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.     

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.     

Mutations in TNFRSF11A, affecting the signal peptide of RANK, cause familial expansile osteolysis

Familial expansile osteolysis (FEO, MIM 174810) is a rare, autosomal dominant bone disorder characterized by focal areas of increased bone remodelling. The osteolytic lesions, which develop usually in the long bones during early adulthood, show increased osteoblast and osteoclast activity. Our previous linkage studies mapped the gene responsible for FEO to an interval of less than 5 cM between D18S64 and D18S51 on chromosome 18q21.2-21.3 in a large Northern Irish family. The gene encoding receptor activator of nuclear factor-kappa B (RANK; ref. 5), TNFRSF11A, maps to this region. RANK is essential in osteoclast formation. We identified two heterozygous insertion mutations in exon 1 of TNFRSF11A in affected members of four families with FEO or familial Paget disease of bone (PDB). One was a duplication of 18 bases and the other a duplication of 27 bases, both of which affected the signal peptide region of the RANK molecule. Expression of recombinant forms of the mutant RANK proteins revealed perturbations in expression levels and lack of normal cleavage of the signal peptide. Both mutations caused an increase in RANK-mediated nuclear factor-kappaB (NF-kappaB) signalling in vitro, consistent with the presence of an activating mutation.     

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.     

TLR4 mutations are associated with endotoxin hyporesponsiveness in humans

There is much variability between individuals in the response to inhaled toxins, but it is not known why certain people develop disease when challenged with environmental agents and others remain healthy. To address this, we investigated whether TLR4 (encoding the toll-like receptor-4), which has been shown to affect lipopolysaccharide (LPS) responsiveness in mice, underlies the variability in airway responsiveness to inhaled LPS in humans. Here we show that common, co-segregating missense mutations (Asp299Gly and Thr399Ile) affecting the extracellular domain of the TLR4 receptor are associated with a blunted response to inhaled LPS in humans. Transfection of THP-1 cells demonstrates that the Asp299Gly mutation (but not the Thr399Ile mutation) interrupts TLR4-mediated LPS signalling. Moreover, the wild-type allele of TLR4 rescues the LPS hyporesponsive phenotype in either primary airway epithelial cells or alveolar macrophages obtained from individuals with the TLR4 mutations. Our findings provide the first genetic evidence that common mutations in TLR4 are associated with differences in LPS responsiveness in humans, and demonstrate that gene-sequence changes can alter the ability of the host to respond to environmental stress.     

Computer-based three-dimensional visualization of developmental gene expression

A broad understanding of the relationship between gene activation, pattern formation and morphogenesis will require adequate tools for three-dimensional and, perhaps four-dimensional, representation and analysis of molecular developmental processes. We present a novel, computer-based method for the 3D visualization of embryonic gene expression and morphological structures from serial sections. The information from these automatically aligned 3D reconstructions exceeds that from single-section and whole-mount visualizations of in situ hybridizations. In addition, these 3D models of gene-expression patterns can become a central component of a future developmental database designed for the collection and presentation of digitized, morphological and gene-expression data. This work is accompanied by a web site (http://www.univie.ac.at/GeneEMAC).