Limb-girdle muscular dystrophy type 2G is caused by mutations in the gene encoding the sarcomeric protein telethonin

Autosomal recessive limb-girdle muscular dystrophies (AR LGMDs) are a genetically heterogeneous group of disorders that affect mainly the proximal musculature. There are eight genetically distinct forms of AR LGMD, LGMD 2A-H (refs 2-10), and the genetic lesions underlying these forms, except for LGMD 2G and 2H, have been identified. LGMD 2A and LGMD 2B are caused by mutations in the genes encoding calpain 3 (ref. 11) and dysferlin, respectively, and are usually associated with a mild phenotype. Mutations in the genes encoding gamma-(ref. 14), alpha-(ref. 5), beta-(refs 6,7) and delta (ref. 15)-sarcoglycans are responsible for LGMD 2C to 2F, respectively. Sarcoglycans, together with sarcospan, dystroglycans, syntrophins and dystrobrevin, constitute the dystrophin-glycoprotein complex (DGC). Patients with LGMD 2C-F predominantly have a severe clinical course. The LGMD 2G locus maps to a 3-cM interval in 17q11-12 in two Brazilian families with a relatively mild form of AR LGMD (ref. 9). To positionally clone the LGMD 2G gene, we constructed a physical map of the 17q11-12 region and refined its localization to an interval of 1.2 Mb. The gene encoding telethonin, a sarcomeric protein, lies within this candidate region. We have found that mutations in the telethonin gene cause LGMD 2G, identifying a new molecular mechanism for AR LGMD.     

ARSACS, a spastic ataxia common in northeastern Québec, is caused by mutations in a new gene encoding an 11.5-kb ORF

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS or SACS) is an early onset neurodegenerative disease with high prevalence (carrier frequency 1/22) in the Charlevoix-Saguenay-Lac-Saint-Jean (CSLSJ) region of Quebec. We previously mapped the gene responsible for ARSACS to chromosome 13q11 and identified two ancestral haplotypes. Here we report the cloning of this gene, SACS, which encodes the protein sacsin. The ORF of SACS is 11,487 bp and is encoded by a single gigantic exon spanning 12,794 bp. This exon is the largest to be identified in any vertebrate organism. The ORF is conserved in human and mouse. The putative protein contains three large segments with sequence similarity to each other and to the predicted protein of an Arabidopsis thaliana ORF. The presence of heat-shock domains suggests a function for sacsin in chaperone-mediated protein folding. SACS is expressed in a variety of tissues, including the central nervous system. We identified two SACSmutations in ARSACS families that lead to protein truncation, consistent with haplotype analysis.     

MLH3: a DNA mismatch repair gene associated with mammalian microsatellite instability

DNA mismatch repair is important because of its role in maintaining genomic integrity and its association with hereditary non-polyposis colon cancer (HNPCC). To identify new human mismatch repair proteins, we probed nuclear extracts with the conserved carboxy-terminal MLH1 interaction domain. Here we describe the cloning and complete genomic sequence of MLH3, which encodes a new DNA mismatch repair protein that interacts with MLH1. MLH3 is more similar to mismatch repair proteins from yeast, plants, worms and bacteria than to any known mammalian protein, suggesting that its conserved sequence may confer unique functions in mice and humans. Cells in culture stably expressing a dominant-negative MLH3 protein exhibit microsatellite instability. Mlh3 is highly expressed in gastrointestinal epithelium and physically maps to the mouse complex trait locus colon cancer susceptibility I (Ccs1). Although we were unable to identify a mutation in the protein-coding region of Mlh3 in the susceptible mouse strain, colon tumours from congenic Ccs1 mice exhibit microsatellite instability. Functional redundancy among Mlh3, Pms1 and Pms2 may explain why neither Pms1 nor Pms2 mutant mice develop colon cancer, and why PMS1 and PMS2 mutations are only rarely found in HNPCC families.     

Genetic and epigenetic incompatibilities underlie hybrid dysgenesis in Peromyscus

Crosses between the two North American rodent species Peromyscus polionotus (PO) and Peromyscus maniculatus (BW) yield parent-of-origin effects on both embryonic and placental growth. The two species are approximately the same size, but a female BW crossed with a male PO produces offspring that are smaller than either parent. In the reciprocal cross, the offspring are oversized and typically die before birth. Rare survivors are exclusively female, consistent with Haldane's rule, which states that in instances of hybrid sterility or inviability, the heterogametic sex tends to be more severely affected. To understand these sex- and parent-of-origin-specific patterns of overgrowth, we analysed reciprocal backcrosses. Our studies reveal that hybrid inviability is partially due to a maternally expressed X-linked PO locus and an imprinted paternally expressed autosomal BW locus. In addition, the hybrids display skewing of X-chromosome inactivation in favour of the expression of the BW X chromosome. The most severe overgrowth is accompanied by widespread relaxation of imprinting of mostly paternally expressed genes. Both genetic and epigenetic mechanisms underlie hybrid inviability in Peromyscus and hence have a role in the establishment and maintenance of reproductive isolation barriers in mammals.     

Mutations in KERA, encoding keratocan, cause cornea plana

Specialized collagens and small leucine-rich proteoglycans (SLRPs) interact to produce the transparent corneal structure. In cornea plana, the forward convex curvature is flattened, leading to a decrease in refraction. A more severe, recessively inherited form (CNA2; MIM 217300) and a milder, dominantly inherited form (CNA1; MIM 121400) exist. CNA2 is a rare disorder with a worldwide distribution, but a high prevalence in the Finnish population. The gene mutated in CNA2 was assigned by linkage analysis to 12q (refs 4, 5), where there is a cluster of several SLRP genes. We cloned two additional SLRP genes highly expressed in cornea: KERA (encoding keratocan) in 12q and OGN (encoding osteoglycin) in 9q. Here we report mutations in KERA in 47 CNA2 patients: 46 Finnish patients are homozygous for a founder missense mutation, leading to the substitution of a highly conserved amino acid; and one American patient is homozygous for a mutation leading to a premature stop codon that truncates the KERA protein. Our data establish that mutations in KERA cause CNA2. CNA1 patients had no mutations in these proteoglycan genes.     

De novo deletions of SNRPN exon 1 in early human and mouse embryos result in a paternal to maternal imprint switch

Prader-Willi syndrome (PWS) is a neurogenetic disease characterized by infantile hypotonia, gonadal hypoplasia, obsessive behaviour and neonatal feeding difficulties followed by hyperphagia, leading to profound obesity. PWS is due to a lack of paternal genetic information at 15q11-q13 (ref. 2). Five imprinted, paternally expressed genes map to the PWS region, MKRN3 (ref. 3), NDN (ref. 4), NDNL1 (ref. 5), SNRPN (refs 6-8 ) and IPW (ref. 9), as well as two poorly characterized framents designated PAR-1 and PAR-5 (ref. 10). Imprinting of this region involves a bipartite 'imprinting centre' (IC), which overlaps SNRPN (refs 10,11). Deletion of the SNRPN promoter/exon 1 region (the PWS IC element) appears to impair the establishment of the paternal imprint in the male germ line and leads to PWS. Here we report a PWS family in which the father is mosaic for an IC deletion on his paternal chromosome. The deletion chromosome has acquired a maternal methylation imprint in his somatic cells. We have made identical findings in chimaeric mice generated from two independent embryonic stem (ES) cell lines harbouring a similar deletion. Our studies demonstrate that the PWS IC element is not only required for the establishment of the paternal imprint, but also for its postzygotic maintenance.     

Systematic variation in gene expression patterns in human cancer cell lines

We used cDNA microarrays to explore the variation in expression of approximately 8,000 unique genes among the 60 cell lines used in the National Cancer Institute's screen for anti-cancer drugs. Classification of the cell lines based solely on the observed patterns of gene expression revealed a correspondence to the ostensible origins of the tumours from which the cell lines were derived. The consistent relationship between the gene expression patterns and the tissue of origin allowed us to recognize outliers whose previous classification appeared incorrect. Specific features of the gene expression patterns appeared to be related to physiological properties of the cell lines, such as their doubling time in culture, drug metabolism or the interferon response. Comparison of gene expression patterns in the cell lines to those observed in normal breast tissue or in breast tumour specimens revealed features of the expression patterns in the tumours that had recognizable counterparts in specific cell lines, reflecting the tumour, stromal and inflammatory components of the tumour tissue. These results provided a novel molecular characterization of this important group of human cell lines and their relationships to tumours in vivo.     

Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation

The composite structure of the mammalian skull, which forms predominantly via intramembranous ossification, requires precise pre- and post-natal growth regulation of individual calvarial elements. Disturbances of this process frequently cause severe clinical manifestations in humans. Enhanced DNA binding by a mutant MSX2 homeodomain results in a gain of function and produces craniosynostosis in humans. Here we show that Msx2-deficient mice have defects of skull ossification and persistent calvarial foramen. This phenotype results from defective proliferation of osteoprogenitors at the osteogenic front during calvarial morphogenesis, and closely resembles that associated with human MSX2 haploinsufficiency in parietal foramina (PFM). Msx2-/- mice also have defects in endochondral bone formation. In the axial and appendicular skeleton, post-natal deficits in Pth/Pthrp receptor (Pthr) signalling and in expression of marker genes for bone differentiation indicate that Msx2 is required for both chondrogenesis and osteogenesis. Consistent with phenotypes associated with PFM, Msx2-mutant mice also display defective tooth, hair follicle and mammary gland development, and seizures, the latter accompanied by abnormal development of the cerebellum. Most Msx2-mutant phenotypes, including calvarial defects, are enhanced by genetic combination with Msx1 loss of function, indicating that Msx gene dosage can modify expression of the PFM phenotype. Our results provide a developmental basis for PFM and demonstrate that Msx2 is essential at multiple sites during organogenesis.