carrying mutations from individuals with KIN rescued the defect, with the exception of the encoded p.Cys871Arg variant, which partially complemented cell survival upon MMC exposure, indicating that it represents a hypomorphic allele of FANl (Fig. 16a). The FANl variant p.Cys44Ala/Cys47Ala that abolishes FANCD2- dependent localization of FANl to sites of DNA damage (Smogorzewska, A. et al, supra) was fully capable of rescuing the MMC resistance defect, indicating that FAN1 activity is independent of the Fanconi anemia pathway in cell lines from individuals with KIN. The p.Glu975Ala/Lys977Ala mutant lacking nuclease activity could not complement the FAN1 defect.

It was previously shown in DT40 chicken cells that deletion of FAN 1 has an effect on

ICL sensitivity additive to the effect seen upon deletion of the Fanconi anemia-associated genes FANCC and FANCJ (Yoshikiyo, K. et al. Proc. Natl. Acad. Sci. USA 107, 21553- 21557 (2010)). To test whether inhibition of the Fanconi anemia pathway in the FAN1- mutant cells led to increased ICL sensitivity, transcripts of FANCD2, SLX4 or the SLX4- associated nucleases XPF and MUS81 were depleted (Fig. 16c,d). Depletion gave rise to profound MMC sensitivity that was greater than in cells with FAN1 deficiency alone, indicating that FAN1 can work independently of the Fanconi anemia pathway to repair ICL damage.

The fibrotic and cystic kidney phenotypes observed in NPHP -related ciliopathies are still mostly unknown (Hildebrandt et al, N. Engl. J. Med. 364, 1533-1543 (2011); Simons, M. et al. Nat. Genet. 37, 537-543 (2005); Huangfu, D. et al. Nature 426, 83-87 (2003)). Because most DDR pathways as well as NPHP -related ciliopathy phenotypes are conserved in zebrafish (Otto, E.A. et al. Nat. Genet. 42, 840-850 (2010); Otto, E.A. et al. Nat. Genet. 34, 413-420 (2003); Zhou et al, Am. J. Physiol. Renal Physiol. 299, F55-F62 (2010);

Schafer, T. et al. Hum. Mol. Genet. 17, 3655-3662 (2008); Sayer, J.A. et al. Nat. Genet. 38, 674-681 (2006)), it was evaluated whether loss of FAN 1 function through morpholino oligonucleotide knockdown of fanl in zebrafish embryos would cause both disturbance of DDR signaling and NPHP-like phenotypes. Injection into 1-4 cell stage embryos of a morpholino (fanlD7) that targets the splice-acceptor site of exon 7 caused the characteristic NPHP-like phenotype of shortened body axis (Fig. 17a,b) but also the DDR phenotypes of microcephaly, microphthalmia and massive cell death throughout the embryo (Fig. 17c). Cell death was from apoptosis, as shown by greater amounts of activated Caspase-3 (Fig. 14d). DDR signaling was activated, with increased signal for γΗ2ΑΧ shown in

immunofluorescence analysis (Fig. 17e). These finding are similar to the ones described for knockdown of the Fanconi anemia-associated gene fancd2 in zebrafish (Liu, T.X. et al. Dev. Cell 5, 903-914 (2003); Zeng et al, Zebrafish 6, 405-415 (2009)). In addition, the fanlD7 morphants showed phenotypes characteristic of NPHP -related ciliopathies (Otto, E.A. et al. Nat. Genet. 42, 840-850 (2010), including ventral body axis curvature and renal cysts when apoptosis was suppressed through knockdown of p53 function (Fig. 17f-h). Specifically, zebrafish embryos injected with fanlD7 and p53 morpho linos at 72 h post- fertilization (h.p.f.) showed pronephric kidney cysts (19 ± 3%) (Fig. 17f,h) and body curvature (45 ± 4%), whereas p53 morpholino alone did not cause pronephric cysts (Fig. 17g,h) and caused body axis curvature in a significantly smaller fraction of embryos (11 ± 2%) (Fig. 17h). Thus, loss of fanl function results in ciliopathy-related phenotypes, which are further revealed when p53 function is inhibited. Masking of the renal cystic phenotype in the presence of p53 function is most likely due to the fact that fanl morphants had embryonic deformity (Fig. 17b,c) and highly elevated levels of apoptosis (Fig. 18d), which prevented the observation of kidney cysts that developed in later embryonic stages. The data show that loss of fanl function in zebrafish embryos leads to a dual set of phenotypes: one representing activation of DDR and apoptosis (microcephaly and microphthalmia) and the other mimicking NPHP- like ciliopathy phenotypes that are revealed when p53 -dependent apoptosis is suppressed. It was found that whereas knockdown of fancd2 in zebrafish led to increased γΗ2ΑΧ staining, it did not cause kidney cysts or other ciliopathy phenotypes seen following fanl knockdown.

The clinical phenotypes caused by FAN1 and FANCD2 mutations differ substantially.

The former causes KIN and histological karyomegaly in the liver and brain, whereas the latter causes Fanconi anemia. To evaluate whether these phenotypic differences can be partially attributed to differential tissue expression, we performed quantitative PCR of 48 different human tissue sources and observed significant differences in expression levels in 25 sources. Notably, FANl expression exceeded that of FANCD2 in parenchymatous organs, including the kidney, liver, neuronal tissue and female reproductive organs (Fig. 18a), whereas FANCD2 expression levels far exceeded those of FANl in six different lymphatic or bone marrow-derived sources, as well as in skin and testis (Fig. 18b). FAN1 mutations cause KIN and karyomegaly in parenchymatous organs in the absence of Fanconi anemia-like blood dyscrasias, whereas FANCD2 mutation causes Fanconi anemia with pancytopenia, skin involvement and male infertility. FAN1 expression by protein blotting was particularly high in the kidney, indicating that the kidney may depend on FAN1 for its normal function.

It was further explored whether a defect in DDR signaling represents a broader pathogenic mechanism that applies also to other forms of CKD. Renal tissue sections from a standard congenic rat model were evaluated for progressive chronic renal failure, the fawn- hooded hypertensive (FHH) rat, which has well-defined physiological parameters. Ten animals were selected on the basis of disease progression at 9-10 months of age, as measured by proteinuria (Koeners et al., Am. J. Physiol. Regul. Integr. Comp. Physiol. 294, R1847- R1855 (2008)). To determine the load of DDR in the kidneys of these animals, the criterion of increased nuclear γΗ2ΑΧ staining in immunohistochemistry was applied (Fig. 18c-e). After staining, whole slides (two to four kidney sections per animal) were digitally scanned, ten randomized cortical fields were analyzed per section for positively stained nuclei using a modified algorithm, and results were calibrated for diagnostic pathology. In total, 2,000- 3,000 cells were scored per animal by a researcher blinded to the disease status of the animals. A positive correlation (R2 = 0.64, P < 0.0054) was found between disease progression and DDR in this assay (Fig. 18c). Having established a quantitative correlation in a well-defined congenic model for kidney disease, genetically heterogeneous affected humans were examined. A small pilot study using renal transplant biopsies from individuals that were clinically well characterized by a nephropathologist and nephrologist supported the correlation found in the rat model. γΗ2ΑΧ immunostaining was examined in a specimen from a transplant kidney 4 months after transplantation (histology report without injury and no proteinuria) (Fig. 18f), a specimen from a transplant kidney 16 years after transplantation (pathology report of chronic damage) (Fig. 18g) and a transplant kidney 10 years after transplantation (pathology report of chronic tissue damage) (Fig. 18h). These data support in human CKD the relationship seen in the FHH rat model between the extent of kidney damage and DNA lesions (γΗ2ΑΧ). They indicate that DDR contributes to renal damage in pediatric NPHP -related ciliopathies and in CKD of adults.

Recessive mutations of FANl were identified as the cause of KIN, which leads to CKD with fibrotic degeneration of the kidney. Thus, DDR, which has a known role in cellular senescence (Mallette, F.A. & Ferbeyre, G. Cell Cycle 6, 1831-1836 (2007)), contributes to the premature aging phenotype of renal fibrosis in NPHP-like diseases. A KIN-like phenotype has also been described in humans and animal models that were exposed to ochratoxin A (Godin, M. et al. Karyomegalic interstitial nephritis. Am. J. Kidney Dis. 27, 166 (1996)), busulfan or pyrrolizidine alkaloids (Burry, A.F. J. Pathol. 113, 147-150 (1974)), all of which cause ICL. Thus, FANl mutation represents the genetic equivalent of environmental genotoxic causes of KIN by the shared pathogenic mechanism of defective ICL repair. In this context, it is notable that, among the 12 individuals with KIN-associated and FANl mutations, CKD presented by a median age at 45 years and that 7 of the 12 individuals with KIN carried 2 truncating mutations or missense mutations affecting the nuclease domain of FANl, which represent null alleles (Fig. 16a and Table 3). A high percentage of adult individuals treated within chronic dialysis programs suffer from fibrotic nephropathy of unknown cause. Furthermore, recessive mutations in FANl confer susceptibility to MMC and DEB, FAN1 mutations may also sensitize to other ICL-causing environmental genotoxins, such as ochratoxin A, which are abundant (Verine et al., Ann. Pathol. 30, 240- 242 (2010)).

Figure imgf000077_0001

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the medical sciences are intended to be within the scope of the following claims.

Claims

1. A kit for detecting gene variants associated with nephronophthisis-related ciliopathies (NPHP-RC) in a subject, consisting essentially of:
a) a first NPHP-RC informative detection reagent for identification of one or more variants in a first gene selected from the group consisting of meiotic recombination 11 homo log (MREl 1), zinc finger protein 423 (ZNF423), and centrosomal protein 164kDa (CEP 164); and
b) a second NPHP-RC informative detection reagent for identification of one or more variants in a second gene selected from the group consisting of MREl 1, ZNF423, CEP 164, wherein said second gene is different than said first gene.
2. The kit of claim 1, wherein said variants result in a loss of function of said one or more genes.
3. The kit of claim 2, wherein said MREl 1 mutation is c.l897C>T.
4. The kit of claim 3, wherein said mutation encodes a p.R633X protein mutation.
5. The kit of claim 2, wherein said ZNF423 mutation is selected from the group consisting of c.2738C>T, cl518delC, and c.3829C>T.
6. The kit of claim 5, wherein said mutation encodes a protein mutation selected from the group consisting of p.P913L, p.P506fzX43, and p.H1277Y.
7. The kit of claim 2, wherein said CEP 164 mutations are selected from the group consisting of c.32A>C, c.277C>T, C.15730T, C.17260T, and c.4383A>G.
8. The kit of claim 7, wherein said mutation encodes a protein mutation selected from the group consisting of p.Ql IP, p.R93W, p.Q525X, p.R576X, and p.X1460W.
9. The kit of claim 1, wherein said first gene is MREl 1 and said second gene is ZNF423.
10. The kit of claim 1, wherein said first gene is MREl 1 and said second gene is CEP 164.
11. The kit of claim 1, wherein said first gene is CEP 164 and said second gene is ZNF423.
12. The kit of claim 1 , further comprising a third reagent that identifies variants in a third distinct gene.
13. The kit of any one of claims 1 to 12, wherein said first and second reagents are nucleotide probes that specifically bind to said variants.
14. The kit of any one of claims 1 to 12, wherein said first and second reagents are antibodies that specifically bind to polypeptides encoded by said variants.
15. The kit of any one of claims 1 to 12, wherein said first reagent is a pair of primers for amplifying said first gene and second reagent is a pair of primer for amplifying said second gene.
16. The kit of any one of claims 1 to 12, wherein said first and second reagents are sequence primers for sequencing said first and second genes.
17. A method for detecting gene variants associated with NPHP-RC in a subject, comprising:
a) contacting a sample from a subject with a variant detection assay comprising the reagents of any one of claims 1 to 16; and
b) diagnosing said subject with NPHP-RC when said variants are present in said sample.
18. A method for detecting gene variants associated with NPHP-RC in a subject, comprising:
a) contacting a sample from a subject with a variant detection assay, wherein said variant detection assay comprises i) a first NPHP-RC informative detection reagent for identification of one or more variants in a first gene selected from the group consisting of MREl 1, ZNF423, and CEP 164; and ii) a second NPHP-RC informative detection reagent for identification of one or more variants in a second gene selected from the group consisting of MRE11, ZNF423, and CEP 164, wherein said second gene is different than said first gene, under conditions that the presence of a variant associated with NPHP-RC is determined; and b) diagnosing said subject with NPHP-RC when said variants are present in said sample.
19. The method of claim 18, wherein said variations result in loss of function mutations in said one or more genes.
20. The method of claim 19, wherein said MREl 1 mutation is c.1897C>T.
21. The method of claim 20, wherein said mutation encodes a p.R633X protein mutation.
22. The method of claim 19, wherein said ZNF423 mutation is selected from the group consisting of c.2738C>T, cl518delC, and c.3829C>T.
23. The method of claim 22, wherein said mutation encodes a protein mutation selected from the group consisting of p.P913L, p.P506fzX43, and p.H1277Y.
24. The method of claim 19, wherein said CEP 164 mutations are selected from the group consisting of c.32A>C, c.277C>T, C.1573C>T, C.1726C>T, and c.4383A>G.
25. The method of claim 24, wherein said mutation encodes a protein mutation selected from the group consisting of p.Q11P, p.R93W, p.Q525X, p.R576X, and p.X1460W.
26. The method of claim 18, wherein said determining comprises detecting variant nucleic acids or polypeptides.
27. The method of claim 26, wherein said detecting variant nucleic acids comprises one or more nucleic acid detection method selected from the group consisting of sequencing, amplification and hybridization.
28. The method of claim 18, wherein said first gene is MREl 1 and said second gene is ZNF423.
29. The method of claim 18, wherein said first gene is MRE11 and said second gene is CEP 164.
30. The method of claim 18, wherein said first gene is CEP 164 and said second gene is ZNF423.
31. The method of claim 18, further comprising a third reagent that identifies variants in a third distinct gene.
32. The method of claim 18, wherein said first and second reagents are nucleotide probes that specifically bind to said variants.
33. The method of claim 18, wherein said first and second reagents are antibodies that specifically bind to polypeptides encoded by said variants.
34. The method of claim 18, wherein said first reagent is a pair of primers for amplifying said first gene and second reagent is a pair of primer for amplifying said second gene.
35. The method of claim 18, wherein said first and second reagents are sequence primers for sequencing said first and second genes.
36. The method of Claim 18, wherein said biological sample is selected from the group consisting of a tissue sample, a cell sample, and a blood sample.
37. The method of claim 18, wherein said determining comprises a computer
implemented method.
38. The method of claim 37, wherein said computer implemented method comprises analyzing variant information and displaying said information to a user.
39. The method of claim 18, further comprising the step of treating said subject for NPHP-RC.
40. The method of claim 18, further comprising the step of treating said subject for NPHP-RC under condition such that at least one symptom of said NPHP-RC is diminished or eliminated.
41. Use of first and second HPHP-RC informative reagents that detect two variant MREl l, ZNF423, or CEP 164 nucleic acid or polypeptide for identifying NPHP-RC in a subject.
42. The use of claim 41, wherein said MREl 1, ZNF423, or CEP 164 variant encodes a loss of function mutation.
43. The use of claim 41 , wherein said MRE 11 mutation is c.18970T, said ZNF423 mutation is selected from the group consisting of c.2738C>T, cl518delC, and c.3829C>T, said CEP 164 mutations are selected from the group consisting of c.32A>C, c.277C>T, c.15730T, c.17260T, and c.4383A>G, and said CEP164 mutations are selected from the group consisting of c.32A>C, c.277C>T, C.15730T, C.17260T, and c.4383A>G.
44. A kit for detecting gene variants associated with congenital abnormalities of the kidney and urinary tract (CAKUT) in a subject, consisting essentially of:
a) a first CAKUT informative detection reagent for identification of one or more variants in a first gene selected from the group consisting of fraser syndrome 1 (FRASl), FRASl related extracellular matrix protein 2 (FREM2), Ret proto-oncogene, (RET), bone morphogenetic protein 4 (BMP4); and
b) a second CAKUT informative detection reagent for identification of one or more variants in a second gene selected from the group consisting of FRASl, FREMl, RET, and BMP4, wherein said second gene is different than said first gene.
45. The kit of claim 44, wherein said variants result in a loss of function of said one or more genes.
46. The kit of claim 45, wherein said FRASl mutations are selected from the group consisting of c.776T>G, c.299G>T, c.981G>A, c.7861C>T, and C.112680A.
47. The kit of claim 46, wherein said mutation encodes a protein mutation selected from the group consisting of p.L259R, p.D998Y, p.R3273H, p.R2621X, and p.H3757Q.
48. The kit of claim 45, wherein said FREM2 mutation is c.7013C>T.
49. The kit of claim 48, wherein said mutation encodes a p.T2338I protein mutation.
50. The kit of claim 45, wherein said RET mutation is c.2110G>T.
51. The kit of claim 50, wherein said mutation encodes a p.V704F protein mutation.
52. The kit of claim 45, wherein said BMP4 mutation is c.362A>G.
53. The kit of claim 52, wherein said mutation encodes a p.H121R protein mutation.
54. The kit of claim 45, wherein said first gene is FRASl and said second gene is FREMl .
55. The kit of claim 45, wherein said first gene is FRASl and said second gene is RET.
56. The kit of claim 45, wherein said first gene is FRASl and said second gene is BMP4.
57. The kit of claim 45, wherein said first gene is FREMl and said second gene is RET. 58. The kit of claim 45, wherein said first gene is FREMl and said second gene is BMP4.
54. The kit of claim 45, wherein said first gene is RET and said second gene is BMP4.
55. The kit of claim 45, further comprising a third reagent that identifies variants in a third distinct gene and optionally a fourth reagent that identifies variants in a fourth distinct gene.
56. The kit of any one of claims 45 to 55, wherein said first and second reagents are nucleotide probes that specifically bind to said variants.
57. The kit of any one of claims 45 to 55, wherein said first and second reagents are antibodies that specifically bind to polypeptides encoded by said variants.
58. The kit of any one of claims 45 to 55, wherein said first reagent is a pair of primers for amplifying said first gene and second reagent is a pair of primer for amplifying said second gene.
59. The kit of any one of claims 45 to 55, wherein said first and second reagents are sequence primers for sequencing said first and second genes.
60. A method for detecting gene variants CAKUT in a subject, comprising:
a) contacting a sample from a subject with a variant detection assay of any one of claims 45 to 59; and
b) diagnosing said subject with CAKUT when said variants are present in said sample.
61. A method for detecting gene variants CAKUT in a subject, comprising:
a) contacting a sample from a subject with a variant detection assay, wherein said variant detection assay comprises i) a first CAKUT informative detection reagent for identification of one or more variants in a first gene selected from the group consisting of FRASl, FREM2, RET, BMP4; and ii) a second CAKUT informative detection reagent for identification of one or more variants in a second gene selected from the group consisting of FRASl, FREMl, RET, and BMP4, wherein said second gene is different than said first gene under conditions that the presence of a variant associated with CAKUT is determined; and b) diagnosing said subject with CAKUT when said variants are present in said sample.
62. The method of claim 62, wherein said variations result in loss of function mutations in said one or more genes.
63. The method of claim 62, wherein said FRASl mutations are selected from the group consisting of c.776T>G, c.299G>T, c.981G>A, c.7861C>T, and C.112680A.
64. The method of claim 63, wherein said mutation encodes a protein mutation selected from the group consisting of p.L259R, p.D998Y, p.R3273H, p.R2621X, and p.H3757Q.
65. The method of claim 62, wherein said FREM2 mutation is c.7013C>T.
66. The method of claim 65, wherein said mutation encodes a p.T2338I protein mutation.
67. The method of claim 62, wherein said RET mutation is c.2110G>T.
68. The method of claim 67, wherein said mutation encodes a p.V704F protein mutation.
69. The method of claim 62, wherein said BMP4 mutation is c.362A>G.
70. The method of claim 69, wherein said mutation encodes a p.H121R protein mutation.
71. The method of claim 61 , wherein said determining comprises detecting variant nucleic acids or polypeptides.
72. The method of claim 61, wherein said detecting variant nucleic acids comprises one or more nucleic acid detection method selected from the group consisting of sequencing, amplification and hybridization.
73. The method of claim 61, wherein said first gene is FRAS1 and said second gene is FREM1.
74. The method of claim 61, wherein said first gene is FRAS1 and said second gene is RET.
75. The method of claim 61, wherein said first gene is FRAS1 and said second gene is BMP4.
76. The method of claim 61, wherein said first gene is FREM1 and said second gene is RET.
77. The method of claim 61, wherein said first gene is FREM1 and said second gene is BMP4.
78. The method of claim 61, wherein said first gene is RET and said second gene is BMP4.
79. The method of claim 61, further comprising a third reagent that identifies variants in a third distinct gene.
80. The method of claim 61, wherein said first and second reagents are nucleotide probes that specifically bind to said variants.
81. The method of claim 61 , wherein said first and second reagents are antibodies that specifically bind to polypeptides encoded by said variants.
82. The method of claim 61, wherein said first reagent is a pair of primers for amplifying said first gene and second reagent is a pair of primer for amplifying said second gene.
83. The method of claim 61, wherein said first and second reagents are sequence primers for sequencing said first and second genes.
84. The method of Claim 61, wherein said biological sample is selected from the group consisting of a tissue sample, a cell sample, and a blood sample.
85. The method of claim 61, wherein said determining comprises a computer
implemented method.
86. The method of claim 85, wherein said computer implemented method comprises analyzing variant information and displaying said information to a user.
87. The method of claim 61, further comprising the step of treating said subject for CAKUT.
88. The method of claim 61, further comprising the step of treating said subject for CAKUT under condition such that at least one symptom of said CAKUT is diminished or eliminated.
89. Use of first and second CAKUT informative reagents that detect two variant FRAS 1 , FREM2, RET, or BMP4 nucleic acid or polypeptide for identifying CAKUT in a subject.
90. The use of claim 36, wherein said FRAS 1 , FREM2, RET, or BMP4 variant encodes a loss of function mutation.
91. The use of claim 37, wherein FRASl mutations are selected from the group consisting of c.776T>G, c.299G>T, c.981G>A, c.7861C>T, and C.112680A, said FREM2 mutation is C.7013C>T, said RET mutation is c.2110G>T, and said BMP4 mutation is c.362A>G.
92. A kit for detecting gene variants associated with karyomegalic interstitial nephritis (KIN) in a subject, consisting essentially of:
a) a first KIN informative detection reagent for identification of a first mutation in a KAN gene; and
b) a second KIN informative detection reagent for identification of a second mutation in a KANl gene, wherein said second mutation is different than said first mutation.
93. The kit of claim 92, wherein said first and second mutations are selected from the group consisting of c. l234+2T>A, c.2036_7delGA, c.2245C>T, C.1375+1G>A, c.2616delA, C.1606C>T, c.2878G>A, c.2120G>A, c.2786A>C, c.2611T>C, c.2774_5delTT, and c.2810G>A.
94. The kit of claim 93, wherein said first mutation is c.l234+2T>A and said second mutation is c.2036_7delGA.
95. The kit of claim 93, wherein said first mutation is c.l234+2T>A and said second mutation is c.2245C>T.
96. The kit of claim 93, wherein said first mutation is C.1375+1G>A and said second mutation is c.2616delA.
97. The kit of claim 93, wherein said first mutation is C.1606C>T and said second mutation is c.2786A>C.
98. The kit of claim 93, wherein said first mutation is C.1606C>T and said second mutation is c.2878G>A.
99. The kit of claim 93, wherein said first mutation is c.2611T>C and said second mutation is c.2878G>A.
100. The kit of claim 93, wherein said first mutation is c.2774_5delTT and said second mutation is c.2810G>A.
101. The kit of any one of claims 92 to 100, wherein said first and second mutations encode a polypeptide mutation selected from the group consisting of p.Arg679Thrfs*5, p.Arg749*, p.Asp873Thrfs* 17, p.Arg536*, p.Cys871Arg, p.Trp707*, p.Leu925Profs*25, p.Gln929Pro, p.Gly937Asp, p.Asp960Asn, and a splice site mutation.
102. The kit of claim 101, wherein said first mutation is a splice site mutation and said second mutation is p.Arg679Thrfs*5.
103. The kit of claim 101, wherein said first mutation is a splice site mutation and said second mutation is p.Arg749*.
104. The kit of claim 101, wherein said first mutation is a splice site mutation and said second mutation is p.Asp873Thrfs* 17.
105. The kit of claim 101, wherein said first mutation is p.Arg536* and said second mutation is p.Gln929Pro.
106. The kit of claim 101, wherein said first mutation is p.Arg536* and said second mutation is p.Asp960Asn.
107. The kit of claim 101, wherein said first mutation is p.Cys871Arg and said second mutation is p.Asp960Asn.
108. The kit of claim 101, wherein said first mutation is p.Leu925Profs*25 and said second mutation is p.Gly937Asp.
109. The kit of any one of claims 92 to 108, wherein said variants result in a loss of function of said KAN1 gene.
110. The kit of any one of claims 92 to 100, wherein said first and second reagents are nucleotide probes that specifically bind to said variants.
111. The kit of any one of claims 100 to 108, wherein said first and second reagents are antibodies that specifically bind to said polypeptides.
112. The kit of any one of claims 92 to 100, wherein said first reagent is a pair of primers for amplifying said first gene and second reagent is a pair of primer for amplifying said second gene.
113. The kit of any one of claims 92 to 100, wherein said first and second reagents are sequence primers for sequencing said first and second genes.
114. A method for detecting gene variants associated with KIN in a subject, comprising: a) contacting a sample from a subject with a variant detection assay, wherein said variant detection assay comprises i) a first KIN informative detection reagent for
identification of a first variant in a KAN gene; and optionally ii) a second KIN informative detection reagent for identification of a second variant in a KANl gene, wherein said second variant is different than said first variant under conditions that the presence of a variant associated with KIN is determined; and b) diagnosing said subject with KIN when said variant is present in said sample.
115. The method of claim 114, wherein said variations result in loss of function mutations in said one or more genes.
116. The method of claim 114, wherein said first and second mutations are selected from the group consisting of c. l234+2T>A, c.2036_7delGA, c.2245C>T, c.1375+1G>A, c.2616delA, C.1606C>T, c.2878G>A, c.2120G>A, c.2786A>C, c.2611T>C, c.2774_5delTT, and c.2810G>A.
117. The method of claim 114, wherein said first mutation is c.1234+2T>A and said second mutation is c.2036_7delGA.
118. The method of claim 114, wherein said first mutation is c.1234+2T>A and said second mutation is c.2245C>T.
119. The method of claim 114, wherein said first mutation is c.1375+1G>A and said second mutation is c.2616delA.
120. The method of claim 114, wherein said first mutation is c.1606C>T and said second mutation is c.2786A>C.
121. The method of claim 114, wherein said first mutation is c.1606C>T and said second mutation is c.2878G>A.
122. The method of claim 114, wherein said first mutation is c.2611T>C and said second mutation is c.2878G>A.
123. The method of claim 114, wherein said first mutation is c.2774_5delTT and said second mutation is c.2810G>A.
124. The method of any one of claims 116 to 123, wherein said first and second mutations encode a polypeptide mutation selected from the group consisting of p.Arg679Thrfs*5, p.Arg749*, p.Asp873Thrfs* 17, p.Arg536*, p.Cys871Arg, p.Trp707*, p.Leu925Profs*25, p.Gln929Pro, p.Gly937Asp, p.Asp960Asn, and a splice site mutation.
125. The method of claim 124, wherein said first mutation is a splice site mutation and said second mutation is p.Arg679Thrfs*5.
126. The method of claim 124, wherein said first mutation is a splice site mutation and said second mutation is p.Arg749*.
127. The method of claim 124, wherein said first mutation is a splice site mutation and said second mutation is p.Asp873Thrfs* 17.
128. The method of claim 124, wherein said first mutation is p.Arg536* and said second mutation is p.Gln929Pro.
129. The method of claim 124, wherein said first mutation is p.Arg536* and said second mutation is p.Asp960Asn.
130. The method of claim 124, wherein said first mutation is p.Cys871Arg and said second mutation is p.Asp960Asn.
131. The method of claim 124, wherein said first mutation is p.Leu925Profs*25 and said second mutation is p . Gly937 Asp .
132. The method of claim 112, wherein said detecting variant nucleic acids comprises one or more nucleic acid detection method selected from the group consisting of sequencing, amplification and hybridization.
133. The method of any one of claims 113 to 123, wherein said first and second reagents are nucleotide probes that specifically bind to said variants.
134. The method of any one of claims 114 to 131, wherein said first and second reagents are antibodies that specifically bind to polypeptides encoded by said variants.
135. The method of any one of claims 113 to 123, wherein said first reagent is a pair of primers for amplifying said first gene and second reagent is a pair of primer for amplifying said second gene.
136. The method of any one of claims 113 to 123, wherein said first and second reagents are sequence primers for sequencing said first and second genes.
137. The method of Claim 112, wherein said biological sample is selected from the group consisting of a tissue sample, a cell sample, and a blood sample.
138. The method of claim 112, wherein said determining comprises a computer
implemented method.
139. The method of claim 138, wherein said computer implemented method comprises analyzing variant information and displaying said information to a user.
140. The method of claim 112, further comprising the step of treating said subject for KIN.
141. The method of claim 140, further comprising the step of treating said subject for KIN under condition such that at least one symptom of said KIN is diminished or eliminated.
142. Use of at least a first KIN informative reagents that detects at least one variant KAN1 nucleic acids or polypeptides for identifying KIN in a subject.
143. The use of claim 142, wherein said KAN1 variant nucleic acids are selected from the group consisting of c. l234+2T>A, c.2036_7delGA, c.2245C>T, C.1375+1G>A, c.2616delA, C.1606C>T, c.2878G>A, c.2120G>A, c.2786A>C, c.2611T>C, c.2774_5delTT, and c.2810G>A.
144. The use of claiml42, wherein said variant KANl polypeptides are selected from the group consisting of p.Arg679Thrfs*5, p.Arg749*, p.Asp873Thrfs* 17, p.Arg536*, p.Cys871Arg, p.Trp707*, p.Leu925Profs*25, p.Gln929Pro, p.Gly937Asp, p.Asp960Asn, and a splice site mutation.
PCT/US2013/048688 2012-06-29 2013-06-28 Methods and biomarkers for detection of kidney disorders WO2014005076A9 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US201261666423 true 2012-06-29 2012-06-29
US61/666,423 2012-06-29
US201261672916 true 2012-07-18 2012-07-18
US61/672,916 2012-07-18

Publications (3)

Publication Number Publication Date
WO2014005076A2 true true WO2014005076A2 (en) 2014-01-03
WO2014005076A3 true WO2014005076A3 (en) 2014-02-20
WO2014005076A9 true WO2014005076A9 (en) 2014-04-03

Family

ID=48790654

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2013/048688 WO2014005076A9 (en) 2012-06-29 2013-06-28 Methods and biomarkers for detection of kidney disorders

Country Status (1)

Country Link
WO (1) WO2014005076A9 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105256378B (en) * 2015-04-13 2018-01-05 卢彦平 Precursory cilia related disease microarray screening for disease genes

Citations (54)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4800159A (en) 1986-02-07 1989-01-24 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences
US4965188A (en) 1986-08-22 1990-10-23 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme
US4981785A (en) 1988-06-06 1991-01-01 Ventrex Laboratories, Inc. Apparatus and method for performing immunoassays
US5130238A (en) 1988-06-24 1992-07-14 Cangene Corporation Enhanced nucleic acid amplification process
US5270184A (en) 1991-11-19 1993-12-14 Becton, Dickinson And Company Nucleic acid target generation
US5283174A (en) 1987-09-21 1994-02-01 Gen-Probe, Incorporated Homogenous protection assay
US5358691A (en) 1992-03-27 1994-10-25 Abbott Laboratories Automated continuous and random access analytical system
US5399491A (en) 1989-07-11 1995-03-21 Gen-Probe Incorporated Nucleic acid sequence amplification methods
US5455166A (en) 1991-01-31 1995-10-03 Becton, Dickinson And Company Strand displacement amplification
EP0684315A1 (en) 1994-04-18 1995-11-29 Becton Dickinson and Company Strand displacement amplification using thermophilic enzymes
US5480784A (en) 1989-07-11 1996-01-02 Gen-Probe Incorporated Nucleic acid sequence amplification methods
US5599677A (en) 1993-12-29 1997-02-04 Abbott Laboratories Immunoassays for prostate specific antigen
US5695934A (en) 1994-10-13 1997-12-09 Lynx Therapeutics, Inc. Massively parallel sequencing of sorted polynucleotides
US5710029A (en) 1995-06-07 1998-01-20 Gen-Probe Incorporated Methods for determining pre-amplification levels of a nucleic acid target sequence from post-amplification levels of product
US5714330A (en) 1994-04-04 1998-02-03 Lynx Therapeutics, Inc. DNA sequencing by stepwise ligation and cleavage
US5750341A (en) 1995-04-17 1998-05-12 Lynx Therapeutics, Inc. DNA sequencing by parallel oligonucleotide extensions
US5814447A (en) 1994-12-01 1998-09-29 Tosoh Corporation Method of detecting specific nucleic acid sequences
US5885530A (en) 1996-06-28 1999-03-23 Dpc Cirrus, Inc. Automated immunoassay analyzer
US5912148A (en) 1994-08-19 1999-06-15 Perkin-Elmer Corporation Applied Biosystems Coupled amplification and ligation method
US5925517A (en) 1993-11-12 1999-07-20 The Public Health Research Institute Of The City Of New York, Inc. Detectably labeled dual conformation oligonucleotide probes, assays and kits
US5928862A (en) 1986-01-10 1999-07-27 Amoco Corporation Competitive homogeneous assay
WO2000018957A1 (en) 1998-09-30 2000-04-06 Applied Research Systems Ars Holding N.V. Methods of nucleic acid amplification and sequencing
US6150097A (en) 1996-04-12 2000-11-21 The Public Health Research Institute Of The City Of New York, Inc. Nucleic acid detection probes having non-FRET fluorescence quenching and kits and assays including such probes
US6159750A (en) 1995-12-22 2000-12-12 Abbott Laboratories Fluorescence polarization immunoassay diagnostic method
US6210891B1 (en) 1996-09-27 2001-04-03 Pyrosequencing Ab Method of sequencing DNA
US6258568B1 (en) 1996-12-23 2001-07-10 Pyrosequencing Ab Method of sequencing DNA based on the detection of the release of pyrophosphate and enzymatic nucleotide degradation
US6303305B1 (en) 1999-03-30 2001-10-16 Roche Diagnostics, Gmbh Method for quantification of an analyte
US6432360B1 (en) 1997-10-10 2002-08-13 President And Fellows Of Harvard College Replica amplification of nucleic acid arrays
US6485944B1 (en) 1997-10-10 2002-11-26 President And Fellows Of Harvard College Replica amplification of nucleic acid arrays
US6511803B1 (en) 1997-10-10 2003-01-28 President And Fellows Of Harvard College Replica amplification of nucleic acid arrays
US6534274B2 (en) 1998-07-02 2003-03-18 Gen-Probe Incorporated Molecular torches
US6541205B1 (en) 1999-05-24 2003-04-01 Tosoh Corporation Method for assaying nucleic acid
US6706162B1 (en) 2000-09-25 2004-03-16 Applera Corporation High speed, high resolution compositions, methods, and kits for capillary electrophoresis
US6787308B2 (en) 1998-07-30 2004-09-07 Solexa Ltd. Arrayed biomolecules and their use in sequencing
US6818395B1 (en) 1999-06-28 2004-11-16 California Institute Of Technology Methods and apparatus for analyzing polynucleotide sequences
US6833246B2 (en) 1999-09-29 2004-12-21 Solexa, Ltd. Polynucleotide sequencing
US20050042638A1 (en) 2003-05-01 2005-02-24 Gen-Probe Incorporated Oligonucleotides comprising a molecular switch
US20050130173A1 (en) 2003-01-29 2005-06-16 Leamon John H. Methods of amplifying and sequencing nucleic acids
US6969488B2 (en) 1998-05-22 2005-11-29 Solexa, Inc. System and apparatus for sequential processing of analytes
US20060046265A1 (en) 2004-08-27 2006-03-02 Gen-Probe Incorporated Single-primer nucleic acid amplification methods
WO2006084132A2 (en) 2005-02-01 2006-08-10 Agencourt Bioscience Corp. Reagents, methods, and libraries for bead-based squencing
US7169560B2 (en) 2003-11-12 2007-01-30 Helicos Biosciences Corporation Short cycle methods for sequencing polynucleotides
US7282337B1 (en) 2006-04-14 2007-10-16 Helicos Biosciences Corporation Methods for increasing accuracy of nucleic acid sequencing
US7329492B2 (en) 2000-07-07 2008-02-12 Visigen Biotechnologies, Inc. Methods for real-time single molecule sequence determination
US7482120B2 (en) 2005-01-28 2009-01-27 Helicos Biosciences Corporation Methods and compositions for improving fidelity in a nucleic acid synthesis reaction
US20090026082A1 (en) 2006-12-14 2009-01-29 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US20090035777A1 (en) 2007-06-19 2009-02-05 Mark Stamatios Kokoris High throughput nucleic acid sequencing by expansion
US7501245B2 (en) 1999-06-28 2009-03-10 Helicos Biosciences Corp. Methods and apparatuses for analyzing polynucleotide sequences
US20090127589A1 (en) 2006-12-14 2009-05-21 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US20100137143A1 (en) 2008-10-22 2010-06-03 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes
US20100301398A1 (en) 2009-05-29 2010-12-02 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes
US8043493B2 (en) 2001-09-28 2011-10-25 Hitachi, Ltd. Multi-capillary array electrophoresis device

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7838231B2 (en) * 2006-04-07 2010-11-23 The Regents Of The University Of Michigan NPH6 nucleic acids and proteins

Patent Citations (64)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683202B1 (en) 1985-03-28 1990-11-27 Cetus Corp
US5928862A (en) 1986-01-10 1999-07-27 Amoco Corporation Competitive homogeneous assay
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683195B1 (en) 1986-01-30 1990-11-27 Cetus Corp
US4800159A (en) 1986-02-07 1989-01-24 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences
US4965188A (en) 1986-08-22 1990-10-23 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme
US5283174A (en) 1987-09-21 1994-02-01 Gen-Probe, Incorporated Homogenous protection assay
US4981785A (en) 1988-06-06 1991-01-01 Ventrex Laboratories, Inc. Apparatus and method for performing immunoassays
US5130238A (en) 1988-06-24 1992-07-14 Cangene Corporation Enhanced nucleic acid amplification process
US5480784A (en) 1989-07-11 1996-01-02 Gen-Probe Incorporated Nucleic acid sequence amplification methods
US5399491A (en) 1989-07-11 1995-03-21 Gen-Probe Incorporated Nucleic acid sequence amplification methods
US5824518A (en) 1989-07-11 1998-10-20 Gen-Probe Incorporated Nucleic acid sequence amplification methods
US5455166A (en) 1991-01-31 1995-10-03 Becton, Dickinson And Company Strand displacement amplification
US5270184A (en) 1991-11-19 1993-12-14 Becton, Dickinson And Company Nucleic acid target generation
US5358691A (en) 1992-03-27 1994-10-25 Abbott Laboratories Automated continuous and random access analytical system
US5925517A (en) 1993-11-12 1999-07-20 The Public Health Research Institute Of The City Of New York, Inc. Detectably labeled dual conformation oligonucleotide probes, assays and kits
US5599677A (en) 1993-12-29 1997-02-04 Abbott Laboratories Immunoassays for prostate specific antigen
US5672480A (en) 1993-12-29 1997-09-30 Abbott Laboratories Immunoassays for prostate specific antigen
US5714330A (en) 1994-04-04 1998-02-03 Lynx Therapeutics, Inc. DNA sequencing by stepwise ligation and cleavage
EP0684315A1 (en) 1994-04-18 1995-11-29 Becton Dickinson and Company Strand displacement amplification using thermophilic enzymes
US5912148A (en) 1994-08-19 1999-06-15 Perkin-Elmer Corporation Applied Biosystems Coupled amplification and ligation method
US6130073A (en) 1994-08-19 2000-10-10 Perkin-Elmer Corp., Applied Biosystems Division Coupled amplification and ligation method
US5695934A (en) 1994-10-13 1997-12-09 Lynx Therapeutics, Inc. Massively parallel sequencing of sorted polynucleotides
US5814447A (en) 1994-12-01 1998-09-29 Tosoh Corporation Method of detecting specific nucleic acid sequences
US6306597B1 (en) 1995-04-17 2001-10-23 Lynx Therapeutics, Inc. DNA sequencing by parallel oligonucleotide extensions
US5750341A (en) 1995-04-17 1998-05-12 Lynx Therapeutics, Inc. DNA sequencing by parallel oligonucleotide extensions
US5710029A (en) 1995-06-07 1998-01-20 Gen-Probe Incorporated Methods for determining pre-amplification levels of a nucleic acid target sequence from post-amplification levels of product
US6159750A (en) 1995-12-22 2000-12-12 Abbott Laboratories Fluorescence polarization immunoassay diagnostic method
US6150097A (en) 1996-04-12 2000-11-21 The Public Health Research Institute Of The City Of New York, Inc. Nucleic acid detection probes having non-FRET fluorescence quenching and kits and assays including such probes
US5885530A (en) 1996-06-28 1999-03-23 Dpc Cirrus, Inc. Automated immunoassay analyzer
US6210891B1 (en) 1996-09-27 2001-04-03 Pyrosequencing Ab Method of sequencing DNA
US6258568B1 (en) 1996-12-23 2001-07-10 Pyrosequencing Ab Method of sequencing DNA based on the detection of the release of pyrophosphate and enzymatic nucleotide degradation
US6432360B1 (en) 1997-10-10 2002-08-13 President And Fellows Of Harvard College Replica amplification of nucleic acid arrays
US6485944B1 (en) 1997-10-10 2002-11-26 President And Fellows Of Harvard College Replica amplification of nucleic acid arrays
US6511803B1 (en) 1997-10-10 2003-01-28 President And Fellows Of Harvard College Replica amplification of nucleic acid arrays
US6969488B2 (en) 1998-05-22 2005-11-29 Solexa, Inc. System and apparatus for sequential processing of analytes
US6534274B2 (en) 1998-07-02 2003-03-18 Gen-Probe Incorporated Molecular torches
US6787308B2 (en) 1998-07-30 2004-09-07 Solexa Ltd. Arrayed biomolecules and their use in sequencing
WO2000018957A1 (en) 1998-09-30 2000-04-06 Applied Research Systems Ars Holding N.V. Methods of nucleic acid amplification and sequencing
US7115400B1 (en) 1998-09-30 2006-10-03 Solexa Ltd. Methods of nucleic acid amplification and sequencing
US6303305B1 (en) 1999-03-30 2001-10-16 Roche Diagnostics, Gmbh Method for quantification of an analyte
US6541205B1 (en) 1999-05-24 2003-04-01 Tosoh Corporation Method for assaying nucleic acid
US7501245B2 (en) 1999-06-28 2009-03-10 Helicos Biosciences Corp. Methods and apparatuses for analyzing polynucleotide sequences
US6911345B2 (en) 1999-06-28 2005-06-28 California Institute Of Technology Methods and apparatus for analyzing polynucleotide sequences
US6818395B1 (en) 1999-06-28 2004-11-16 California Institute Of Technology Methods and apparatus for analyzing polynucleotide sequences
US6833246B2 (en) 1999-09-29 2004-12-21 Solexa, Ltd. Polynucleotide sequencing
US7329492B2 (en) 2000-07-07 2008-02-12 Visigen Biotechnologies, Inc. Methods for real-time single molecule sequence determination
US6706162B1 (en) 2000-09-25 2004-03-16 Applera Corporation High speed, high resolution compositions, methods, and kits for capillary electrophoresis
US8043493B2 (en) 2001-09-28 2011-10-25 Hitachi, Ltd. Multi-capillary array electrophoresis device
US20050130173A1 (en) 2003-01-29 2005-06-16 Leamon John H. Methods of amplifying and sequencing nucleic acids
US20050042638A1 (en) 2003-05-01 2005-02-24 Gen-Probe Incorporated Oligonucleotides comprising a molecular switch
US7169560B2 (en) 2003-11-12 2007-01-30 Helicos Biosciences Corporation Short cycle methods for sequencing polynucleotides
US20060046265A1 (en) 2004-08-27 2006-03-02 Gen-Probe Incorporated Single-primer nucleic acid amplification methods
US7482120B2 (en) 2005-01-28 2009-01-27 Helicos Biosciences Corporation Methods and compositions for improving fidelity in a nucleic acid synthesis reaction
WO2006084132A2 (en) 2005-02-01 2006-08-10 Agencourt Bioscience Corp. Reagents, methods, and libraries for bead-based squencing
US7282337B1 (en) 2006-04-14 2007-10-16 Helicos Biosciences Corporation Methods for increasing accuracy of nucleic acid sequencing
US20090026082A1 (en) 2006-12-14 2009-01-29 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US20090127589A1 (en) 2006-12-14 2009-05-21 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale FET arrays
US20100188073A1 (en) 2006-12-14 2010-07-29 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale fet arrays
US20100197507A1 (en) 2006-12-14 2010-08-05 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes using large scale fet arrays
US20090035777A1 (en) 2007-06-19 2009-02-05 Mark Stamatios Kokoris High throughput nucleic acid sequencing by expansion
US20100137143A1 (en) 2008-10-22 2010-06-03 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes
US20100301398A1 (en) 2009-05-29 2010-12-02 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes

Non-Patent Citations (150)

* Cited by examiner, † Cited by third party
Title
ADESSI ET AL., NUCLEIC ACID RES., vol. 28, 2000, pages E87
ALCARAZ ET AL., HUM MOL GENET, 2011
ALCARAZ ET AL., PROC NATL ACAD SCI U S A, vol. 103, 2006, pages 19424 - 19429
ANDERSEN, NATURE, vol. 426, 2003, pages 570 - 574
ANGERS, NAT CELL BIOL, vol. 8, 2006, pages 348 - 357
ANSLEY ET AL., NATURE, vol. 425, 2003, pages 628 - 633
ASTIER ET AL., J. AM. CHEM. SOC., vol. 128, no. 5, 8 February 2006 (2006-02-08), pages 1705 - 10
ATTANASIO ET AL., NAT GENET, vol. 39, 2007, pages 1018 - 1024
AUCRBACH, A.D.; WOLMAN, S.R., NATURE, vol. 261, 1976, pages 494 - 496
AUERBACH, A.D., MUTAT. RES., vol. 668, 2009, pages 4 - 10
AUERBACH, A.D.; WOLMAN, NATURE, vol. 261, 1976, pages 494 - 496
BABA ET AL., PATHOL. RES. PRACT., vol. 202, 2006, pages 555 - 559
BENNETT ET AL., PHARMACOGENOMICS, vol. 6, 2005, pages 373 - 382
BENTLEY ET AL., NATURE, vol. 456, 2008, pages 53 - 59
BENTLEY, D.R. ET AL., NATURE, vol. 456, 2008, pages 53 - 59
BIRREN ET AL.: "Genome Analysis: Analyzing DNA", vol. 1, COLD SPRING HARBOR
BRANTON ET AL., NAT. BIOTECHNOL., vol. 26, no. 10, 2008, pages 1146 - 53
BRENNER ET AL., NAT. BIOTECHNOL., vol. 18, 2000, pages 630 - 634
BRYJA ET AL., J CELL SCI, vol. 120, 2007, pages 586 - 595
BRYJA ET AL., PROC NATL ACAD SCI USA, vol. 104, 2007, pages 6690 - 6695
BUKANOV ET AL., NATURE, vol. 444, 2006, pages 949 - 952
BURRY, A.F., J. PATHOL., vol. 113, 1974, pages 147 - 150
BURRY, J. PATHOL., vol. 113, 1974, pages 147 - 150
CHAKI ET AL., KIDNEY INT, 2011
CHANG ET AL., HUM MOL GENET, vol. 15, 2006, pages 1847 - 1857
CHENG ET AL., DEV BIOL, vol. 307, 2007, pages 43 - 52
CICCIA; ELLEDGE, MOL CELL, vol. 40, 2010, pages 179 - 204
COENE ET AL., HUM MOL GENET, vol. 20, 2011, pages 3592 - 3605
DELOUS ET AL., NAT GENET, vol. 39, 2007, pages 875 - 881
DRMANAC ET AL., NAT. BIOTECHNOL., vol. 16, 1998, pages 54 - 58
EDMOND ET AL., EMBO J., 2010
EDMOND, EMBO J., 2010
EID ET AL., SCIENCE, vol. 323, 2009, pages 133 - 138
ELIAS ET AL., EXP CELL RES, vol. 291, 2003, pages 176 - 188
GENOMICS, vol. 92, 2008, pages 255
GERMINO, NAT GENET, vol. 37, 2005, pages 455 - 457
GODIN, M. ET AL., AM. J. KIDNEY DIS., vol. 27, 1996, pages 166
GODIN, M. ET AL.: "Karyomegalic interstitial nephritis", AM. J. KIDNEY DIS., vol. 27, 1996, pages 166
GRASCR, J CELL BIOL, vol. 179, 2007, pages 321 - 330
GUATELLI ET AL., PROC. NATL. ACAD. SCI. USA, vol. 87, 1990, pages 1874
GUDBJARTSSON ET AL., NAT GENET, vol. 25, 2000, pages 12 - 13
HABBIG ET AL., J CELL BIOL, vol. 193, 2011, pages 633 - 642
HARRIS ET AL., SCIENCE, vol. 320, 2008, pages 106 - 109
HCLOU, J MED GENET, vol. 44, 2007, pages 657 - 663
HILDEBRANDT ET AL., N ENGL J MED, vol. 364, 2011, pages 1533 - 1543
HILDEBRANDT ET AL., N. ENGL. J. MED., vol. 364, 2011, pages 1533 - 1543
HILDEBRANDT ET AL., NAT GENET, vol. 17, 1997, pages 149 - 153
HILDEBRANDT ET AL., PLOS GENETICS, vol. 5, 2009, pages 31000353
HILDEBRANDT, F. ET AL., PLOS GENET., vol. 5, 2009, pages E1000353
HILDEBRANDT, F. ET AL., PLOS GENET., vol. 5, 2009, pages EL 000353
HILDEBRANDT, F. ET AL., PLOS GENET., vol. 5, 2009, pages EL000353
HOLLANDER ET AL., AM J HUM GENET, vol. 79, 2006, pages 556 - 561
HUANGFU ET AL., NATURE, vol. 426, 2003, pages 83 - 87
HUANGFU, D. ET AL., NATURE, vol. 426, 2003, pages 83 - 87
HUANGFU; ANDERSON, PROC NATL ACAD SCI U S A, vol. 102, 2005, pages 11325 - 11330
JADEJA ET AL., NAT GENET, vol. 37, 2005, pages 520 - 52
JANDEROVA-ROSSMEISLOVA ET AL., J STRUCT BIOL, vol. 159, 2007, pages 56 - 70
KATO, INT. J. CLIN. EXP. MED., vol. 2, 2009, pages 193 - 202
KNIPSCHEER, P. ET AL., SCIENCE, vol. 326, 2009, pages 1698 - 1701
KOENERS ET AL., AM. J. PHYSIOL. REGUL. INTEGR. COMP. PHYSIOL., vol. 294, 2008, pages R1847 - R1855
KORLACH, PROC. NATL. ACAD. SCI. USA, vol. 105, 2008, pages 1176 - 1181
KRATZ, K. ET AL., CELL, vol. 142, 2010, pages 77 - 88
KRUGLYAK ET AL., AM. J. HUM. GENET., vol. 58, 1996, pages 1347 - 1363
KRUGLYAK, AM J HUM GENET, vol. 58, 1996, pages 1347 - 1363
KU ET AL., BIOCHEM BIOPHYS RES COMMUN, vol. 311, 2003, pages 702 - 707
KWOH ET AL., PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 1173
LETTEBOER; ROEPMAN, METHODS MOL BIOL, vol. 484, 2008, pages 145 - 159
LEVENE ET AL., SCIENCE, vol. 299, 2003, pages 682 - 686
LIEBAU ET AL., J BIOL CHEM, vol. 286, 2011, pages 14237 - 14245
LIU ET AL., SCIENCE, vol. 329, 2010, pages 693 - 696
LIU, MOL CELL PROTEOMICS, vol. 6, 2007, pages 1299 - 1317
LIU, T.X. ET AL., DEV. CELL, vol. 5, 2003, pages 903 - 914
LIZARDI ET AL., BIOTECHNOL., vol. 6, 1988, pages 1197
MACKAY, C. ET AL., CELL, vol. 142, 2010, pages 65 - 76
MACLEAN ET AL., NATURE REV. MICROBIOL., vol. 7, pages 287 - 296
MALLETTE, F.A.; FERBEYRE, G., CELL CYCLE, vol. 6, 2007, pages 1831 - 1836
MARGULIES ET AL., NATURE, vol. 437, 2005, pages 376 - 380
MAUDE; ENDERS, CANCER RES, vol. 65, 2005, pages 780 - 786
MAXAM ET AL., PROC. NATL. ACAD. SCI. USA, vol. 74, 1977, pages 560 - 564
MCGREGOR ET AL., NAT GENET, vol. 34, 2003, pages 203 - 208
MITRA ET AL., ANALYTICAL BIOCHEMISTRY, vol. 320, 2003, pages 55 - 65
MOCH ET AL., PATHOLOGE, vol. 15, 1994, pages 44 - 48
MOLLET ET AL., NAT GENET, vol. 32, 2002, pages 300 - 305
MULLIS ET AL., METH. ENZYMOL., vol. 155, 1987, pages 335
MURAKAWA ET AL., DNA, vol. 7, 1988, pages 287
NG ET AL., NATURE, vol. 461, 2009, pages 272 - 276
NORMAN C. NELSON ET AL.: "Nonisotopic Probing, Blotting, and Sequencing, 2d ed.", 1995
OLBRICH ET AL., NAT GENET, vol. 34, 2003, pages 455 - 459
OTTO ET AL., AM J HUM GENET, vol. 71, 2002, pages 1167 - 1171
OTTO ET AL., J MED GENET, 2010
OTTO ET AL., J. AM SOC NEPHROL, vol. 19, 2008, pages 587 - 592
OTTO ET AL., J. MED GENET, 2010
OTTO ET AL., NAT GENET, vol. 34, 2003, pages 413 - 420
OTTO ET AL., NAT GENET, vol. 34, 2010, pages 413 - 420
OTTO ET AL., NAT GENET, vol. 42, 2010, pages 840 - 850
OTTO, E.A. ET AL., HUM. MUTAT., vol. 29, 2008, pages 418 - 426
OTTO, E.A. ET AL., NAT. GENET., vol. 34, 2003, pages 413 - 420
OTTO, E.A. ET AL., NAT. GENET., vol. 42, 2010, pages 840 - 850
OTTO, NAT GENET, vol. 37, 2005, pages 282 - 288
OTTO, NAT GENET, vol. 42, 2010, pages 840 - 850
PALMER ET AL., DIAGN. CYTOPATHOL., vol. 35, 2007, pages 179 - 182
PAN; LEE, CELL CYCLE, vol. 8, 2009, pages 655 - 664
PERSING, DAVID H. ET AL.: "Diagnostic Medical Microbiology: Principles and Applications", 1993, AMERICAN SOCIETY FOR MICROBIOLOGY, article "In Vitro Nucleic Acid Amplification Techniques", pages: 51 - 87
RAMENSKY ET AL., NUCLEIC ACIDS RES, vol. 30, 2002, pages 3894 - 3900
REINHARDT, CELL CYCLE, vol. 10, 2011, pages 23 - 27
ROBU ET AL., PLOS GENET, vol. 3, 2007, pages E78
RONAGHI ET AL., ANAL. BIOCHEM., vol. 242, 1996, pages 84 - 89
RUPAREL ET AL., PROC. NATL. ACAD. SCI. USA, vol. 102, 2005, pages 5932 - 5937
SANG ET AL., CELL, vol. 145, 2011, pages 513 - 528
SANGER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 74, 1997, pages 5463 - 5467
SAYER ET AL., NAT GENET, vol. 38, 2006, pages 674 - 681
SAYER, J.A. ET AL., NAT. GENET., vol. 38, 2006, pages 674 - 681
SCHAFER, T. ET AL., HUM. MOL. GENET., vol. 17, 2008, pages 3655 - 3662
SCIENCE, vol. 327, no. 5970, 2010, pages 1190
SHAFEGHATI ET AL., AM J MED GENET A, vol. 146A, 2008, pages 529 - 531
SHENDURE ET AL., SCIENCE, vol. 309, 2005, pages 1728 - 1732
SIMONS ET AL., NAT GENET, vol. 37, 2005, pages 537 - 543
SIMONS, M. ET AL., NAT. GENET., vol. 37, 2005, pages 537 - 543
SIVASUBRAMANIAM ET AL., GENES DEV, vol. 22, 2008, pages 587 - 600
SLAVOTINEK ET AL., J MED GENET, vol. 39, 2002, pages 623 - 633
SMITH ET AL., J AM SOC NEPHROL, vol. 17, 2006, pages 2821 - 2831
SMITH ET AL., JASN, vol. 17, 2006, pages 2821 - 283 1
SMOGORZEWSKA ET AL., CELL, vol. 129, 2007, pages 289 - 301
SMOGORZEWSKA, A. ET AL., MOL. CELL, vol. 39, 2010, pages 36 - 47
SPOENDLIN ET AL., AM. J. KIDNEY DIS., vol. 25, 1995, pages 242 - 252
SPOENDLIN, M. ET AL., AM. J. KIDNEY DIS., vol. 25, 1995, pages 242 - 252
SPOENDLIN, M. ET AL.: "Karyomegalic interstitial nephritis: further support for a distinct entity and evidence for a genetic defect", AM. J. KIDNEY DIS., vol. 25, 1995, pages 242 - 252
STRAUCH ET AL., AM J HUM GENET, vol. 66, 2000, pages 1945 - 1957
STRAUCH, K., AM. J. HUM. GENET., vol. 66, 2000, pages 1945 - 1957
SULTAN ET AL., BIOL CHEM, vol. 286, 2011, pages 4566 - 4575
SZCZERBAL; BRIDGER, CHROMOSOME RES, vol. 18, 2010, pages 887 - 895
TSAI; REED, MOL CELL BIOL, vol. 18, 1998, pages 6447 - 6456
VALCNTC, NAT GENET, vol. 38, 2006, pages 623 - 625
VALENTE ET AL., NAT GENET, vol. 38, 2006, pages 623 - 625
VAN HAELST ET AL., AM J MED GENET A, vol. 143A, 2007, pages 3194 - 3203
VAN HAELST ET AL., AM J MED GENET A, vol. 146A, 2008, pages 2252 - 2257
VCRINC, ANN. PATHOL., vol. 30, 2010, pages 240 - 242
VERINE ET AL., ANN. PATHOL., vol. 30, 2010, pages 240 - 242
VOCLKCRDING ET AL., CLINICAL CHEM., vol. 55, 2009, pages 641 - 658
VOELKERDING ET AL., CLINICAL CHEM., vol. 55, 2009, pages 641 - 658
VOELKERDING ET AL., CLINICAL CHERN., vol. 55, 2009, pages 641 - 58
VRONTOU ET AL., NAT GENET, vol. 34, 2003, pages 209 - 214
WALKER, G. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 392 - 396
WEISS, R., SCIENCE, vol. 254, 1991, pages 1292
XIAO ET AL., MOL CELL BIOL, vol. 27, 2007, pages 5393 - 5402
YOSHIKIYO, K. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 107, 2010, pages 21553 - 21557
ZENG ET AL., ZEBRAFISH, vol. 6, 2009, pages 405 - 415
ZHOU ET AL., AM J PHYSIOL RENAL PHYSIOL, vol. 299, 2010, pages F55 - 62
ZHOU, AM. J. PHYSIOL. RENAL PHYSIOL., vol. 299, 2010, pages F55 - F62
ZOLLINGER, H.U. ET AL., HELV. PAEDIATR. ACTA, vol. 35, 1980, pages 509 - 530

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105256378B (en) * 2015-04-13 2018-01-05 卢彦平 Precursory cilia related disease microarray screening for disease genes

Also Published As

Publication number Publication date Type
WO2014005076A9 (en) 2014-04-03 application
WO2014005076A3 (en) 2014-02-20 application

Similar Documents

Publication Publication Date Title
Cheng et al. Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood
Prensner et al. The long noncoding RNA SChLAP1 promotes aggressive prostate cancer and antagonizes the SWI/SNF complex
Graser et al. Cep68 and Cep215 (Cdk5rap2) are required for centrosome cohesion
Willer et al. ISPD loss-of-function mutations disrupt dystroglycan O-mannosylation and cause Walker-Warburg syndrome
Weng et al. MicroRNA profiling of clear cell renal cell carcinoma by whole‐genome small RNA deep sequencing of paired frozen and formalin‐fixed, paraffin‐embedded tissue specimens
Bahn et al. The landscape of microRNA, Piwi-interacting RNA, and circular RNA in human saliva
Liu et al. Identification of RNF213 as a susceptibility gene for moyamoya disease and its possible role in vascular development
Stuart et al. Exome sequencing links mutations in PARN and RTEL1 with familial pulmonary fibrosis and telomere shortening
Memczak et al. Identification and characterization of circular RNAs as a new class of putative biomarkers in human blood
Lau et al. Alteration of the microRNA network during the progression of Alzheimer's disease
Kornblum et al. Loss-of-function mutations in MGME1 impair mtDNA replication and cause multisystemic mitochondrial disease
Veeramah et al. Exome sequencing reveals new causal mutations in children with epileptic encephalopathies
Chen-Plotkin et al. TMEM106B, the risk gene for frontotemporal dementia, is regulated by the microRNA-132/212 cluster and affects progranulin pathways
Griffith et al. Mutations in pericentrin cause Seckel syndrome with defective ATR-dependent DNA damage signaling
Słabicki et al. A genome-scale DNA repair RNAi screen identifies SPG48 as a novel gene associated with hereditary spastic paraplegia
Majewski et al. Identification of recurrent FGFR3 fusion genes in lung cancer through kinome‐centred RNA sequencing
Krumm et al. A de novo convergence of autism genetics and molecular neuroscience
Chaki et al. Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling
Roberts Transcriptional regulation by WT1 in development
Rivière et al. De novo mutations in the actin genes ACTB and ACTG1 cause Baraitser-Winter syndrome
Otto et al. Candidate exome capture identifies mutation of SDCCAG8 as the cause of a retinal-renal ciliopathy
Palmer et al. Specificity of cytoplasmic dynein subunits in discrete membrane-trafficking steps
Oram et al. TSPY potentiates cell proliferation and tumorigenesis by promoting cell cycle progression in HeLa and NIH3T3 cells
Cappello et al. Mutations in genes encoding the cadherin receptor-ligand pair DCHS1 and FAT4 disrupt cerebral cortical development
Rademakers et al. Advances in understanding the molecular basis of frontotemporal dementia

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13737089

Country of ref document: EP

Kind code of ref document: A2

122 Ep: pct application non-entry in european phase

Ref document number: 13737089

Country of ref document: EP

Kind code of ref document: A2