Meiotic stability and genotype-phenotype correlation of the trinucleotide repeat in X-linked spinal and bulbar muscular atrophy.

Expansion of the trinucleotide repeat (CAG)n in the first exon of the androgen receptor gene is associated with a rare motor neuron disorder, X-linked spinal and bulbar muscular atrophy. We have found that expanded (CAG)n alleles undergo alteration in length when transmitted from parent to offspring. Of 45 meioses examined, 12 (27%) demonstrated a change in CAG repeat number. Both expansions and contractions were observed, although their magnitude was small. There was a greater rate of instability in male meiosis than in female meiosis. We also found evidence for a correlation between disease severity and CAG repeat length, but other factors seem to contribute to the phenotypic variability in this disorder.     

CTCF-binding sites flank CTG/CAG repeats and form a methylation-sensitive insulator at the DM1 locus

An expansion of a CTG repeat at the DM1 locus causes myotonic dystrophy (DM) by altering the expression of the two adjacent genes, DMPK and SIX5, and through a toxic effect of the repeat-containing RNA. Here we identify two CTCF-binding sites that flank the CTG repeat and form an insulator element between DMPK and SIX5. Methylation of these sites prevents binding of CTCF, indicating that the DM1 locus methylation in congenital DM would disrupt insulator function. Furthermore, CTCF-binding sites are associated with CTG/CAG repeats at several other loci. We suggest a general role for CTG/CAG repeats as components of insulator elements at multiple sites in the human genome.     

Msh2 deficiency prevents in vivo somatic instability of the CAG repeat in Huntington disease transgenic mice

Huntington disease (HD), an autosomal dominant, progressive neurodegenerative disorder, is caused by an expanded CAG repeat sequence leading to an increase in the number of glutamine residues in the encoded protein. The normal CAG repeat range is 5-36, whereas 38 or more repeats are found in the diseased state; the severity of disease is roughly proportional to the number of CAG repeats. HD shows anticipation, in which subsequent generations display earlier disease onsets due to intergenerational repeat expansion. For longer repeat lengths, somatic instability of the repeat size has been observed both in human cases at autopsy and in transgenic mouse models containing either a genomic fragment of human HD exon 1 (ref. 9) or an expanded repeat inserted into the endogenous mouse gene Hdh (ref. 10). With increasing repeat number, the protein changes conformation and becomes increasingly prone to aggregation, suggesting important functional correlations between repeat length and pathology. Because dinucleotide repeat instability is known to increase when the mismatch repair enzyme MSH2 is missing, we examined instability of the HD CAG repeat by crossing transgenic mice carrying exon 1 of human HD (ref. 16) with Msh2-/- mice. Our results show that Msh2 is required for somatic instability of the CAG repeat.     

Trinucleotide expansion in haploid germ cells by gap repair

Huntington disease (HD) is one of eight progressive neurodegenerative disorders in which the underlying mutation is a CAG expansion encoding a polyglutamine tract. The mechanism of trinucleotide expansion is poorly understood. Expansion is mediated by misaligned pairing of repeats and the inappropriate formation of DNA secondary structure as the duplex unpairs. It has never been clear, however, whether duplex unpairing occurs during mitotic replication or during strand-break repair. In simple organisms, trinucleotide expansion arises by replication slippage on either the leading or the lagging strand, homologous recombination, gene conversion, double-strand break repair and base excision repair; it is not clear which of these mechanisms is used in mammalian cells in vivo. We have followed heritable changes in CAG length in male transgenic mice. In germ cells, expansion is limited to the post-meiotic, haploid cell and therefore cannot involve mitotic replication or recombination between a homologous chromosome or a sister chromatid. Our data support a model in which expansion in the germ cells arises by gap repair and depends on a complex containing Msh2. Expansion occurs during gap-filling synthesis when DNA loops comprising the CAG trinucleotide repeats are sealed into the DNA strand.     

An untranslated CTG expansion causes a novel form of spinocerebellar ataxia (SCA8)

Myotonic dystrophy (DM) is the only disease reported to be caused by a CTG expansion. We now report that a non-coding CTG expansion causes a novel form of spinocerebellar ataxia (SCA8). This expansion, located on chromosome 13q21, was isolated directly from the genomic DNA of an ataxia patient by RAPID cloning. SCA8 patients have expansions similar in size (107-127 CTG repeats) to those found among adult-onset DM patients. SCA8 is the first example of a dominant SCA not caused by a CAG expansion translated as a polyglutamine tract.     

Correlation between CTG trinucleotide repeat length and frequency of severe congenital myotonic dystrophy.

The myotonic dystrophy (DM) mutation has recently been identified as an unstable trinucleotide CTG repeat which is present 5-30 times in the normal population but which is amplified up to 2,000 times in DM. We have determined the status of the CTG repeat in 272 DM individuals. Infants with severe congenital DM, as well as their mothers, are shown to have on average a greater amplification of the CTG repeat than is seen in the noncongenital DM population. This fact, when viewed in conjunction with the tendency to increased CTG repeat length in our DM kindreds, provides evidence for the existence of genetic anticipation in the transmission of DM.     

Large expansion of the ATTCT pentanucleotide repeat in spinocerebellar ataxia type 10

Spinocerebellar ataxia type 10 (SCA10; MIM 603516; refs 1,2) is an autosomal dominant disorder characterized by cerebellar ataxia and seizures. The gene SCA10 maps to a 3.8-cM interval on human chromosome 22q13-qter (refs 1,2). Because several other SCA subtypes show trinucleotide repeat expansions, we examined microsatellites in this region. We found an expansion of a pentanucleotide (ATTCT) repeat in intron 9 of SCA10 in all patients in five Mexican SCA10 families. There was an inverse correlation between the expansion size, up to 22.5 kb larger than the normal allele, and the age of onset (r2=0.34, P=0.018). Analysis of 562 chromosomes from unaffected individuals of various ethnic origins (including 242 chromosomes from Mexican persons) showed a range of 10 to 22 ATTCT repeats with no evidence of expansions. Our data indicate that the new SCA10 intronic ATTCT pentanucleotide repeat in SCA10 patients is unstable and represents the largest microsatellite expansion found so far in the human genome.     

Nuclear localization or inclusion body formation of ataxin-2 are not necessary for SCA2 pathogenesis in mouse or human

Instability of CAG DNA trinucleotide repeats is the mutational mechanism for several neurodegenerative diseases resulting in the expansion of a polyglutamine (polyQ) tract. Proteins with long polyQ tracts have an increased tendency to aggregate, often as truncated fragments forming ubiquitinated intranuclear inclusion bodies. We examined whether similar features define spinocerebellar ataxia type 2 (SCA2) pathogenesis using cultured cells, human brains and transgenic mouse lines. In SCA2 brains, we found cytoplasmic, but not nuclear, microaggregates. Mice expressing ataxin-2 with Q58 showed progressive functional deficits accompanied by loss of the Purkinje cell dendritic arbor and finally loss of Purkinje cells. Despite similar functional deficits and anatomical changes observed in ataxin-1[Q80] transgenic lines, ataxin-2[Q58] remained cytoplasmic without detectable ubiquitination.     

Meiotic instability of human minisatellite CEB1 in yeast requires DNA double-strand breaks.

Minisatellites are tandemly repeated DNA sequences of 10-100-bp units. Some minisatellite loci are highly unstable in the human germ line, and structural analysis of mutant alleles has suggested that repeat instability results from a recombination-based process. To provide insights into the molecular mechanism of human minisatellite instability, we developed Saccharomyces cerevisiae strains carrying alleles of the most unstable human minisatellite locus, CEB1 (ref. 2). We observed that CEB1 is destabilized in meiosis, resulting in a variety of intra- and inter-allelic gains or losses of repeat units, similar to rearrangements described in humans. Using mutations affecting the initiation of recombination (spo11) or mismatch repair (msh2 pms1 ), we demonstrate that meiotic destabilization depends on the initiation of homologous recombination at nearby DNA double-strand break (DSBs) sites and involves a 'rearranged heteroduplex' intermediate. Most of the human and yeast data can be explained and unified in the context of DSB repair models.     

The NOTCH4 locus is associated with susceptibility to schizophrenia

Linkage disequilibrium mapping of the MHC region in 80 British parent-offspring trios showed that NOTCH4 was highly associated with schizophrenia. The A-->G substitution in the promoter region and the (CTG)n repeat in exon 1 of NOTCH4 may be candidate sites conferring susceptibility to schizophrenia.     

A repeat expansion in the gene encoding junctophilin-3 is associated with Huntington disease-like 2.

We recently described a disorder termed Huntington disease-like 2 (HDL2) that completely segregates with an unidentified CAG/CTG expansion in a large pedigree (W). We now report the cloning of this expansion and its localization to a variably spliced exon of JPH3 (encoding junctophilin-3), a gene involved in the formation of junctional membrane structures.     

Heterogeneous mutation processes in human microsatellite DNA sequences

Although microsatellite polymorphisms are one of the most commonly used tools in genetic analyses, it remains to be understood how microsatellite DNA has evolved as a ubiquitous and highly abundant class of repetitive sequences in eukaryotic genomes. On the basis of analyses of spontaneous human microsatellite mutations of germline origin, I show here that different mutation biases underlie the evolution of microsatellite repeats. The within-locus mutation rate increases with allele length, but is not affected by the size difference between an individual's two alleles (allele span). Within loci, long alleles tend to mutate to shorter lengths, thereby acting to prevent infinite growth. Expansions are more common than contractions among dinucleotide repeats, whereas no such trend is evident among tetranucleotide repeats. This observation is consistent with the longer repeat lengths and higher frequency of di- compared with tetranucleotide repeats. An excess of paternally transmitted mutations (male-to-female ratio of 4.9) supports a male-biased mutation rate in the human genome.     

Molecular mechanism for duplication 17p11.2- the homologous recombination reciprocal of the Smith-Magenis microdeletion

Recombination between repeated sequences at various loci of the human genome are known to give rise to DNA rearrangements associated with many genetic disorders. Perhaps the most extensively characterized genomic region prone to rearrangement is 17p12, which is associated with the peripheral neuropathies, hereditary neuropathy with liability to pressure palsies (HNPP) and Charcot-Marie-Tooth disease type 1A (CMT1A;ref. 2). Homologous recombination between 24-kb flanking repeats, termed CMT1A-REPs, results in a 1.5-Mb deletion that is associated with HNPP, and the reciprocal duplication product is associated with CMT1A (ref. 2). Smith-Magenis syndrome (SMS) is a multiple congenital anomalies, mental retardation syndrome associated with a chromosome 17 microdeletion, del(17)(p11.2p11.2) (ref. 3,4). Most patients (>90%) carry deletions of the same genetic markers and define a common deletion. We report seven unrelated patients with de novo duplications of the same region deleted in SMS. A unique junction fragment, of the same apparent size, was identified in each patient by pulsed field gel electrophoresis (PFGE). Further molecular analyses suggest that the de novo17p11.2 duplication is preferentially paternal in origin, arises from unequal crossing over due to homologous recombination between flanking repeat gene clusters and probably represents the reciprocal recombination product of the SMS deletion. The clinical phenotype resulting from duplication [dup(17)(p11.2p11.2)] is milder than that associated with deficiency of this genomic region. This mechanism of reciprocal deletion and duplication via homologous recombination may not only pertain to the 17p11.2 region, but may also be common to other regions of the genome where interstitial microdeletion syndromes have been defined.     

Intragenic tandem repeats generate functional variability

Tandemly repeated DNA sequences are highly dynamic components of genomes. Most repeats are in intergenic regions, but some are in coding sequences or pseudogenes. In humans, expansion of intragenic triplet repeats is associated with various diseases, including Huntington chorea and fragile X syndrome. The persistence of intragenic repeats in genomes suggests that there is a compensating benefit. Here we show that in the genome of Saccharomyces cerevisiae, most genes containing intragenic repeats encode cell-wall proteins. The repeats trigger frequent recombination events in the gene or between the gene and a pseudogene, causing expansion and contraction in the gene size. This size variation creates quantitative alterations in phenotypes (e.g., adhesion, flocculation or biofilm formation). We propose that variation in intragenic repeat number provides the functional diversity of cell surface antigens that, in fungi and other pathogens, allows rapid adaptation to the environment and elusion of the host immune system.     

Regulation of premeiotic S phase and recombination-related double-strand DNA breaks during meiosis in fission yeast.

The meiotic cell cycle is characterized by high levels of recombination induced by DNA double-strand breaks (DSBs), which appear after completion of premeiotic S phase, leading to the view that initiation of recombination depends on meiotic DNA replication. It has also been indicated that DNA replication initiation proteins may differ between the meiotic and mitotic cell cycles, giving rise to an altered S phase, which could contribute to the high level of recombination during meiosis. We have investigated these possibilities in the fission yeast Schizosaccharomyces pombe and found that core DNA replication initiation proteins used during the mitotic cell cycle, including Cdc18p (budding yeast Cdc6p), Cdc19p (Mcm2p), Cdc21p (Mcm4p) and Orp1p (Orc1p), are also required for premeiotic S phase. Reduced activity of these proteins prevents completion of DNA replication but not formation of DSBs. We conclude that recombination-related DSB formation does not depend on the completion of meiotic DNA replication and we propose two parallel developmental sequences during the meiotic cell cycle: one for premeiotic S phase and the other for initiating recombination.