JrMed Genet 1997;34:411-413 Short reports Meiotic instability associated with the CAGR1 trinucleotide repeat at 13q13 Nicholas T Potter Abstract CAGR1 is a recently characterised poly- morphic trinucleotide repeat localised to 13qI3, which has been suggested as a pos- sible candidate gene for neurological dis- orders that manifest genetic anticipation. To provide evidence in support of this hypothesis, a large number of chromo- somes (n=928) from patients with a wide variety of neurological diseases were screened for evidence of repeat expansion and meiotic instability. One person with a CAGR1 repeat number of 50 was identi- fied (normal range 9-29). Subsequent molecular analyses of CAGR1 repeat number in additional family members showed meiotic instability of a (CAG)45 allele through three generations. While CAGRI repeat number did not correlate with a readily discernible phenotype in this family, the finding of meiotic stability and mendelian inheritance of normal CAG alleles and meiotic instability of larger repeats fulfil several criteria thought essential for pathologically rel- evant mutations of this type. Thus, these data strengthen the hypothesis for a role of CAGR1 in the development of an as yet molecularly uncharacterised human neurological disease. (i Med Genet 1997;34:411-413) Keywords: CAGRI; trinucleotide repeat; meiotic insta- bility Developmental and Genetic Center, The University of Tennessee Medical Center, 1924 Alcoa Highway, Knoxville, TN 37920, USA N T Potter Received 15 October 1996 Revised version accepted for publication 12 December 1996 The expansion of reiterated CAG sequences is associated with phenotypic expression in at least six inherited neurological disorders' and mutations of this type are suspected candidates for several other neuropsychiatric and neuro- degenerative disorders which manifest genetic anticipation.2' Efforts to identify and charac- terise candidate genes containing trinucleotide repeats have generally relied upon the screen- ing ofhuman brain cDNA libraries and human expressed sequence tags (ESTs) and the use of repeat expansion detection (RED) or FISH for the identification of abnormally long or highly polymorphic sequences,5-'0 and the causative mutation in at least one neurodegenerative dis- ease, dentatorubral-pallidoluysian atrophy, was subsequently identified using one of these approaches.6 1 12 Recently Margolis et al,'3 screening a human retinal cDNA library, iden- tified a highly polymorphic CAG repeat (called CAGRI) contained within the 5' untranslated region of a gene that shares sequence homology with the Caenorhabditis elegans cell fate deter- mining protein mab-21. The highly polymor- phic nature of this repeat (normal repeat range from 6-31 copies), its high observed heterozy- gote frequency, its predominant expression in brain (cerebellum), and their subsequent iden- tification of a single allele with 46 repeats in a person with an idiopathic movement disorder and an affective disorder provide compelling arguments for consideration of this locus as a candidate gene for molecularly uncharacter- ised inherited neurodegenerative, neuropsychi- atric, or neurodevelopmental disorders. Whereas previous studies have elucidated the polymorphic nature of this repeat,'3 '4 there have not been any reports on the identification of meiotically unstable CAGR1 alleles, an essential feature for any trinucleotide repeat locus presumed to be involved in the aetiology of disorders of this type. 400 r 300 oo CN a) 11 In a, 200 a) Co 0 100 0 10 20 30 40 50 60 CAGR1 repeat number Figure 1 Distribution of CAGRI repeat length from the 928 alleles. (CAGRI),3 represented the most common allele (33% of all alleles tested) followed by (CAGRI),9 (11% of alleles). Excluding the one patient with the (CAGRI),s there were no differences in allele distribution between the nine different patient groups. 411 o n A p ril 5 , 2 0 2 1 b y g u e st. P ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / J M e d G e n e t: first p u b lish e d a s 1 0 .1 1 3 6 /jm g .3 4 .5 .4 1 1 o n 1 M a y 1 9 9 7 . D o w n lo a d e d fro m http://jmg.bmj.com/ Potter ;:. ..f I..... WIF Figure 2 Six percent sequencing gel analysis showing meiotic stability an inheritance ofCAGR1 repeats in the normal range through 14 meioses in t pedigree 884. PCR primers for CAGR1 and conditions for analysis were es described in Margolis et all. with the exception that 2P-dCTP was incorpo) reaction and the annealing temperature for CAGJM-5'/CAG7M-3' (10 pi reaction) was lowered to 50°C. Similar analysis of the segregation of a stab through five meioses was also performed on DNA samples obtainedfrom ft (data not shown). Allele sizes were as follows: 1 (10/19); L2 (16125);IL. (13119);III.2 (13125);III.3 (13125);III.4 (13125);III.5 (20125);III.6 (13125);III.8 (19120);III.9 (20125);III.10 (19120);III.11 (13125);III (13120); L3 (20120);L.4 (13125). DNA samples from the CEPH/Amish, family DE0005 were obtainedfrom the NIGMS Human Genetic Mutant Coriell Institute for Medical Research, Camden, NJ 08103. a Figure 3 Meiotic instability of a CAG than 45 through three meioses. DNA w. peripheral blood lymphocytes. Allele size (22151); II. 2 (22147); III. I (13150). A were as described in fig 2. In an effort to identify such alleles, a total of 928 chromosomes were studied for the deter- mination of CAGRI repeat number. This included people with inherited neurodegenera- tive disorders of unknown aetiology who tested negative for CAG expansions at the HD, DRPLA, SCA-1, SCA-2, and SCA-3 loci (n=62), patients with Parkinson's disease (n=14), progressive supranuclear palsy (n=6), multiple system atrophy (n=6), nigrostriatal degeneration (n=6), multiple sclerosis (n=10), Alzheimer's disease (n= 17), molecularly nega- * tive fragile XA patients with developmental delay/mental retardation (n=295), and normal controls (n=48). The distribution of normal CAG repeat size in this cohort was 9-29 (fig 1), with a modal repeat length of 13 and heterozy- gosity of 89%. One person from the fragile XA negative group was found to have an allele with 50 CAGR1 repeats (fig 3, III.1). As this allele was almost twice the size of the largest normal allele found in this cohort, additional DNA samples were obtained from other family members to examine for evidence of meiotic instability. In contrast to the stable inheritance of CAG repeat lengths in the normal range (fig !d mendelian 2), this family clearly showed meiotic instability the CEPH/Amish for CAGR1 alleles of 45 or larger with a ACAG ssentially as of at least five repeats observed through three)rated into the Mol of each/25 Ild generations (fig 3). ble (GCAG)20 repeat Although the index case in this family had a tinily DE0005 :1 (19125); III. 1 phenotype of idiopathic mental retardation (20125); III. 7 with an emergent attention deficit/ F.12 (19120);1H.2 hyperactivity disorder, there are several reasons pedigree 884 andpCell Repository why it may be premature to presume his phenotype is related to the presence of the (CAGR1)50 allele. First, and most significant, is the lack of any readily discernible phenotype in his grandmother, mother, or aunt despite the presence of similarly sized CAGRI alleles. Sec- ond, the localisation of the repeat in an untranslated region of the gene, the small increase in CAG length over three generations in a manner consistent with the "slippage mediated model" for repeat sequence instability,'5 and the finding of meiotically unstable alleles in the 40-50 range collectively suggest that the CAGR1 expansion has mo- lecular characteristics reminiscent of the pre- mutations found in the type II repeat expan- sion mutation diseases fragile XA and E, myotonic dystrophy, and Jacobsen syndrome.'6 Whether CAGR1 alleles are prone to further expansion into a pathological size range is cur- rently unknown, although our finding of CAGR1 locus instability and the recent identi- fication of a large (3.0 kb) expansion in a novel CAG repeat found in the non-coding region of HC 18q21-22 segregating in a family with schizophrenia'7 suggests that larger expansions are possible. In summary, our data provide evidence of meiotic stability for normal sized CAGR1 alle- les and meiotic instability for larger alleles. Collectively these data strengthen the 3RI repeat greater possibility that CAGRI expansion may be eas: olate(d2445)II I involved in the development of an as yet 4ssay conditions molecularly uncharacterised human neurological disease. I! 412 i, o n A p ril 5 , 2 0 2 1 b y g u e st. P ro te cte d b y co p yrig h t. h ttp ://jm g .b m j.co m / J M e d G e n e t: first p u b lish e d a s 1 0 .1 1 3 6 /jm g .3 4 .5 .4 1 1 o n 1 M a y 1 9 9 7 . D o w n lo a d e d fro m http://jmg.bmj.com/ Meiotic instability associated with the CAGRI trinucleotide repeat at 13q13 I would like to thank Richard Greene, MD and Virginia Frye, EdD for their assistance with the evaluation of the index case and Enid Bowlin for her technical assistance with the fragile XA studies. I would also like to thank the other family members for their participation in this study. Postmortem tissue samples were obtained from the Harvard Brain Tissue Resource Center, Bel- mont, MA 02178 which is supported by NIH award RO1-MH31862 and the National Neurological Research Specimen Bank, VAMC, Los Angeles, CA 90073, which is sponsored by NINDS/NIMH, National Multiple Sclerosis Society, Hereditary Disease Foundation, Comprehensive Epi- lepsy Program, Tourette Syndrome Association, Dystonia Medical Research Foundation, and Veterans Health Services and Research Administration, Department of Veterans Affairs. This study was supported in part by grants from the Physicians' Medical and Education Research Foundation (PMERF), Knoxville, TN and the State of Tennessee. 1 Zoghbi HY The expanding world of ataxins. Nat Genet 1996;14:237-8. 2 Ross CA, McInnis MG, Margolis RL, Li SH. Genes with triplet repeats: candidate mediators of neuropsychiatric disorders. Trends Neurosci 1993;16:254-60. 3 O'Donovan MC, Guy C, Craddock N, et al. 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