Suzanne L. Winfield

The structure and expression of the human neuroligin-3 gene.

The neuroligins are a family of proteins that are thought to mediate cell to cell interactions between neurons. During the sequencing at an Xq13 locus associated with a mental retardation syndrome in some studies, we discovered a portion of the human orthologue of the rat neuroligin-3 gene. We now report the structure and the expression of that gene. The gene spans approximately 30kb and contains eight exons. Unlike the rat gene, it codes for at least two mRNAs and at least one of which is expressed outside the CNS. Interestingly, the putative promoter for the gene overlaps the last exon of the neighboring HOPA gene and is located less than 1kb from an OPA element in which a polymorphism associated with mental retardation is found. These findings suggest a possible role for the neuroligin gene in mental retardation and that the role of the gene in humans may differ from its role in rats.

Isolation of cDNA clones for human β-glucocerebrosidase using the λgtll expression system

Two cDNA clones (λGC-1 and λGC-2) for human β-glucocerebrosidase [EC 3.2.1.45] have been isolated from a human hepatoma library in λgtll by immunological screening using monospecific polyclonal antibody for β-glucocerebrosidase. Restriction endonuclease mapping indicates that these clones are probably identical in size, each with a 1900 bp insert. The 50 kDa size of the insert-encoded polypeptide produced by these clones in fusion with β-galactosidase of λgtll in E.coli BNN103 is consistent with the size of the nascent form of β-glucocerebrosidase. These fusion proteins are shown by Western blotting to react with antibody to β-glucocerebrosidase. Amino acid sequence deduced from the nucleotide sequence of the insert ir pGC-1 is identical to known amino acid sequence of β-glucocerebrosidase, and thus, confirms that the clones are specific for β-glucocerebrosidase.

Association of an X-chromosome dodecamer insertional variant allele with mental retardation

Mental retardation is a prominent feature of many neurodevelopmental syndromes. In an attempt to identify genetic components of these illnesses, we isolated and sequenced a large number of human genomic cosmid inserts containing large trinucleotide repeats. One of these cosmids, Cos-4, maps to the X-chromosome and contains the sequence of a 7.3-kb mRNA. Initial polymorphism analysis across a region of repetitive DNA in this gene revealed a rare 12-bp exonic variation (1% in non-ill males) having an increased prevalence in non-Fragile X males with mental retardation (4%, P <0.04, n = 81). This variant was not present in the highly conserved mouse homologue that has 100% amino acid identity to the human sequence near the polymorphism. Subsequent screening of two additional independent cohorts of non-Fragile X mentally retarded patients and ethnically matched controls demonstrated an even higher prevalence of the 12-bp variant in males with mental retardation (8%, P <0.0003, n = 125, and 14%, P <0.10, n = 36) vs the controls. Multivariate analysis was conducted in an effort to identify other phenotypic components in affected individuals, and the findings suggested an increased incidence of histories of hypothyroidism (P <0.001) and treatment with antidepressants (P <0.001). we conclude that the presence of this 12-bp variant confers significant susceptibility for mental retardation.

Two novel polymorphic sequences in the glucocerebrosidase gene region enhance mutational screening and founder effect studies of patients with Gaucher disease

Gaucher disease, an inherited glycolipid storage disorder, is caused by a deficiency of the catabolic enzyme glucocerebrosidase (EC 3.2.1.45). The gene for human glucocerebrosidase is located on chromosome 1q21 and has a highly homologous pseudogene situated 16 kb downstream. We report two novel polymorphic sequences in the glucocerebrosidase gene region: the first consists of a variable number of dinucleotide (CT) repeats located 3.2 kb upstream from the glucocerebrosidase gene, and the second is a tetranucleotide (AAAT) repeat found between the glucocerebrosidase gene and its pseudogene, 9.8 kb downstream from the functional gene. These polymorphic sequences, along with a previously reported PvuII polymorphism in intron 6 of the glucocerebrosidase gene, were analyzed in patients with Gaucher disease (n=106) and in two normal control populations, one of Ashkenazi Jewish ancestry (n=72) and the second comprising non-Jewish individuals (n=46). In these samples, strong linkage disequilibrium was found between mutations N370S, c.84–85insG, and R463C and specific haplotypes; no significant linkage disequilibrium was found when examining haplotypes of patients with the L444P mutation. Studies of these polymorphic sites in several instances also led to the recognition of genotyping errors and the identification of unusual recombinant alleles. These new polymorphic sites provide additional tools for mutational screening and founder effect studies of Gaucher disease.

Analysis of Two Novel Polymorphic Sites in the Glucocerebrosidase Gene Region in Patients with Gaucher Disease 739

Gaucher disease is caused by the inherited deficiency of the enzyme glucocerebrosidase (EC 3.2.1.45). The gene for glucocerebrosidase is located on chromosome 1q21 and a highly homologous pseudogene is situated 16kb downstream. Two novel polymorphisms were identified in the glucocerebrosidase gene region: the first consists of a variable number of tetranucleotide (AAAT) repeats located upstream of the glucocerebrosidase gene, and the second is a series of dinucleotide (CT) repeats found in the intergenic region between the glucocerebrosidase gene and its pseudogene. These two polymorphisms, along with the previously reported PvuII polymorphism in intron 6 of the glucocerebrosidase gene, were analyzed in an affected population (n=102) and in two normal control populations, one consisting of non-Jewish individuals of mixed ethnic background (n=46) and the second of Ashkenazi Jewish individuals(n=72). Strong linkage disequilibrium was found between mutations N370S, 84GG, and R463C and specific haplotypes; no significant linkage disequilibrium was found in patients carrying mutation L444P. Unexpected haplotypes lead to the recognition of several genotyping errors and complex alleles. These two novel polymorphic sites provide a new tool for the identification of mutant alleles in patients and for the study of founder effects in populations with Gaucher disease.