CN104487575A

CN104487575A - Nmnat1 mutant gene, primers, kit, and method for detecting same and use thereof

Info

Publication number: CN104487575A
Application number: CN201280074552.6A
Authority: CN
Inventors: 祁鸣; 易鑫; 王娟; 陈洋; 陈演华; 钟晶; 吴仁花; 杨焕明
Original assignee: BGI Shenzhen Co Ltd
Current assignee: BGI Shenzhen Co Ltd
Priority date: 2012-07-28
Filing date: 2012-09-26
Publication date: 2015-04-01
Anticipated expiration: 2032-09-26
Also published as: WO2014019108A1; CN104487575B

Abstract

Provided is a mutant gene relating to a disease, in particular a mutant gene relating to Leber congenital amaurosis. Provided are the mutant gene, primers, kit, and method for detecting same and the use thereof.

Description

Nmnat1 mutant gene, primers, kit, and method for detecting same and use thereof

The mutators of V A 7, the primer for detecting it, kit and method with and application thereof

Technical field is the present invention relates to disease related mutation gene, in particular to leber's congenital amaurosis related mutation gene.Background technology

Leber's congenital amaurosis（Leber congenital amaurosis, LCA) it is a kind of autosomal recessive malnutritive to retina, it shows as genetic heterogeneity.Leber's congenital amaurosis is one of most common reason of children's heredity blindness¹, it is a kind of serious malnutritive to retina, shows as at birth or infancy low visual acuity and nystagmus.So far, it is found that LCA is relevant with least 17 genes.20-30% examination to LCA cases in lack identifiable molecular disease because showing still to have not by existing LCA Disease-causing genes²—⁴.Present clinical test generally leads to t^ and finds molecular defect because replacing, or by oral 9- cis retinals bypasses metabolic block when RPE65 and LRAT are not enough.

Therefore, this area still needs the gene mutation for finding that LCA is related, and detects primer, kit and the method for related mutation gene.The content of the invention

The extron group for suffering from LCA individuals has been sequenced in inventor, identifies the nonsense mutation in NMNAT1 genes（ c.507G>A, p.Trpl69*) and missense mutation（ c.769G>A, p.Glu257Lys), it is found that this mutator is relevant with LCA.

Therefore, in a first aspect, the present invention relates to LCA biomarker, that is, the NMNAT1 genes or NMNAT1 albumen being mutated, the biomarker are with NMNAT1 genes or NMNAT1 albumen selected from following mutation：

Nonsense mutation（ c.507G>A, p.Trpl69*);

Missense mutation（ c.769G>A, p.Glu257Lys).

In one embodiment, the sequence of the genes of mutation Λ Λ 7 of the invention is the SEQ D with following mutation) NO:l:

Nonsense mutation is c.507G>A;And/or

Missense mutation is c.769G>A. In another embodiment, the sequence of the NMNAT1 albumen of mutation of the invention is the SEQ D with following mutation) NO:2:

Nonsense mutation p.Trpl69*;And/or

Missense mutation p.Glu257Lys.

In second aspect, the present invention relates to a kind of detection LCA method, methods described includes whether there is mutational site in the NMNAT1 genes or NMNAT1 albumen of detection subject, if mutational site, then subject's quilt:It is accredited as with LCA or is susceptible to suffer from LCA, or its offspring can suffer from LCA or be susceptible to suffer from LCA, the mutational site is selected from following any or its combination：

Nonsense mutation（c.507G>A, p.Trpl69*);With

Missense mutation（ c.769G>A, p.Glu257Lys).

In one embodiment, the method for the detection LCA includes step：

The DNA sample of subject is extracted,

Enter performing PCR amplification to the DNA sample and obtain amplified production, obtain NMNAT1 gene extrons and exon/intron border,

The amplified production that above-mentioned PCR is expanded carries out sequencing and obtains sequencing result,

The sequencing result is analyzed, determine to whether there is mutational site in NMNAT1 genes, if mutational site, then the subject is accredited as with LCA or is susceptible to suffer from LCA, or its offspring can suffer from LCA or be susceptible to suffer from LCA, the mutational site is selected from following any or its combination：

Nonsense mutation（c.507G>A, p.Trpl69*);With

Missense mutation（ c.769G>A, p.Glu257Lys).

In a further preferred embodiment, PCR uses KAPA2G Robust HotStart

(KAPABIOSYSTEMS, Woburn, MA) is carried out.

In a further preferred embodiment, the primer of PCR amplifications is designed with reference to human genome, such as following at least one set of primer：

SEQ ID NO:3 and SEQ ID NO:4;

SEQ ID NO:5 and SEQ ID NO:6;

SEQ ID NO:7 and SEQ ID NO:8;

SEQ ID NO:9 and SEQ ID NO:10.

In a further preferred embodiment, sequencing is Sanger sequencings.

In a further preferred embodiment, sequence analysis is carried out using Mutation Surveyor program (State College, PA).

In one embodiment, NMNAT1 albumen is SEQ D) NO:2 sequence is represented. In another embodiment, V J genes are SEQ ID NO:L sequence is represented.In still another embodiment, detection LCA of the invention method includes the step of following at least one set of primer is expanded：

SEQ ID NO:3 and SEQ ID NO:4;

SEQ ID NO:5 and SEQ ID NO:6;

SEQ ID NO:7 and SEQ ID NO:8;

SEQ ID NO:9 and SEQ ID NO:10.

In a further embodiment, detect mutational site by being carried out selected from following technology in detection LCA of the invention method：Sequencing, electrophoresis, nucleic acid hybridization, in situ hybridization, PCR, reverse transcriptase chain reaction and denaturing high-performance chromatography.

In the method for second aspect of the present invention, the genes of mutation Λ Γ Λ 7 of homozygosis are preferably detected.In the third aspect, the present invention relates to a kind of method for detecting mutation NMNAT1 genes or NMNAT1 albumen, methods described includes whether there is mutational site in the NMNAT1 genes or NMNAT1 albumen of detection subject, and the mutational site is selected from following any or its combination：

Nonsense mutation（c.507G>A, p.Trpl69*);With

Missense mutation（ c.769G>A, p.Glu257Lys).

In one embodiment, the method for the genes of detection mutation V A 7 of the invention includes step：The DNA sample of subject is extracted,

The amplified production of more above-mentioned PCR amplifications carries out sequencing and obtains sequencing result,

The sequencing result is analyzed, determines to whether there is mutational site in NMNAT1 genes, the mutational site is selected from following any or its combination：

Nonsense mutation（c.507G>A, p.Trpl69*);With

Missense mutation（ c.769G>A, p.Glu257Lys).

In a further preferred embodiment, PCR uses KAPA2G Robust HotStart

(KAPABIOSYSTEMS, Woburn, MA) is carried out.

SEQ ID NO:3 and SEQ ID NO:4;

SEQ ID NO:5 and SEQ ID NO:6;

SEQ ID NO:7 and SEQ ID NO:8; SEQ ID NO:9 and SEQ ID NO:10.

In a further preferred embodiment, sequencing is Sanger sequencings.

In a further preferred embodiment, sequence analysis is carried out using Mutation Surveyor program (State College, PA).In one embodiment, NMNAT1 albumen is SEQ ID NO:2 sequence is represented.

In another embodiment, V J genes are SEQ ID NO:L sequence is represented.In still another embodiment, the method for detection mutation NMNAT1 genes of the invention or NMNAT1 albumen includes the step of following at least one set of primer is expanded：

SEQ ID NO:3 and SEQ ID NO:4;

SEQ ID NO:5 and SEQ ID NO:6;

SEQ ID NO:7 and SEQ ID NO:8;

SEQ ID NO:9 and SEQ ID NO:10.

In a further embodiment, detection mutational site is logical in the method for detection mutation NMNAT1 genes of the invention or NMNAT1 albumen is carried out from following technology：Sequencing, electrophoresis, nucleic acid hybridization, in situ hybridization, PCR, reverse transcriptase chain reaction and denaturing high-performance chromatography.

The method of detection mutation NMNAT1 genes or NMNAT1 albumen described in third aspect present invention can be used for the purpose for diagnosing LCA, can be used for the purpose of non-diagnostic disease.In some embodiments, the purpose of heretofore described non-diagnostic disease includes but is not limited to research SNP distributions and polymorphism, develops for family and studies.Such application be it will be appreciated by those skilled in the art that.

According to description herein as can be seen that some individuals carry the genes of mutation Λ Λ 7 of the present invention but do not suffer from LCA, for example only item chromosome carries the heterozygous genotypes of the mutation.Detection to this part population can not be related to any purpose diagnosed the illness, because these individuals itself are not ill.But the result detected for them can be used as useful information, for example as the important indicator of inspection before educating, fertility is instructed, either the instrument or tracking gene mutation for carriers of mutation examination or as SNP distributions and polymorphic Journal of Sex Research or family develop.Such application be also it will be appreciated by those skilled in the art that.Therefore, the method for detection mutation Λ Λ/gene or NMNAT1 albumen is related to detection heterozygous mutant described in third aspect present invention.

But the method for detection mutation NMNAT1 genes or NMNAT1 albumen also includes detection homozygous mutation described in third aspect present invention.

In fourth aspect, the present invention relates to the primer pair used in mutation NMNAT1 genes or NMNAT1 albumen is detected by PCR, the mutation is selected from following any or its combination：

Nonsense mutation（c.507G>A, p.Trpl69*);And/or Missense mutation（ c.769G>A, p.Glu257Lys),

Wherein described primer pair is based respectively on to be designed before and after following position is on genome sequence or cDNA sequence so that expand the position：CDNA sequence the 507th and the 769th.

In one embodiment, the primer pair is：

SEQ ID NO:3 and SEQ ID NO:4;

SEQ ID NO:5 and SEQ ID NO:6;

SEQ ID NO:7 and SEQ ID NO:8;Or

SEQ ID NO:9 and SEQ ID NO:10.

At the 5th aspect, the present invention relates to the nucleic acid probe with being mutated the gene complementations of Λ Γ Λ 7, the mutation is selected from following any or its combination：

Nonsense mutation is c.507G>A;And/or

Missense mutation is c.769G>Position in A, cDNA sequence：CDNA sequence the 507th and the 769th.

At the 6th aspect, the present invention relates to detection mutation NMNAT1 genes or the kit of NMNAT1 albumen, comprising one or more groups of primer pairs, wherein the mutation is selected from following any or its combination：Nonsense mutation（c.507G>A, p.Trpl69*);With

Missense mutation（ c.769G>A, p.Glu257Lys),

Wherein described primer pair is based respectively on to be designed selected from following position on genome sequence or cDNA sequence so that its amplified production covers the position：CDNA sequence the 507th and the 769th.

In one embodiment, the kit of the detection mutation NMNAT1 genes or NMNAT1 albumen is included selected from following at least one set of primer：

SEQ ID NO:3 and SEQ ID NO:4;

SEQ ID NO:5 and SEQ ID NO:6;

SEQ ID NO:7 and SEQ ID NO:8;

SEQ ID NO:9 and SEQ ID NO:10.

At the 7th aspect, the present invention relates to the kit of the genes of detection mutation Λ Γ Λ 7, comprising one or more nucleic acid probes, the mutation is selected from following any or its combination：

Nonsense mutation is c.507G>A;With

Missense mutation is c.769G>A,

The probe is with including the regional complementarity on genome sequence or cDNA sequence selected from following location on mutation NMNAT1 genes：CDNA sequence the 507th and the 769th. In eighth aspect, the present invention relates to a kind of method for detecting gene extron and the mutation of exon/intron border, including step：

1. DNA sample is extracted from subject,

2. the extron group of pair above-mentioned DNA sample and all exon/intron border sequences be sequenced obtain that fragment is sequenced,

3. above-mentioned sequencing fragment is compared with reference sequences, gene extron and the mutation of exon/intron border are obtained.

In one embodiment, the capture of extron group is carried out to collect the CDS areas of human gene group DNA to above-mentioned sample before second step, the DNA library for being enriched with extron is subjected to the second library construction, for sequencing.

In one embodiment, the sequencing in second step is performed as follows：

1) sample DNA is broken into 200-300 bp or so fragment at random, then connecting top connection respectively at fragment two ends prepares Hybrid Library；

2) PCR (LM-PCR) mediated after library is purified by coupled reaction linear amplification and the DNA library of biotin labeling carry out hybridization enrichment, upper machine sequencing is carried out after LM-PCR linear amplification again, reading length is 90 bp, and the average sequencing depth of each sample is at least 50.

In one embodiment, removed in third step and repeat sequencing fragment.Brief description of the drawings

The retina of Fig. 1 patient 1 is apparent, shows that retinal vessel is reduced, macula retinae pigmentation and atrophic macula lutea are damaged.

The schematic diagram for the mutation that Fig. 2 are identified in NMNAT1 genes.The gene mutations of Sanger sequencing examination Λ Γ Λ 7 are passed through to the LCA patient of the mutation without former identification.Point analysis of variance of genomic DNA is also carried out for available kinsfolk.Close sheep product from patient 6 are unavailable.The only current sample of patient 9 and 10 can be obtained.

Fig. 3 pass through Sanger sequencing examination NMNAT1 gene mutations to the LCA patient of the mutation without former identification.Point analysis of variance of genomic DNA is also carried out for available kinsfolk.Parental animal from patient 6 is unavailable.The only current sample of patient 9 and 10 can be obtained.

Fig. 4 graphical representations show the residue related to the LCA mutation identified in people's NMNAT1 protomer structures.General structure appears dimmed ribbon, with reference to NAD be orange club.All 8 mutational sites are shown as club（Nonsense blue-green, missense green）.Red dotted line shows three Embodiment

In the present invention, gene mutation and protein mutant are represented using representation generally in the art.In the present invention, c.507G>A represents that the 507th nucleotides of cDNA becomes A by G;The codon of p.Trpl69* and p.Trpl69stop all the 169th, presentation code protein becomes terminator codon by the codon for encoding Trp； c.769G>A represents that the 769th nucleotides of cDNA becomes A by G;The bit codon of p.Glu257Lys presentation codes protein the 169th is become to encode Lys codon by the codon for encoding Glu, or represents that the 169th, protein becomes Lys by Glu.

Therefore, in one embodiment, the codon of the amino acids of coding the 169th of the genes of mutation Λ Γ Λ 7 of the invention can be AAA or AAG.

The cDNA sequence of the genes of wild type UV 7 such as SEQ ID Ν Ο:Shown in 1.The ^ row such as SEQ ID NO of wild type human NMNAT1 albumen:Shown in 2.The mRNA sequence such as SEQ ID NO of wild type Α Λ Λ/gene:Shown in 11.

For in description of the invention and claims, carrying because of sequence, it will be appreciated by those skilled in the art that actual include any one or two of complementary strand.For convenience, in the present specification and claims, although only give a chain in most cases, but actually also disclose that another complementary therewith chain.The cDNA sequence of the genes of SJV 7 is for example put forward, actually including the sequence and its complementary series.For example, referring to SEQ ID NO:L, it is actual to include its complementary series.Those skilled in the art are further appreciated that can detect another chain using a ^^, and vice versa.

Gene order in the application includes DNA form or rna form, open one of which, it is meant that another to be also disclosed.The cDNA sequence of the genes of SJV 7 is for example put forward, it is actual also to include corresponding RNA sequence.

In the present invention, exon/intron border refers to the 1-200 since exon-intron boundaries, such as such as 10-100, such as 20-80,30-70, such as 40,50,60 introne nucleotides.

In order to find the mutation in known LCA related genes, the code area and all exon/intron borders of inventor's LCA related genes known to 17（At least 50 introne nucleotides since exon-intron boundaries）It is sequenced, 17 genes arePZJ, CABP4, CEP290, CRB1, CRX, GUCY2D. IQCB1, LCA5. LRAT, RD3, RDH12, RPE65, RPGRIPU SPATA7. TULP IMPDH1 and OTX2, the relevant information of these genes can be from Mendel's human inheritance's database（OMIM) obtain.In the 220 LCA individuals studied, mutant allele is found that on two chromosomes of 160 cases, a mutant equipotential is found in 10 cases Gene.

Because not identifying known LCA^ in patient 1 (face ^^ type information and see below literary table 5) because having carried out full extron group sequence analysis to it（Referring to table 1 and 2 and embodiment）.2460 are identified altogether not report because of variation.Preferred 10 genes of inventor, are each respectively provided with the nonsense variation do not reported in the past, it is contemplated that can truncate product, these genes are further analyzed.GeneCards shows that 5 in this 10 genes express in retina, and including V 7, the gene genetic collection of illustrative plates is in the areas of 1 ρ 36.The genetic map in Pakistani family of the same clan was in the ρ 36 of chromosome 1 in the past⁵。

Gene Λ Λ/encoding nicotinamide adenine-dinucleotide（NAD a kind of) enzyme in biosynthesis pathway, skin is thought can anti-axonal degeneration.

Nonsense is identified in Λ Λ/gene by extron group sequencing to make a variation（c.507G>A, p.Trpl69*) and missense mutation variation（ c.769G>A, p.Glu257Lys), and pass through Sanger sequencings（PCR primer is provided in ^3）Confirmed.Parent's test determines that male parent carries the heterozygote of the nonsense variation, and female parent carries the heterozygote of the missense variation, and the LCA caused by NMNAT1 gene mutations is autosomal recessive hereditary diseases, therefore father and mother will not cause a disease.But the two variations of filial generation heredity simultaneously, therefore cause NMNAT1 gene functions abnormal, so that cause a disease.Analysis shows, unaffected born of the same parents parent does not carry variation.In a word, these results support that the genes of 7 Λ Λ 1 are LCA candidate genes.Table 1. passes through sequencing of extron group examination LCA genes

The new nonsense mutation that table 2. is identified by sequencing of extron group.Rna expression pattern is analyzed by GeneCards.

The relevant information of said gene can be from Mendel's human inheritance's database（OMIM www.omim.or) obtain.

X represents translation termination.

^ PCR primer sequences

Acted on to further assess V J genes in LCA, identified 50 irrelevant LCA cases of mutation without before in known LCA related genes by Sanger Sequence analysis.Other 10 patients are accredited has compound heterozygous mutant in the genes of Λ Λ 1（Fig. 2 and table 4).It is interesting to note that c.769G all 11 patients carry common missense mutation mutation>A (p.Glu257Lys), this is one that identified two become in heteroallele.As shown in pedigree analysis, the mutation of the identification is all isolated with LCA phenotypes（Fig. 3). The country source for the mutation # patient that table 4. is identified and race's summary.

# provides the PolyPhen-2 predictions that identified missense makes a variation.The maximum of the damage variation of prediction is 1. 0.However, it should be appreciated that data are explained.Variation P. Glu257Lys are initially predicted possible damage, and score value is 0. 903.However, last prediction shows, the variation is benign, and score value is 0. 089.On the contrary, it is benign that variation p. Met35Thr, which are initially predicted, score value is 0. 059.Finally prediction may be damaged, and score value is 0. 601.

NMNAT1 (NMNAT1) includes 4 encoded exons, encodes 279 residues proteins, plays an important roll in NAD+ biosynthesis, and wherein it is catalyzed from nicotinamide mononucleotide（NMN) and Α formation NAD+.Do not associated before the genes of VMA 7 with LCA, but it is relevant with axonal degeneration⁶.The TVw knock out mice of homozygosis can not live to birth, and the nmnat gene mutations Drosophila melanogaster (DrosophUa melanogaster) of homozygosis is also lethal；These results support Vw a genes the viewpoint of required function in some species⁷.The genetic map of Λ Λ/gene is being previously determined near the LCA9 locus of genetic map in 1 ρ 36.22.Impacted individual has the inborn serious impaired vision of non-progressive in the LCA9 families⁵.Similarly, the 11 LCD cases reported herein all have the serious vision loss of very early hair（Table 5).

By finding disease related mutation totally in 9 unrelated families and in 11 cases from 4 country variants, W open there is provided Λ Λ/gene be the gene for drawing ^ LCA evidence（Table 4). Clinically, all 11 cases have serious presentation（Table 5).Although seldom, disease severity may be relevant with the property of mutation for data.Terms used herein " allele is clamped（Allele clamping) " it is related to following process：Check that there is identical variation in the case where there is the second change heteroallele by the process（Such as p.Glu257Lys) individual phenotype, to infer the effect of second allele.All 4 individuals for carrying nonsense mutation p.Trpl69* are all blinded at birth, however it remains light sensation is to different degrees of.5 individuals for only carrying missense variation, vision is reduced in the several years after birth.The retina of all affected individuals has edge macula lutea outward appearance and the infringement of atrophic macular coloboma sample

(Fig. 1;It is macula lutea cicatrization described in patient 8）.The expansion of macula lutea infringement is recorded in follow-up examination at 2 age of patient 6-9 month and 4 age of patient 5-7 Sui.Patient 4 has two change heteroalleles on the second chromosome, there is 20/200 OD (right eyes at 8 years old）With 20/400 OS (left eyes）Visual acuity, be unique electroretinogram（ERG the case that primary cone dysfunction rather than all reactions all depth are lost) is shown；It is malnutritive that the individual diagnosis is corrected for cone retinal rod.Patient 5 occurred left eye exudative retinitis at 3 years old（Coats'disease)-patient 6 is unique carries c.451G>T

(p.Vall51Phe) individual of allele, in the 18-24 Sui lasting most of vision relevant with light sensation of forfeiture.

c.769G>A (p.Glu257Lys) gene frequency estimation is 0.001 (dbSNP: rsl50726175 ).Herein, c.769G the present inventor has not had found individual for homozygosis>A (p.Glu257Lys) allele.Other variants of the present inventor's identification are reported not in any public database.P.Trpl69* variations are appeared in 4 of 9 families；The reason for such high rate, is unknown.

It is related to several different biological multinomial researchs that the function of NMNAT1 genes is associated with axonal degeneration⁶.In s mouse models of hideing, by being protection cut-out extron by the effect of Ube4b sequences and the hybrid protein of Nmnatl protein fusions formation^{9 1ϋ}.Meanwhile, Nmnat gene functions, which are lost, in drosophila eye can cause serious neurodegeneration phenotype, but catalysis is not that photoreceptor differentiation or development are required.On the contrary, Nmnat albumen participates in maintenance and the integrality of native neurons.Drosophila photoreceptor Nmnat gene functions, which are lost, causes the neuronal cell developed to be degenerated --- a kind of phenotype for being considered malnutritive to retina^u.Before the application, VMA 7 is not associated with human disease.Therefore, the related organ of biology that eye has been accredited as In vivo study NMNAT1 functions by inventor.

NMNAT1 genes be it is general reach, catalysis NAD+ biosynthesis final step in show highest activity specific.People NMNAT1 is a kind of with six aggressiveness, and each protomer includes central six strands of parallel β-pleated sheets, is several spirals sideways^{12 13}(Fig. 4).Herein, all 11 individuals with LCA all carry p.Glu257Lys variations.According to people NMNAT1 crystal structure, Glu257 is located at short Outer surface in C-terminal spiral.It is worth noting that, it is upright stone tablet polyadenylation sites that adjacent S er256, which is predicted, and the Physical interaction with for example many (ADP- ribose) polymerases of other related proteins or protein kinase may be participated in¹²' ¹³.The electrostatic property on surface may be changed by replacing negatively charged glutamic acid with positively charged lysine, so as to influence proposed Physical interaction.Asn273 is located at last short spiral（Residue 267-274) in, participate in coordinating active site hydrone^{12 i3}.With acidic residues（Aspartic acid）Replacing the residue will be likely to influence enzymatic activity.Met35 and Vall51 be located at the albumen it is thin in the minds of, and Val98 and Leul53 are very close to ligand binding site（Distance about 6-7 A) 4 kinds of variations（P.Met35Thr, p.Val98Gly. p.Vall51Phe and p.Leul53Val) local interaction is most possibly disturbed, consequence is to change active site, so as to influence enzymatic activity.A new direction can be provided.Due to Λ Λ 7 less, it is that gene replacement therapy is hopeful target.Because NMNAT1 has effect in neuroprotein and is a kind of enzyme, medicine intervention is also possible.These treatment methods can be conducive to by limiting the clinical manifestation to retina.The nearest progress in the retinal function that gene therapy is mediated is found¹⁴' ¹⁵The importance of the genetic cause of all individual behinds for suffering from LCA of discovery is highlighted.

The patient's phenotype details of table 5. are summarized.

Patient describes in detail

LCA diagnosis is to be based on clinical assessment（Lack limited imaging, eye quiver with baby's pupil it is weak reaction, funduscopy it is consistent with diagnosis）, suspected case uses electroretinogram again（ERG) confirm.Patient is diagnosed as macular coloboma, and this is that atrophic is damaged, rather than really ectrogeny.Patient 1-6 and patient 11 are single cases.Patient 7 and 8 is born of the same parents parent.Patient 9 and 10 is born of the same parents parent.Patient 1,7 and 8 comes from USA;Patient 2,3 and 5 is from Brazil；Patient 4,6,9 and 10 comes from.Patient 11 is from Australia.Written informed consent form is signed by their father and mother or guardian, and the research has obtained BGI and OHSU (P-WC) Ethics Committee（The Institutional Ethnics Committee) approval.

Patient 1 comes from Children's Hospital of Los Angeles

The male infant, its mother is Che Luoji American Indians, Spaniard and Caucasian offspring, and its father is Asian（Chinese）, Spaniard and Caucasian offspring, full-term pregnancy production is accompanied with the Subchorionic hematoma of rupture, it is necessary to lie in bed until 7 months.The mother notices lacks obvious vision after child's birth in a few hours.Within some months, Claremont Eye Associates paediatrics oculists and Children's Hospital Los Angeles retina specialists enter to break discovery electroretinogram to child（ERG) do not recorded more than noise.Based on shortage vision response, view film outward appearance（Fig. 1) tested with ERG, patient's skin is diagnosed as LCA.Patient 1 may still possess light sensation.Patient 1 is the unique patient of family.His elder brother acts normally.

Patient 2 is provided by JS

The male is Brazilian Caucasoid baby, and its maternal ancestor comes from Poland, Portugal, Lebanon and Italy, and paternal ancestor comes from Turkey, Germany, Italy and Portugal, is normal pregnancy and Cesarean esction birth.Father and mother noticed that child's eye quivers at first 3 months.At 6 months, because eye quivers and without Paulo hereditary experts of imaging consulting Sao, the expert had found child's eyes light sensation, macular dystrophy（So-called macular coloboma）It is calm with the thin macular pigment of retina, therefore confirm as LCA.To September age, photo shows the increase of macular dystrophy area.Patient 2, without born of the same parents parent, is the patient that family uniquely suffers from LCA.In one-year-old period, no light perception.

Patient 3 is provided by LG

Patient 3 is Brazilian girl baby, and its mother notices that it pressed eye at 4 months.Check find Teller cards detection no visual, there are Franceschetti chirals million, eye to quiver, the spiced salt pigmentation of Sunken orbital socket, retina and the peened copper presentation of macula lutea presentation.In children, she has blinded.She is family unique patient. Patient 4 is provided by EH

Patient 4 is that its ancestors is western male, there is a born of the same parents parent, is unique ill kinsfolk.The western ancestors of close consanguinity-less relation.At its 2 months, discovery level eye quivered feels with amblyopia.6 monthly ages, which check, shows that pigment reduces macula lutea infringement, and patient is diagnosed as LCA.At 5 years old, adjustment chin head pose can be clearly visible color.Outer boundaries hyperpigmentation and side atrophic macula lutea infringement (defect）.At 7 years old, night vision dies down.Check display, the infringement increase of hyperpigmentation atrophic macula lutea, retinal vasculature atrophy.About 145 degree of the visual field.ERG shows cone main function of system obstacle, there is slight retinal rod-cone b ripples latency delay, and the b ripples of amplitude and the cone separation of all bright reactions are all reduced and the delay of 30Hz scintillation responses.At 8 years old, at a distance regarding quick

20/200 OD and 20/400 OS, bilateral is 20/100 on hand, and the visual field is reduced to 95 degree.Diagnosis is changed to retinal cone-rod dystrophy, rather than LCA.Still it can be seen that color.Parents ERG is normal.One parental generation has uncertain significant middle periphery pigment spot.

Patient 5 is provided by JS

Patient 5 is Brazilian male, and its maternal ancestor is Spaniard, Caucasian, and paternal background is African and Portuguese.His ^ L months gestation and normal birth.At 3 monthly age, its mother observes that he does not see she and any object.Sao Paulo Hospital, which are sent to, at 1 year old is diagnosed as LCA.Initial retina shows that macular dystrophy and thin retinal pigment are calm.At 4 years old, he has exudative retinitis by left eye.The atrophic macular coloboma sample infringement of patient 5 is similar with patient 2.Patient 5 is unique kinsfolk for suffering from LCA.

Patient 6 is provided by EH

Retinal pigment degeneration was diagnosed as at 6 months, LCA is changed to later.Most of vision was lost at 18 years old.Color and shape can only be can see at 20 years old, at this moment patient begins to use seeing-eye dog.

Lose the ability for seeing color within 22 years old, lose within 24 years old the ability for seeing shape.It can only distinguish between brightness within 26 years old.Eye test shows motility eye movement, macular dystrophy infringement, blood vessel die back and has the pigment of bone spicule is heavy to write disease phenomenon.Without family history.Patient is uniquely to suffer from LCA kinsfolk.Patient has 5 born of the same parents parents.Patient has Greek ancestors, is not close relative.Father has 2 born of the same parents parents, and mother has 8 born of the same parents parents.The relatives of her grand parents have dysopia.

Patient 7 and 8:There is provided by DW

Patient 7 is women, and at 10 years old age, after her younger brother's skin is diagnosed as LCA, she is conveyed to Retina Foundation and carries out visual performance test and assessment.In test, eyes are shown in that sensitivity is assessed as LP.Full visual field ERG, which is shown, to react without retinal rod single blue light that dodges, and reaction of the cone to 30 Hz passage of scintillation light declines 99% and have delay.These symptoms and LCA-cause.The record sheet for the clinician that she goes to a doctor It is bright, during 2 years old half eye examination, big atrophia choroideae et retinae infringement in the atrophy of retina of both eyes blood vessel height, macula lutea（There is deterioration compared with previous visit）And peripheral retina is normal, initial diagnosis is macular degeneration.

The brother of patient 7（Patient 8) 1 years old when found when Retina Foundation are tested it is ill.He had found at 4.5 months because touring mesh H is dynamic and lacks cutification pediatrician.Visual acuity is LP.The full visual field ERG of left eye does not react for retinal rod reaction detection, and glistened to 30 Hz amplitude reduction 99% and b ripples latency of cone reaction significantly postpones.

Patient 9 and 10:There is provided by EH

Patient 9 is Caucasian male, and he is just very weak from infancy vision, childhood be diagnosed as leber's congenital amaurosis（LCA ).He can only point out light tone in toddler, but lose the ability quickly afterwards.At 21 years old, retina ^ looks into that display eyes macula lutea cicatrization, optic papilla be pale and retinal blood shrink tube.At 22 years old, visual acuity report has light sensation.When carrying out genetic evaluation within 25 years old, it is found that pupillary reaction is dull, the reaction is sometimes unusual, wherein pupil becomes big when illumination is come in.In the presence of the well-balanced nystagmus of the vertically and horizontally high frequency, small amplitude of mixing.Retina, which expands, to be checked and shows that the infringement of macular coloboma sample, retinal vessel are reduced, through nipple is small and pale and surrounding pigment conglomeration.Intraocular pressure is normal.Double suitable ERG of light that secretly accommodate are stimulated more than noise without testing result all.He can not complete the test of the Goldmann visuals field.Health and intelligence etc. other facilitate normal.One brother, 30 years old, with doubtful LCA;- individual sisters are not also ill.Father and mother ancestors are Englishman and Dutchman.Mother has 1 sisters and 2 brothers.Father is adopted, there is 1 brother and 1 sisters.

Patient 11:There is provided by CB

The female infants, its mother is British descendant, and its father is Australian descendant, full-term pregnancy production, no complication.At 8 weeks, she can not watch attentively and follow, and observe nystagmus and touring eye movement.Then, paediatrics oculist is diagnosed as doubtful LCA.Other aspects of baby are healthy.The serious side retinal dysfunction of ERG detection displays is carried out, has light sensation but is perceived without figure.Typical AVM is normal.At 7 months 2 years old, there is certain light sensation.Patient has two not ill elder brothers, does not have LCA family histories.Similar with the clinical presentation of patient 1,2 and 3, serious LCA is presented in patient 11.Importantly, also observing macular dystrophy in its father and mother（Macula lutea cicatrization or macular coloboma）.In fact, suspecting there is Λ Λ/gene mutation, the genes of V 7 are tested based on the analysis and suggestion to fundus imaging.

Embodiment

Method

1. sample preparation Patient and related relatives' peripheral blood are gathered, the genome in peripheral white blood cells is extracted using kit

DNA (RelaxGene Blood DNA System DP319-02. TIANGEN, China), DNA concentration and purity (Thermo Scientific is measured using NanoDrop 2000, USA), the OD260/OD280 values of each sample genomic DNA of gained are respectively positioned between 1.7-2.0, concentration is no less than lOO ng/ μ Ι, and total amount is no less than 30 μ 1.

2. sequencing

Then, the extron group sequence to above-mentioned sample is sequenced.

Use Agilent 38M (Agilent companies）Chip, carries out the capture of extron group to collect the CDS areas of human gene group DNA to above-mentioned sample.Then, the DNA library for being enriched with extron is subjected to the second library construction, for Illumina GA sequencings.Microarray dataset is Illumina Genome Analyzer Π, and storehouse specification is built according to Illumina/Solexa standards（Referring to http://www.illumina.com/) it is sequenced, it is summarized as follows：

3. information analysis

People refers to and is downloaded from UCSC databases (http under genome and its gene annotation://genome.ucsc.edu/), version hgl9 (build 37).Sequence from LCA Patient Sample As is compared with BWA (Burrows-Wheeler Alignment) with canonical sequence, then except fragment is sequenced in deduplication（ reads ).SNP is called in and uses default SOAPsnp components（ Li R, Li Y, Fang X, Yang H, et al, SNP detection for massively parallel whole-genome resequencing. Genome Res 2009, 19(6):1124-1132 ).By insertion and deletion（Indel Dindel (Albers CA, Lunter Q MacArthur DQ McVean G, Ouwehand WH, Durbin R., Dindel) are called in: accurate indel calls from short-read data. Genome Res. 2011 Jun;21(6):961-73 ).Under wealthy value bag ^ for filtering SNP：1) consistant mass value is necessary for>=20 (mass value is Phred values, is generated by program SOAPsnp, and accuracy is called on the basis that mass value 20 represents 99%）；2) number of SNP unique match sequencing fragment is supported to be necessary for>= 4；3) copy number of estimation is not more than 2;4) the distance between two SNP should be greater than 5.For indel, only extract have by Dindel " PASS " labels provided those, this table Bright high reliability.

4. the method for confirming extron sequencing result and identifying NMNAT1 gene mutations in other LCA patients：

( >50 nt) two-way Sanger determined dna sequences progress.PCR is carried out using KAPA2G Robust HotStart (KAPABIOSYSTEMS, Woburn, MA).Design of primers refers to human genome, and the PCR primer of NMNAT1 genes is shown in Table 3.Sanger sequencings are reacted in SeqWright

(Houston, TX) is carried out.Sequence analysis is carried out using Mutation Surveyor program (State College, PA).

PCR amplification system：

The μ 1 of 10* Ex Taq Slow fliud flushings 3

dNTP ( 2.5 mM ) 2 μ1

Forward primer（lOmM ) 1 μΐ

Reverse primer（lOmM ) 1 μΐ

DNA 2 μ1

Ex Taq 0.2 μΐ

Η₂0 20.8 μΐ

Amount to 30 μ

PCR ^ answer condition：

94 °C:5 minutes；

30 circulations：94 °C 30 seconds, 55 °C 30 seconds, 72 °C 45 seconds;

72 "C:10 minutes；

12°C:Continue.

Sequence table explanation

SEQ ID NO:l:The cDNA sequence of the genes of wild type A V 7（Wherein underscore represents that the position may undergo mutation）（ http：〃 www.ncbi.nlm.nih.gov/nuccore/95113669 ) ATGGAA AATTCCGAGA AGACTGAAGT GGTTCTCCTT GCTTGTGGTT CATTCAATCC CATCACCAAC ATGCACCTCA GGTTGTTTGA

GCTGGCCAAG GACTACATGA ATGGAACAGG AAGGTACACA GTTGTCAAAG GCATCATCTC TCCTGTTGGT GATGCCTACA AGAAGAAAGG ACTCATTCCT GCCTATCACC GGGTCATCAT GGCAGAACTT GCTACCAAGA ATTCTAAATG GGTGGAAGTT 81

V0WV310V3 313W33V3V V33VV33100 33VVV3V101 0V100V010V V3V03V00V0 18

1V31310V31 3V1033V331 Shang WV3V3W0 Shang V00V0V3W 103133VV31 010V3V3VVV IZ

V011013VVV 3331V0V1V3 113VV33133 31VVV1011V V3VV00V103 19£

The ￡ of Shang Shang 0W3V033 1V01V01333 00V01V1003 10011V010V 33VVV3VV3V V0V10031V3 10

1331131001 0101V01V03 3VVV013113 V0V0V133VV 33V0VV331V V31V0V10V3

The L8 L of 33 V0100V031V 0VV00V01V0 OOIVVOIIVO of 3VV0033103 V3 3 Shang Shang of Shang Shang Shang 1133131103

1100101133 13VV310V3V V3V300 Shang Shang W VV331V00V1 10113VV011 0W0VV01V3 IZ l

Vmvoo Shang Shang Shang 13V0V01313 3V333VV0VV 0V3030333V 313V0V3303 300333010V 19

03V1330010 1V31331030 0333001303 3103010V00 1131V3V300 V30313V3V3 I

Fontanel UVMWM ^ ^: Π:ΟΝ QI 03S

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^^ soil ψ Π ： z:ON ai 0as

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The Shang Shang WX303XVOO of Shang Shang Shang 3

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DIDIYDDDIY Shang V Shang 3IV Shang Shang Shang Shang 3D Shang Shang VWOO Shang

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330 Shang O Shang：) W W3VWW33 OVOVX333XV Shang O s

The Shang Shang Shang DIDYYODYDY VOOWOO Shang 33 of V Shang 3

The Shang 3V Shang of OOVWOV Shang 3) 3V3 Shang 3V VOV30V33V3 Shang Shang O Shang Shang

Shang DOOVOi) Shang XVWOVOW3 XV33V3VOW Shang) O Shang Shang

The Shang OVW DDDIYDYIYD of 3 Shang DDIDYDDYYD Shang Shang 3

ITC100/Z10ZN3/X3d 80Ϊ6Ϊ0/ 0Ζ OAV 61

101003VV00 0V11001101 VO Shang Shang Shang 0333V V031V03V01 33V100031V Z ￡

33W33VV3V 13V3V0VV0V V101V33V31 V01V003311 111111031V V3VVV1133V IQ I£ i

111331V333 31V003V311 V3V3V13333 VV31V31331 1001111033 13VV3V3VV1 IZ I£

The ε of 003 3V03V0VV31 31V Shang Shang of V3V0 Shang Shang Shang, 03 Shang Shang Shang 313V133131 Shang Shang 003V3V33 1,V31,V33,011 190

V01101V033 11V3110013 VVVV13V031 OOVVVOVVVO 3313VVV311 1W131311V LOOS

The mz of 0V110000V1 Shang Shang 000V13 Shang Shang 3131V11113 3,333,103,1V3 1,113,111,310 0331113111

The Shang Shang Shang V01V0 L882 Of of 03 Shang Shang Shang WIV of 131331031V 313131VVV3 3V3V Shang Shang Shang WO 1310V13111 Shang 00

The Shang Shang VOO 31V1VVVV3V V3130V310V 31VV11V1VV V1VV3333V0 of 3100100011 V Shang Shang Shang 3

The L9Z.2 of V0111VV31V V0VV3V113V V331VV1VV3 Shang Shang Shang OVOOllO W3V0V33VV 0100131333

The iOLZ of Shang Shang Shang Shang VV1V03 V1VV130V00 31031V3113 1031V33V30 0,30V,V00,010 1111103311

The VV0VV3VVV0 V131110VV0 W03V331V1 Shang Shang V10V03 Shang Shang z of 03W0VV3V3 V0V13 Shang Shang Shang 03

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331V3V3V13 V11111V131 Shang Shang Shang VV10330 0131V00V10 V000V033V0 V11V333103 mz

The 100300100V V0310V0103 of V13V310310 03V0100310 01011VV03V VO Shang Shang 333000

The Shang Shang mz of 00101V3031 3313V0313V 331033V000 10131010V0 1013V331V3 V3 Shang Shang Shang Shang Shang 3

The ￡ of 3 Shang Shang V10 01V31VV0V1 333VV13310 003V33V031 1000V33331 31V033VV0V of Shang Shang Shang Shang 0

3310V0100V 33V0100V31 0V10V31310 3V01031000 313100313V V3V010V100 iZZZ

The WV3V0310V VV130V0131 mz of 110031301V 10V1V01010 110VVV3310 31VV00 Shang Shang Shang 3

The Shang Shang 0V33 Shang Shang 1VV310VV3V 01V103VV31 z of 3VV3101V01 13V100V0V1 0,VVV,333,101 00

V3W03V3V1 V31W13VVV V310013V33 VV3113V3VV 1113V33110 Shang Shang 03V Shang Shang 001

0010 Shang Shang Shang Shang VO 1333V31V3V OVIVOIVVVO 1003V33103 3VVV313V11 3W003101V Yang ζ

001V33V011 133V03V103 V0V31003VV VVVVVVVVVV W0101V001 0V3V303V3V

The 3V0010V031 1V0031301V 3VV003VV13 V031133V3V 033V333100 Hidden of 0V3 Shang 333100

W3 Shang Shang 0301V V3V33V033V 31033V3331 10V103V000 13V1310010 Hidden

V03V3103V1 1VVWVV0V1 VV3VV10V10 101V0000VV V31331V1VV 00331003V0

0V3VV0113V 3VV0133V31 30V01V3313 3313VV1003 Shang Shang Shang OV 03V0001VV1 Hidden oz

3100V0V010 300V3VVVVV VVVVVVV3V3 WV0101V00 Shang 0WW03VV

VVW033310 03V0010V03 Shang 0V00313 Shang Shang 33VV003V31 3V001133V0 V033V33300 L9S L

0VV3110301 VV3V33V033 V31033V333 Shang Shang 0V103V00 OIVVIVIOVV 1V03003133 LOS L

The Shang Shang Shang i of 31 Shang Shang V Shang Shang Shang of 1V033V003V Shang Shang VWWVOV Shang VVVVVIOVI 01013V331V 3V3 Shang Shang Shang Shang Shang 3

3111113VV1 WWIOSVV Shang 331VV01VVV VV13V111VV VVVVVVVVVV VW0101310

10V3VV03V3 V0V3033100 3V0010V031 Shang V00V03 Shang Shang V 3VV003V313 V031133V33 IZ ￡ 1

133133V000 0V3110V01V V3V33V033V 31033V3331 10V103V000 13V1310031 L92 L

30V0331331 31313103V1 1VVVVV0V1V VVVV10V101 01V0000VVV 31331V1VV0 L02 L

The Shang Shang Shang 0V03V of 0331001V00 V3V301V3V3 VV0133330V 01V33133V0 3,3V3,103,3V3 33

0001VV1310 030V010331 3V0V033310 1301VVVVV1 3V1113VVVV Shang Shang 03VW10V ■ 01

VV10031131 01V313113V V3VVV33331 OVVIVVIISV 333101VV0V VV3101V333

3111V00011 0V3V0 Shang Shang Shang VI V31V03V0V1 011VV33V1V 0V3VV103VV 3V0310V0VV L96

The V013333103 1VV33V0V3V V313V3V310 Shang 03V0V13 Shang Shang L06 of V3V3V03 Shang Shang Shang 0000331001

1VV1V03VVV V311V0V1VV 3VV0013110 1V3V00V133 Shang Shang 0V1030 Shang Shang V03V3V0033

33W3V0100 03V3V33001 VVVVV0V001 V0101V0V31 W10301V33 1W31VV313 IQL ς

3130V011V0 VV03V3300V 0VVV331310 3131V3301V V31V101V11 13VV3V0103 IZL

The Shang OV Shang Shang 3131V1V010 3331V10VV0 0331301VVV 000V01V0V3 L99 of 1V31VVV331 03330

The L09 of 313 0113103111 0013V3311V 111V3V0333 of Shang Shang Shang WOO of VV313V3VV3 3131310310

3VV0133VVV 00313103VV VVOVVVVVOO 3V3V10001V W3VVVV013 V1011V3VV0

ITC100/Z10ZN3/X3d 80Ϊ6Ϊ0/ 0Ζ OAV 3301 CTTTTCACTT TTCTAGAAGT CCAGAAGTTG TTATATGATG AAATAGCCTC CTTTAACGTT

3361 TATTTCTGGG TGCCAACGGA GGCCCATTCC TCTAACATTC TCATAATTTT TCTCAAAGGC

3421 CTATGATCTA AACATTTCAC CACGGCATCC ACTCAGCTGT GAGGCTGCGT ACACAGTCTC

3481 CACCTCTGAA ATCTGAACTT CGTTTACCAG TGGTGCTGTT TGAACTTCAT AATGTCAGCA

3541 CTTCCTGAAC ACTTACTGTG TGCTTGGCTT GTGCTCCTGA GTGCCTTATA TCATAAGGAA

3601 ACGGCAAAAT CAGGGGACTG GTATAAATGG TGAGCTGAGC TTGAATCTAA GCTTTGTCTT

3661 CAGAGCCAGT ACCCCTAATC TCTCTTTCTG TAAAATATTA CTTTTCAAAG AATGAAGTTG

3721 TAGCCAAATC TTGAAATTTT TCATTTACCC TAAGTGAGGA CAAATAAAGC TTTCAACAAC

3781 A

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Claims

Claims

1. a kind of have the genes of V 7 or NMNAT1 albumen selected from following mutation：Nonsense mutation（c.507G>A, p.Trpl69*);With

Missense mutation（ c.769G>A, p.Glu257Lys).

2. the NMNAT1 genes of claim 1, are SEQ Π) Ν Ο:There is following mutation in 1 sequence：

Nonsense mutation（c.507G>A, p.Trpl69*);With

Missense mutation（ c.769G>A, p.Glu257Lys).

3. the NMNAT1 albumen of claim 1, is SEQ D) NO:There is following mutation in 2 sequence：

Nonsense mutation（c.507G>A, p.Trpl69*);With

Missense mutation（ c.769G>A, p.Glu257Lys).

4. a kind of method for detecting the mutation genes of V 7 or NMNAT1 albumen, methods described includes whether there is mutational site in detection NMNAT1 genes or NMNAT1 albumen, the mutational site is selected from following any or its combination：

Nonsense mutation（c.507G>A, p.Trpl69*);With

Missense mutation（ c.769G>A, p.Glu257Lys).

5. the method for claim 4, including following at least one set of primer is the step of expand：

SEQ ID NO:3 and SEQ ID NO:4;

SEQ ID NO:5 and SEQ ID NO:6;

SEQ ID NO:7 and SEQ ID NO:8;

SEQ ID NO:9 and SEQ ID NO:10.

6. detection mutation ^^^ the primer pair that uses in 7 genes or NMNAT1 albumen, the mutation is selected from following any or its combination：

Nonsense mutation（c.507G>A, p.Trpl69*);And/or

Missense mutation（ c.769G>A, p.Glu257Lys),

Wherein described primer pair is based respectively on to be designed before and after following position is on genome sequence or cDNA sequence so that expand the position：CDNA sequence the 507th and the 769th.

7. the nucleic acid probe with mutation NMNAT1 gene complementations, the mutation is selected from following any or its combination：

Nonsense mutation is c.507G>A;And/or Missense mutation is c.769G>Position in A, cDNA sequence：CDNA sequence the 507th and the 769th.

8. the kit of the genes of detection mutation Λ Γ Λ 7 or NMNAT1 albumen, comprising one or more groups of primer pairs, wherein the mutation is selected from following any or its combination：

Nonsense mutation（c.507G>A, p.Trpl69*);With

Missense mutation（ c.769G>A, p.Glu257Lys),

Wherein described primer pair is based respectively on to be designed selected from following position on genome sequence or cDNA sequence so that its amplified production covers the position：CDNA sequence the 507th and the 769th.

9. the kit of claim 8, comprising selected from following at least one set of primer：

SEQ ID NO:3 and SEQ ID NO:4;

SEQ ID NO:5 and SEQ ID NO:6;

SEQ ID NO:7 and SEQ ID NO:8;

SEQ ID NO:9 and SEQ ID NO:10.

10. the kit of the detection mutation genes of V 7, comprising one or more nucleic acid probes, the mutation is selected from following any or its combination：

Nonsense mutation is c.507G>A;With

Missense mutation is c.769G>A,

The probe is with including the regional complementarity on genome sequence or cDNA sequence selected from following location on mutation NMNAT1 genes：CDNA sequence the 507th and the 769th.