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.     

Evidence of cis-acting factors in replication-mediated trinucleotide repeat instability in primate cells

The mechanism of disease-associated trinucleotide repeat instability involves cis-acting factors (cis-elements) in the vicinity of the repeat, but the nature of these elements is unknown. One cis-element may be the location of the replication origin relative to the repeat. We have used an SV40 DNA replication system to investigate the effect of the location of replication initiation on (CTG)(n)*(CAG)(n) stability in primate cells. Depending on the distance between the SV40 replication origin and the repeat tract, templates with 79 repeats yield predominantly expansions or predominantly deletions or remain intact. All templates with 17 repeats are stable. Thus, cis-elements that affect the sites of Okazaki fragment initiation relative to the repeat are crucial determinants of instability. This model system recapitulates the bias for expansions observed in many of the diseases associated with trinucleotide repeats. Our results might explain the variable amounts of CTG/CAG instability that are observed in different chromosomal contexts.     

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.     

Mapping of a gene predisposing to early-onset Alzheimer's disease to chromosome 14q24.3.

Genetic linkage studies with chromosome 21 DNA markers and mutation analysis of the beta-amyloid protein precursor gene located in 21q21.3 have indicated that early-onset Alzheimer's disease (EOAD) is a heterogeneous disorder for which at least one other chromosomal locus exists. We examined two extended histopathologically confirmed EOAD pedigrees, AD/A and AD/B, with highly informative short tandem repeat (STR) polymorphisms and found complete linkage of the disease to a (CA)n dinucleotide repeat polymorphism at locus D14S43 in 14q24.3 (Zmax = 13.25 at theta = 0.0). Using additional chromosome 14 STR polymorphisms we were able to delineate the region containing the EOAD gene to an area of, at most, 8.9 centiMorgans between D14S42 and D14S53, flanking D14S43 on both sides.     

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.     

Fragile X syndrome without CCG amplification has an FMR1 deletion.

We describe a patient with typical clinical features of the fragile X syndrome, but without cytogenetic expression of the fragile X or an amplified CCG trinucleotide repeat fragment. The patient has a previously uncharacterized submicroscopic deletion encompassing the CCG repeat, the entire FMR1 gene and about 2.5 megabases of flanking sequences. This finding confirms that the fragile X phenotype can exist, without amplification of the CCG repeat or cytogenetic expression of the fragile X, and that fragile X syndrome is a genetically homogeneous disorder involving FMR1. We also found random X-inactivation in the mother of the patient who was shown to be a carrier of this deletion.     

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.     

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.     

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.     

Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase

Autosomal recessive juvenile parkinsonism (AR-JP), one of the most common familial forms of Parkinson disease, is characterized by selective dopaminergic neural cell death and the absence of the Lewy body, a cytoplasmic inclusion body consisting of aggregates of abnormally accumulated proteins. We previously cloned PARK2, mutations of which cause AR-JP (ref. 2), but the function of the gene product, parkin, remains unknown. We report here that parkin is involved in protein degradation as a ubiquitin-protein ligase collaborating with the ubiquitin-conjugating enzyme UbcH7, and that mutant parkins from AR-JP patients show loss of the ubiquitin-protein ligase activity. Our findings indicate that accumulation of proteins that have yet to be identified causes a selective neural cell death without formation of Lewy bodies. Our findings should enhance the exploration of the molecular mechanisms of neurodegeneration in Parkinson disease as well as in other neurodegenerative diseases that are characterized by involvement of abnormal protein ubiquitination, including Alzheimer disease, other tauopathies, CAG triplet repeat disorders and amyotrophic lateral sclerosis.     

WDR62 is associated with the spindle pole and is mutated in human microcephaly

Autosomal recessive primary microcephaly (MCPH) is a disorder of neurodevelopment resulting in a small brain. We identified WDR62 as the second most common cause of MCPH after finding homozygous missense and frame-shifting mutations in seven MCPH families. In human cell lines, we found that WDR62 is a spindle pole protein, as are ASPM and STIL, the MCPH7 and MCHP7 proteins. Mutant WDR62 proteins failed to localize to the mitotic spindle pole. In human and mouse embryonic brain, we found that WDR62 expression was restricted to neural precursors undergoing mitosis. These data lend support to the hypothesis that the exquisite control of the cleavage furrow orientation in mammalian neural precursor cell mitosis, controlled in great part by the centrosomes and spindle poles, is critical both in causing MCPH when perturbed and, when modulated, generating the evolutionarily enlarged human brain.     

Genome-wide association and linkage identify modifier loci of lung disease severity in cystic fibrosis at 11p13 and 20q13.2

A combined genome-wide association and linkage study was used to identify loci causing variation in cystic fibrosis lung disease severity. We identified a significant association (P = 3.34 × 10(-8)) near EHF and APIP (chr11p13) in p.Phe508del homozygotes (n = 1,978). The association replicated in p.Phe508del homozygotes (P = 0.006) from a separate family based study (n = 557), with P = 1.49 × 10(-9) for the three-study joint meta-analysis. Linkage analysis of 486 sibling pairs from the family based study identified a significant quantitative trait locus on chromosome 20q13.2 (log(10) odds = 5.03). Our findings provide insight into the causes of variation in lung disease severity in cystic fibrosis and suggest new therapeutic targets for this life-limiting disorder.     

Linkage of Rieger syndrome to the region of the epidermal growth factor gene on chromosome 4.

Rieger syndrome is an autosomal dominant disorder of morphogenesis in which previous cytogenetic arrangements have suggested chromosome 4 as a candidate chromosome. Using a group of highly polymorphic short tandem repeat polymorphisms (STRP), including a new tetranucleotide repeat for epidermal growth factor (EGF), significant linkage of Rieger syndrome to 4q markers has been identified. Tight linkage to EGF supports its role as a candidate gene, although a recombinant in an unaffected individual has been identified. This study demonstrates the utility of using polymorphic STRP markers when only a limited number of small families are available for study.     

Charcot-Marie-Tooth type 1A duplication appears to arise from recombination at repeat sequences flanking the 1.5 Mb monomer unit.

We have constructed a 3.1 megabase (Mb) physical map of chromosome 17p11.2-p12, which contains a submicroscopic duplication in patients with Charcot-Marie-Tooth disease type 1A (CMT1A). We find that the CMT1A duplication is a tandem repeat of 1.5 Mb of DNA. A YAC contig encompassing the CMT1A duplication and spanning the endpoints was also developed. Several low copy repeats in 17p11.2-p12 were identified including the large (> 17 kb) CMT1A-REP unit which may be part of a mosaic repeat. CMT1A-REP flanks the 1.5 Mb CMT1A monomer unit on normal chromosome 17 and is present in an additional copy on the CMT1A duplicated chromosome. We propose that the de novo CMT1A duplication arises from unequal crossing over due to misalignment at these CMT1A-REP repeat sequences during meiosis.     

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.