Positional cloning of an X;8 translocation (p22.13;q22.1) associated with multiple exostoses and autism

Autism is characterized by qualitative impairments in communication and reciprocal social interaction. About 3 in 10,000 in the general population suffer from this neurodevelopmental disorder. The majority of patients also manifest mental retardation and about 20-45% epilepsy. Multiple exostoses is the commonest form of all skeletal dysplasias, affecting 1 in 50,000 live births. The condition, characterized by cartilaginous protuberances at the ends of the diaphyses, affects the extremities causing skeletal deformities and short stature. Autism and multiple exostoses are considered to be inherited disorders, but the underlying biochemical defects of the disorders are unknown. Both of these conditions allow for survival but considerably diminish the quality of life. An X;8 translocation was identified in a female patient, ML, with autism and multiple exostoses. Her phenotypic manifestations are likely due to the chromosomal abnormality. A positional candidate cloning strategy was used to investigate the genes involved in the translocation. The translocation breakpoint was first isolated in Yeast Artificial Chromosomes (YACs), then in cosmid and plasmid clones. The translocation was reciprocal within a 5'-GGCA-3' sequence found on both X and 8 chromosomes without gain or loss of a single nucleotide. The translocation breakpoint on the X chromosome occurred in the first intron of the gastrin releasing peptide receptor (GRPR) gene and on chromosome 8 approximately 30 kb distal to the 3' end of the Syndecan-2 gene (SDC2), also known as human heparan sulfate proteoglycan or fibroglycan. Although the GRPR gene was shown to escape X-inactivation and the coding region of the SDC2 gene was not disrupted, a dosage effect of the GRPR gene and a position effect of the SDC2 gene may, however, have contributed to the phenotype observed in this patient. The orientation of these genes with respect to the translocation was incompatible with the formation of a fusion gene. The GRPR and SDC2 genes may provide insight into the biochemical nature of autism and multiple exostoses. Investigation of mutations in these two genes in unrelated patients with either autism or multiple exostoses as well as linkage and association studies are needed to validate them as candidate genes.</p

Fluorescence in situ hybridisation of multiple probes on a single microscope slide.

We report a method to analyse multiple samples by fluorescence in situ hybridisation on a single glass microscope slide. Wells were formed in which independent hybridisation reactions could proceed by sealing a silicon rubber gasket to the slide. In the largest format tested, different probes were hybridised simultaneously by applying them directly from a 96-well microtitre dish which was inverted on a glass plate. This technique will increase the rate of analysis of multiple probes against a standard set of chromosomes and could also be used to analyse different karyotypes using a panel of probes such as single chromosome paints during a single operation. It should be useful for both chromosomal mapping projects and screening for chromosome abnormalities in clinical diagnostic laboratories

Characterization of a 1.0 Mb YAC contig spanning two chromosome breakpoints related to Menkes disease.

Menkes disease, an X-linked recessive disorder of copper metabolism, has recently been mapped to Xq13.3 by two Menkes patients carrying chromosome rearrangements within this region. The breakpoints have been investigated by nonisotopic in situ suppression hybridization using YACs isolated from this region with the flanking markers DXS56 and PGK1. Three YACs were extending over the breakpoints at Xq13.3 and were shown to be overlapping by partial digest restriction maps, IRS-PCR fingerprinting and by the presence of common cosmid clones. These cosmids were subcloned and one of the single copy probes detected both breakpoints using rare-cutting restriction enzyme digests of the patients. All the results together localize the breakpoints to about 100 kb within the overlapping region of the YACs. Mapping of both breakpoints in a 1 Mb YAC contig implies that these YACs contain at least partially, the gene responsible for Menkes disease

Autism, mental retardation, multiple exostoses and short stature in a female with 46,X,t(X;8)(p22.13;q22.1).

A young adult female with multiple exostoses, short stature, autism, mental retardation and 46,X,t(X;8)(p22.13;q22.1) is described. Although the clinical features and translocation breakpoints raise the possibility of a number of specific conditions, the constellation of problems is not consistent with any previously reported genetic syndrome. It is argued that her clinical disorder is likely due to the chromosomal abnormality and that further detailed molecular genetic investigation may shed light on the genetic basis to various components of her phenotype including the autism

Construction of a YAC contig spanning the Xq13.3 subband.

The loci involved in several X-linked mental retardation syndromes have been linked to the pericentromeric region of the X chromosome long arm (Xq12-q21). To isolate candidate genes for these diseases, we set up the construction of YAC contigs spanning this region. Two of these syndromes (the Juberg-Marsidi syndrome and the alpha-thalessemia mental retardation syndrome) have been recently linked, with high lod scores, to polymorphic probes previously assigned to Xq13.3. We therefore constructed a first YAC contig, encompassing this band, from DXS441 to PGK1. The physical map, deduced from the isolated clones, extends over 2.1 Mb of genomic DNA. Restriction analysis of the YAC contig allowed us to map precisely the loci previously assigned to that chromosomal region and to define their relative order. The validity of this physical map has been checked by comparing Sfi I digests of the YACs to genomic fragments obtained with the same enzyme. A cDNA selection approach, already performed with a previous partial contig, has been extended to cover the whole region

Isolation of a candidate gene for Menkes disease that encodes a potential heavy metal binding protein.

Menkes disease is a lethal-X linked recessive disorder associated with copper metabolism disturbance. We have recently mapped two chromosome breakpoints related to this disease in a 1 megabase yeast artificial chromosome contig at Xq13.3. We now report the construction of a phage contig and the isolation of candidate partial cDNAs for the Menkes disease gene. The candidate gene expresses an 8 kb message in all investigated tissues, and deletions were detected in 16% of 100 unrelated Menkes patients. The deduced partial protein sequence shared the GMTCXXC motif with bacterial metal resistance operons, suggesting a potential heavy metal binding protein. These findings should lead to more accurate prenatal diagnosis of this severe disease and a better understanding of the cellular homeostasis of essential heavy metals

High ice nucleation activity located in blueberry stem bark is linked to primary freeze initiation and adaptive freezing behaviour of the bark

Controlled ice nucleation is an important mechanism in cold-hardy plant tissues for avoiding excessive supercooling of the protoplasm, for inducing extracellular freezing and/or for accommodating ice crystals in specific tissues. To understand its nature, it is necessary to characterize the ice nucleation activity (INA), defined as the ability of a tissue to induce heterogeneous ice nucleation. Few studies have addressed the precise localization of INA in wintering plant tissues in respect of its function. For this purpose, we recently revised a test tube INA assay and examined INA in various tissues of over 600 species. Extremely high levels of INA (−1 to −4 °C) in two wintering blueberry cultivars of contrasting freezing tolerance were found. Their INA was much greater than in other cold-hardy species and was found to be evenly distributed along the stems of the current year's growth. Concentrations of active ice nuclei in the stem were estimated from quantitative analyses. Stem INA was localized mainly in the bark while the xylem and pith had much lower INA. Bark INA was located mostly in the cell wall fraction (cell walls and intercellular structural components). Intracellular fractions had much less INA. Some cultivar differences were identified. The results corresponded closely with the intrinsic freezing behaviour (extracellular freezing) of the bark, icicle accumulation in the bark and initial ice nucleation in the stem under dry surface conditions. Stem INA was resistant to various antimicrobial treatments. These properties and specific localization imply that high INA in blueberry stems is of intrinsic origin and contributes to the spontaneous initiation of freezing in extracellular spaces of the bark by acting as a subfreezing temperature sensor

Identification of YAC and cosmid clones encompassing the ZFX-POLA region using irradiation hybrid cell lines.

The human Xp21.3-p22.1 region is poorly mapped relative to other X chromosome regions. To target cosmid and YAC clones specifically from Xp21.3-p22.1 for rapid contig construction, a hybridization-based screening approach using irradiation hybrids has been used. Alu-PCR products generated from hybrid lines containing small overlapping fragments from Xp21-p22 were hybridized to an X chromosome cosmid library, and cosmids predicted by their hybridization pattern to map to the region of interest were analyzed by fluorescence in situ hybridization (FISH). Hybridization of the cosmids in pools to gridded YAC libraries identified 15 YACs, which were verified and tested for chimerism by FISH. Cosmid content analysis of the YACs defined two contigs, one with 12 YACs covering about 1.5 Mb and one with 3 YACs. Five YACs from the 12-YAC cluster had been previously recognized by DNA polymerase alpha (POLA). ZFX identified a single YAC; hence, the physical linkage of ZFX and POLA was demonstrated within the contig. Four YACs had been isolated previously with ZFX and these extend the contig to 2 Mb. Restriction mapping of several YACs demonstrates that ZFX and POLA are about 700 kb apart, a distance similar to that reported in the mouse between Zfx and Pola. The order of these two loci and two additional loci identified by homologous mouse linking clones was found to be conserved between human and mouse: tel-ZFX-DXCrc57-DXCrc140-POLA-cen. We have shown that YAC contigs can be rapidly constructed from targeted regions without the need for time-consuming YAC end rescue and chromosomal walking.(ABSTRACT TRUNCATED AT 250 WORDS