WO2000078943A2

WO2000078943A2 - EXPANSION OF A NOVEL CAG TRINUCLEOTIDE REPEAT IN THE 5' REGION OF THE PROTEIN PHOSPHATASE 2A PPP2R2Bβ GENE IS ASSOCIATED WITH SCA12

Info

Publication number: WO2000078943A2
Application number: PCT/US2000/040213
Authority: WO
Inventors: Russell L. Margolis; Christopher A. Ross; Susan E. Holmes
Original assignee: The Johns Hopkins University
Priority date: 1999-06-18
Filing date: 2000-06-16
Publication date: 2000-12-28
Also published as: WO2000078943A3; AU6540000A

Abstract

Probes and primers useful for detecting changes in a particular microsatellite marker are provided. Expansions of the microsatellite are associated with a neurological disease with late onset. Thus determining expansions from a 'normal' number can be used to track the inheritance of the disease within affected families.

Description

EXPANSION OF A NOVEL CAG TRINUCLEOTIDE REPEAT

IN THE 5' REGION OF THE

PROTEIN PHOSPHATASE 2A PPP2R2Bβ GENE

IS ASSOCIATED WITH SCA12

This work was supported by grants from the U.S. government, including NIH NS 16375, MHO 1275, and MH50763. The U.S. government therefore retains certain rights in the invention. TECHNICAL FIELP OF THE INVENTION

This invention is related to diagnosis and prognosis of inherited mutations which are associated with a neurological disease.

BACKGROUND OF THE INVENTION

The spinocerebellar ataxias are a heterogeneous group of degenerative neurological disorders with variable clinical features resulting from degeneration of the cerebellum, brain stem, and spinocerebellar tracts. The clinical symptoms include ataxia, dysarthria, ophthalmoparesis, and variable degrees of motor weakness. The sypmptoms usually begin during the third or fourth decade of life, however, juvenile onset has been identified. Typcially, the disease worsens gradually, often resulting in complete disability and death 10-20 years after the onset of symptoms. Individuals with juvenile onset spinocerebellar ataxias, however, typically have more rapid progression of the phenotype than the late onset cases. See U.S. 5741645.

The genetic etiologies of at least 20% of autosomal dominant spinocerebellar ataxias (SCAs) have yet to be elucidated '. Thus there is a need in the art for such elucidation, so that pre-symptomatic identification of affected individuals can be achieved. SUMMARY OF THE INVENTION

It is an object of the present invention to provide an isolated and purified polynucleotide comprising a microsatelhte marker. It is another object of the present invention to provide a pair of primers for amplifying a microsateUite marker associated with SCA12.

It is an object of the present invention to provide a method for diagnosing spinocerebellar ataxia 12 (SCA12) .

It is still another object of the invention to provide probes for detecting microsateUite expansions.

These and other objects of the invention are achieved by providing a method for diagnosing spinocerebellar ataxia 12 (SCA12). The number of CAG trinucleotide repeats in a region 133 nt upstream of a previously published transcription start site of an allele of PPP2R2Bβ is determined. The PP2A-PR55β protein is a brain specific regulatory subunit of protein phosphatase PP2A, If a number of trinucleotide repeats greater than 40 is determined, an expansion which is associated with SCA12 is indicated.

According to another embodiment of the invention, a pair of primers for amplifying a microsateUite marker associated with SCA12 is provided. Each primer of the pair is complementary to at least 12 consecutive nucleotides which are within 500 nucleotides of a CAG trinucleotide repeat located on human chromosome 5q31-33 between markers D5S402 and WI- 6763. Each primer of the pair is complementary to opposite strands of the marker.

According to still another embodiment of the invention, an isolated and purified polynucleotide comprising a microsateUite marker is provided. The marker is located on human chromosome 5q31-33 between markers D5S402 and WI-6763. The polynucleotide comprises a variable number of CAG trinucleotide repeats, depending on the person or tissue from which it is extracted. The poynucleotide comprises a sequence:

A(CAG)_nB, or the complement of A(CAG)_nB. The variables are defined as n=5-100, A= 5'-TCCAGCCTCCTG-3' and B= 5'-CTGCGAGTGCGC-3'. According to still another embodiment of the invention, a probe is provided. The probe is useful for detecting expansions in a microsatelhte marker located on human chromosome 5q31-33 between markers D5S402 and WI-6763. The probe comprises a sequence A(CAG)_n, (CAG)_nB, A(CAG)_nB, or the complement thereof. The variables are defined as n=5- 100, A= 5'-TCCAGCCTCCTG-3' and B= 5'-CTGCGAGTGCGC-3\

The invention thus provides the art with methods and tools for diagnosing, prognosing, and following microsateUite markers which segregate with a neurological disease. BRTFF DESCRIPTION OF THE DRAWINGS Figures la. and lb. CAG expansion in pedigree R. Fig la. CAG RED results of 11 members of pedigree R. The RED products of the proband are in the far right lane. Two unaffected individuals without an expansion of the PPP2R2Bβ CAG repeat have an expansion of about 60 triplets in the ERDAl/Dirl locus, explaining the short expansion detected in the RED assay.

Fig. lb. Association of expansion with disease phenotype. Filled diamonds—affected. Age of onset is noted to the upper left of the symbols and allele sizes to the lower right. The proband is indicated by an arrow. All affecteds used in the analyses, and six of the eight unaffected, were examined by the inventors. A neurological history from multiple informants was gathered for the family members unavailable for examination or deceased. PCR products containing the novel CAG repeat region of members of pedigree R were blotted and probed with a radiolabeled (CAG)₁₅ oligonucleotide. The results were confirmed by automated genotyping of fluorescently labeled products and by sequence of PCR products for six of the affected individuals. The proband has a major signal representing a repeat of 72 triplets detected on the Southern blot, whereas the repeat length measured by automated genotyping was 92 triplets and by direct sequencing of a genomic clone 93 triplets; this variability is consistent with somatic mosaicism and was not observed on other family members. Repeat length is unstable, with slight contractions (average change is -1 repeat) in 3 of 4 vertical transmissions, and variability in each of the two sibships. A faint band representing 10 triplets visible in the sixth lane from the right is an artifact of lane spillover. One affected offspring of the proband and the three unaffected individuals younger than age 50 are not shown.

Figure 2a and 2b. Analysis of CAG repeat. Fig. 2a. Distribution of allele length. Repeat length was determined in 394 normal subjects, 324 subjects with Parkinson's disease (70 familial, 60 early onset), 27 subjects with Huntington's disease, and 748 subjects with ataxia (40% familial). The distribution of repeat length was the same in all groups, which were therefore combined. The two longest repeats, of 27 and 28 triplets, were found in normal controls. Heterozygosity was 60.3%.

Normal and disease controls were primarily Caucasian, with a majority of German origin.

Fig. 2b. Location of CAG repeat. Schematic representation of the genomic sequence containing the expanded CAG repeat and the putative first exon ofPPP2R2Bβ, aligned with the PPP2R2Bβ cDNA. Horizontal arrows indicate the positions of the oligonucleotide primers used in PCR. The CAG repeat lies 133 nt 5' to the published 5' end of the PPP2R2Bβ cDNA sequence (M64930/NM004576). PPP2R2Bβ has previously been mapped to chromosome 5q31-33, between markers D5S436 and D5S470. Radiation hybrid results, using primers J and K, localized clone YB between markers D5S402 and WI-6763, consistent with the established position of PPP2R2Bβ. Primers A (5' TGCTGGGAAAGAGTCGTG) and B (5' GCCAGCGCACTCACCCTC) flanking the CAG tract were used to determine repeat lengths and to isolate a PAC (#21933, Genome Systems), that extended beyond the YB clone (delineated by EcoRI sites) in both directions. Primers C and Ε were used for 5' RACE on human brain cDNAs. 10 clones were sequenced and aligned with the genomic clone (vertical lines indicate 5' end of RACE products). The most 5' sequence ended 9 nt 3' to the CAG repeat. Mouse EST AU051098 containing an interrupted CAG repeat, (CAG)₂GAG(CAG)₂CAC(CAG)₃, begins 22 nt 5' to the human CAG repeat and extends 3' into the protein coding region, and has 91% homology to the human sequence exclusive of the repeat. The hatched region 3' to the published exon 1 includes these multiple possible alternative transcription start sites.

DETAILED DESCRIPTION OF THE INVENTION

We have recently identified a novel form of autosomal dominant SCA, termed SCA 12, in a large pedigree ("R") of German descent. Age of onset ranges from 8 to 55 years. Most individuals present in middle age with upper extremity tremor, progressing over several decades to include head tremor, gait ataxia, dysmetria, hyperreflexia, paucity of movement, abnormal eye movements, and, in the eldest subjects, dementia. MRI or CT scans of five cases indicate both cortical and cerebellar atrophy. Repeat expansion detection (RED)² was used to identify an expanded CAG repeat in the proband and other affected family members (Fig la). PCR analysis demonstrated that the expansion was not one of the eight CAG repeats associated with a neurodegenerative disease or one of the three CAG repeats known to undergo nonpathogenic expansions³.

An "isolated and purified" polynucleotide according to the present invention is one which is removed from other chromosomal sequences with which it is found in a human cell. Thus it is free of other human chromosomes and free of other human chromosome arms. Moreover, it is typically 10 kb or less, more preferably less than 5 kb or 2.5 kb, and most preferably less than 1 kb. In some embodiments, it may be desirable to have polynucleotides which are less than 100 kb. The polynucleotide of the present invention comprises a microsatelhte marker located on human chromosome 5q31-33 between markers D5S402 and WI-6763. Preferably the polynucleotide does not extend beyond these markers. The polynucleotide comprises a variable number of CAG trinucleotide repeats. The polynucleotide can be made, twter alia, when the microsatelhte marker is amplified in an effort to diagnose whether an expansion has occurred or been inherited. The poynucleotide comprises a sequence A(CAG)_nB or the complement of A(CAG)_nB. The variables are defined as n=5-100, A= 5'- TCCAGCCTCCTG-3' and B= 5'-CTGCGAGT-GCGC-3'. The number of trinucleotide repeats typically varies between 7 and 28 in unaffected individuals. In affected individuals, the number of trinucleotide repeats can reach from 65 to 93.

Primer pairs can be used for amplifying the microsatelhte marker associated with SCA12. Each primer is complementary to at least about 12 consecutive nucleotides which are within 500 nucleotides of a CAG trinucleotide repeat located on human chromosome 5q31-33 between markers D5S402 and WI-6763. Suitable primers include those which comprise sequences 5'-TCCAGCCTCCTG-3', 5'-CTGCGAGTGCGC-3', and complements thereof. Preferred primers according to the invention are those which comprise sequences 5'- TGCTGGGAA-AGAGTCGTG-3' and

5'-GCCAGCGCACTCACCCT-3 or complements thereof.

Spinocerebellar ataxia 12 (SCA 12) can be diagnosed or predicted on the basis of the prsence of the expanded trinucleotide repeat on chromosome 5q31-33. The determination can be made pre- or post-natally. It can be made pre- or post-symptomatically. However, because of its infrequence in the population, it is likely to be most useful in families where the disease has already been diagnosed. The expansion can be determined by testing any cells in the body, preferably somatic cells. Suitable cell types include, but are in no way limited to lymphoblastoid cells and brain cells. The same number of repeats may not be found in all cells of an affected individual's body, however, due to secondary events, such as errors in the fidelity of replication in somatic cells. The number of CAG trinucleotide repeats is determined in a region containing a CAG trinucleotide repeat about 133 nt upstream of a previously published transcription start site of an allele of PPP2R2Bβ. PP2A-PR55β is a brain specific regulatory subunit of protein phosphatase PP2A. If a number of repeats greater than 40, 50, 60, or even 65, or 70 is determined, an expansion which is associated with SCA12 is indicated. Unaffected individuals typically have less than 30, 20, or even 10 repeats at the marker. Any of many techniques known in the art for determining nucleotide repeats can be used, including using a polymerase chain reaction. Suitable primers for use in such reactions include 5'-TCCAGCCTCCTG-3', 5'- CTGCGAGTGCGC-3', TGCTGGGAAA-GAGTCGTG-3', 5'- GCCAGCGCACTCACCCT-3', and complements thereof. However, any primer which is complementary to sequences within about 0 to 500, or 500 to 1,000 nucleotides on either side of the trinucleotide repeat can be used. Flanking sequences on one or both primers can be used. Southern blotting can also be used to determine repeat numbers. Probes comprising (CAG)_5. _loo_, preferable (CAG)₁₂.₂₀ can readily be used, for example. Probes preferably comprise flanking sequences on one or both sides as well to render them specific for the particular marker. Another technique which can be readily used is nucleotide sequencing, whether automated or manual, whether enzymatic or by chemical degradation.

EXAMPLE 1

A variation of the method described by Koob et al. was used to clone a 2.5 kb genomic clone (termed YB) from the proband that contained a repeat of 93 uninterrupted CAGs. Primers flanking the repeat were used to determine repeat length (Fig. lb). All ten known living affected family members have a repeat expansion. Eight unaffected offspring of affected family members, five older than age 60 and three age 42-49, were also tested. Seven had no expansion and one 49-year-old with no signs or symptoms of SCA12 had an expansion.

Linkage analysis was performed using the software package LINKAGE⁵. Disease allele frequency was set at 0.0001 and allele size was collapsed and dichotomized into either unexpanded (<29) or expanded (>65). An age-dependent penetrance function was derived based on the age of onset of family members (penetrance: 0.4 at ages ≤ 30, 0.7 at ages 31-40, 0.8 at ages 41-60, and 0.95 at ages >60). The LOD score was 4.61 at θ = 0. There is no apparent correlation between repeat size and age of onset, though the range of expanded alleles is relatively narrow (66-78) and the precise age of the onset of tremor, typically the first symptom, is difficult to define in this disorder.

EXAMPLE 2 CAG repeat length was assessed in 394 unrelated neurologically normal individuals and 1099 individuals with neurological diseases.

Unexpanded alleles ranged in size from 7 to 28 repeats, and there was no difference in allele distribution among the groups (Fig. 2a).

The CAG tract lies 133 nt upstream of the reported transcription start site of the gene encoding PP2A-PR55β (also termed PP2AB55β,

PP2ABβ, and PPP2R2B), a brain-specific regulatory subunit of the protein phosphatase PP2A^6"8 (Fig 2b). 5' RACE on human fetal and cerebellar cDNAs suggests that the transcription start site is upstream of the previously published 5' end of the cDNA, and a mouse brain EST (AU051098) indicates that at least one transcription start site lies 5' to the repeat. A MED-1 sequence (GCTCCC)⁹ is located 65 nt 3' to the repeat, consistent with the presence of multiple transcription start sites. A DRE consensus sequence¹⁰, RGWCGTG, is found 251 nt and 69 nt 5' to the start of the CAG repeat. EXAMPLE 3

We attempted to determine the effect of the expansion on PPP2R2Bβ expression using the only tissue available from affected subjects, lymphoblastoid cell lines. There was no detectable PPP2R2Bβ RNA or protein in these cell lines or cell lines from unaffected controls, consistent with previous studies of PPP2R2Bβ expression⁶'⁷.

Though the region surrounding the CAG tract shows no homology with any additional genes in the database, it could lie within an unidentified gene overlapping or adjacent to PPP2R2Bβ. While this possibility cannot be excluded, an antibody probe" did not detect polyglutamine expansions in protein derived from lymphoblastoid cell lines of affected family members.

Our results indicate linkage between SCA 12 and a CAG repeat expansion in the 5' UTR of PPP2R2B-β. It is possible that the expansion mutation is in linkage disequilibrium with a second, as yet unidentified, causative mutation. However, the correlation between repeat expansion and disease in pedigree R, the lack of expansions in controls, and the known capacity of expansion mutations outside of protein coding regions to cause disease suggest that the expansion is causative. While the precise role of the PR55β subunit in regulating PP2A activity remains to be determined, the trimeric holoenzyme PP2A has been implicated in a number of cellular functions⁸, including modulation of cell-cycle progression¹², tau phosphorylation¹³, and apoptosis¹⁴'¹⁵. We speculate that the CAG repeat expansion may affect PPP2R2Bβ expression, perhaps altering PP2A function in the brain.

EXAMPLE 4 Two-hundred-forty-seven index cases, including 145 families with autosomal dominant cerebvellar ataxia (ADCA), were screened for exapansion of a CAG repeat in the PPP2R2B gene. An expanded repeat ranging from 55 to 61 triplets was detected in six affected and three unaffected individuals at risk in a single family from India. The phenotype is characterized by cerebellar ataxia and pyramidal signs that become manifest in the fifth decade. The association of the PPP2R2B CAG repeat expansion with disease in this new family provides additional evidence that the mutation is causative.

PCR products amplified from expanded but not normal alleles contained seuqences of different lengths, suggesting mosaicism in blood cells. The CAG repeat was slightly unstable. The allele transmitted maternally to three sibships was not altered, whereas differences of three and five CAG repeats were found in the two sibships with paternal transmission. References

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Claims

1. An isolated and purified polynucleotide comprising a microsateUite marker located on human chromosome 5q31-33 between markers D5S402 and WI-6763, said polynucleotide comprising a variable number of CAG trinucleotide repeats, wherein the poynucleotide comprises a sequence:

A(CAG)_πB, or the complement of A(CAG)_nB, wherein n=5-100, A= 5'- TCCAGCCTCCTG-3' and B= 5'-CTGCGAGTGCGC-3\

2. The polynucleotide of claim 1 wherein n=7-28.

3. The polynucleotide of claim 1 wherein n=65-93.

4. A pair of primers for amplifying a microsatelhte marker associated with SCA 12, wherein each primer is complementary to at least 12 consecutive nucleotides which are within 500 nucleotides of a CAG trinucleotide repeat located on human chromosome 5q31-33 between markers D5S402 and WI-6763.

5. The pair of primers of claim 4 which are selected from those which comprise sequences 5'-TCCAGCCTCCTG-3', 5'-

CTGCGAGTGCGC-3', and complements thereof.

6. The pair of primers of claim 4 which comprise sequences 5'-

TGCTGGGAAAGAGTCGTG-3' and 5'-GCCAGCGCACTCACCCT-3 or complements thereof.

7. A method for diagnosing spinocerebellar ataxia 12 (SCA 12) comprising the steps of: determining number of CAG trinucleotide repeats in a 5'- untranslated region of an allele of PPP2R2Bβ, a brain specific regulatory subunit of protein phosphatase PP2A, wherein if a number greater than 40 is determined, an expansion which is associated with SCA 12 is indicated.

8. The method of claim 7 wherein an expansion associated with SCA 12 is indicated if a number greater than 50 is indicated.

9. The method of claim 7 wherein an expansion associated with SCA 12 is indicated if a number greater than 60 is indicated.

10. The method of claim 7 wherein an expansion associated with

SCA 12 is indicated if a number greater than 65 is indicated.

11. The method of claim 7 wherein the allele is isolated from lymphoblastoid cells.

12. The method of claim 7 wherein the allele is isolated from brain cells.

13. The method of claim 7 wherein the number of CAG trinucleotide repeats is determined using a polymerase chain reaction.

14. The method of claim 7 wherein the number of CAG trinucleotide repeats is determined using Southern blotting.

15. The method of claim 7 wherein the number of CAG trinucleotide repeats is determined using a nucleotide sequencing reaction.

16. The method of claim 14 wherein a probe comprising (CAG)_12.20 is used.

17. The method of claim 13 wherein a primer is used selected from the group consisting of: 5'-TCCAGCCTCCTG-3', 5'- CTGCGAGTGCGC-3', TGCTGGGAAAGAGTCGTG-3', 5'-

GCCAGCGCACTCACCCT-3', and complements thereof.

18. A probe for detecting expansions in a microsatelhte marker located on human chromosome 5q31-33 between markers D5S402 and WI- 6763, said probe comprising a sequence:

A(CAG)_n) (CAG)_nB, A(CAG)_nB, or the complement thereof, wherein n=5-100, A= 5'-TCCAGCCTCCTG-3' and B= 5'-CTGCGAGTGCGC-3'.

19. The probe of claim 18 which is labeled.