Publications

Abstract

Family and twin studies of developmental dyslexia have consistently shown that there is a significant heritable component for this disorder. However, any genetic basis for the trait is likely to be complex, involving reduced penetrance, phenocopy, heterogeneity and oligogenic inheritance. This complexity results in reduced power for traditional parametric linkage analysis, where specification of the correct genetic model is important. One strategy is to focus on large multigenerational pedigrees with severe phenotypes and/or apparent simple Mendelian inheritance, as has been successfully demonstrated for speech and language impairment. This approach is limited by the scarcity of such families. An alternative which has recently become feasible due to the development of high-throughput genotyping techniques is the analysis of large numbers of sib-pairs using allele-sharing methodology. This paper outlines our strategy for conducting a systematic genome-wide search for genes involved in dyslexia in a large number of affected sib-pair familites from the UK. We use a series of psychometric tests to obtain different quantitative measures of reading deficit, which should correlate with different components of the dyslexia phenotype, such as phonological awareness and orthographic coding ability. This enable us to use QTL (quantitative trait locus) mapping as a powerful tool for localising genes which may contribute to reading and spelling disability.

Abstract

Recent application of nonparametric-linkage analysis to reading disability has implicated a putative quantitative-trait locus (QTL) on the short arm of chromosome 6. In the present study, we use QTL methods to evaluate linkage to the 6p25-21.3 region in a sample of 181 sib pairs from 82 nuclear families that were selected on the basis of a dyslexic proband. We have assessed linkage directly for several quantitative measures that should correlate with different components of the phenotype, rather than using a single composite measure or employing categorical definitions of subtypes. Our measures include the traditional IQ/reading discrepancy score, as well as tests of word recognition, irregular-word reading, and nonword reading. Pointwise analysis by means of sib-pair trait differences suggests the presence, in 6p21.3, of a QTL influencing multiple components of dyslexia, in particular the reading of irregular words (P=.0016) and nonwords (P=.0024). A complementary statistical approach involving estimation of variance components supports these findings (irregular words, P=.007; nonwords, P=.0004). Multipoint analyses place the QTL within the D6S422-D6S291 interval, with a peak around markers D6S276 and D6S105 consistently identified by approaches based on trait differences (irregular words, P=.00035; nonwords, P=.0035) and variance components (irregular words, P=.007; nonwords, P=.0038). Our findings indicate that the QTL affects both phonological and orthographic skills and is not specific to phoneme awareness, as has been previously suggested. Further studies will be necessary to obtain a more precise localization of this QTL, which may lead to the isolation of one of the genes involved in developmental dyslexia.

Abstract

The murine homologue of the human chloride channel gene, CLCN5, defects in which are responsible for Dent disease, has been cloned and characterized. We isolated the entire coding region of mouse Clcn5 cDNA and approximately 45 kb of genomic sequence embracing the gene. To study its transcriptional control, the 5' upstream sequences of the mouse Clcn5 gene were cloned into a luciferase reporter vector. Deletion analysis of 1.5 kb of the 5' flanking sequence defined an active promoter region within 128 bp of the putative transcription start site, which is associated with a TATA motif but lacks a CAAT consensus. Within this sequence, there is a motif with homology to a purine-rich sequence responsible for the kidney-specific promoter activity of the rat CLC-K1 gene, another member of the chloride-channel gene family expressed in kidney. An enhancer element that confers a 10- to 20-fold increase in the promoter activity of the mouse Clcn5 gene was found within the first intron. The organization of the human CLCN5 and mouse Clcn5 gene structures is highly conserved, and the sequence of the murine protein is 98% similar to that of human, with its highest expression seen in the kidney. This study thus provides the first identification of the transcriptional control region of, and the basis for an understanding of the regulatory mechanism that controls, this kidney-specific, chloride-channel gene.

Abstract

The human Xp11.23-p11.22 interval has been implicated in several inherited diseases including Wiskott-Aldrich syndrome; three forms of X-linked hypercalciuric nephrolithiaisis; and the eye disorders retinitis pigmentosa 2, congenital stationary night blindness, and Aland Island eye disease. In constructing YAC contigs spanning Xp11. 23-p11.22, we have previously shown that the region around the synaptophysin (SYP) gene is refractory to cloning in YACs, but highly stable in cosmids. Preliminary analysis of the latter suggested that this might reflect a high density of coding sequences and we therefore undertook the complete sequencing of a SYP-containing cosmid. Sequence data were extensively analyzed using computer programs such as CENSOR (to mask repeats), BLAST (for homology searches), and GRAIL and GENE-ID (to predict exons). This revealed the presence of 29 putative exons, organized into three genes, in addition to the 7 exons of the complete SYP coding region, all mapping within a 44-kb interval. Two genes are novel, one (CACNA1F) showing high homology to alpha1 subunits of calcium channels, the other (LMO6) encoding a product with significant similarity to LIM-domain proteins. RT-PCR and Northern blot studies confirmed that these loci are indeed transcribed. The third locus is the previously described, but not previously localized, A4 differentiation-dependent gene. Given that the intron-exon boundaries predicted by the analysis are consistent with previous information where available, we have been able to suggest the genomic organization of the novel genes with some confidence. The region has an elevated GC content (>53%), and we identified CpG islands associated with the 5' ends of SYP, A4, and LMO6. The order of loci was Xpter-A4-LMO6-SYP-CACNA1F-Xcen, with intergenic distances ranging from approximately 300 bp to approximately 5 kb. The density of transcribed sequences in this area (>80%) is comparable to that found in the highly gene-rich chromosomal band Xq28. Further studies may aid our understanding of the long-range organization surrounding such gene-enriched regions.

Abstract

Mutations of the renal-specific chloride channel (CLCN5) gene, which is located on chromosome Xp11.22, are associated with hypercalciuric nephrolithiasis (kidney stones) in the Northern European and Japanese populations. CLCN5 encodes a 746 amino acid channel (CLC-5) that has approximately 12 transmembrane domains, and heterologous expression of wild-type CLC-5 in Xenopus oocytes has yielded outwardly rectifying chloride currents that were markedly reduced or abolished by these mutations. In order to assess further the structural and functional relationships of this recently cloned chloride channel, additional CLCN5 mutations have been identified in five unrelated families with this disorder. Three of these mutations were missense (G57V, G512R and E527D), one was a nonsense (R648Stop) and one was an insertion (30:H insertion). In addition, two of the mutations (30:H insertion and E527D) were demonstrated to be de novo, and the G57V and E527D mutations were identified in families of Afro-American and Indian origin, respectively. The G57V and 30:H insertion mutations represent the first CLCN5 mutations to be identified in the N-terminus region, and the R648Stop mutation, which has been observed previously in an unrelated family, suggests that this codon may be particularly prone to mutations. Heterologous expression of the mutations resulted in a marked reduction or abolition of the chloride currents, thereby establishing their functional importance. These results help to elucidate further the structure-function relationships of this renal chloride channel.

Abstract

The murine homologues of the loci for McLeod syndrome (XK), Dent's disease (CICN5), and synaptophysin (SYP) have been mapped to the proximal region of the mouse X chromosome and positioned with respect to other conserved loci in this region using a total of 948 progeny from two separate Mus musculus x Mus spretus backcrosses. In the mouse, the order of loci and evolutionary breakpoints (EB) has been established as centromere-(DXWas70, DXHXF34h)-EB-Clcn5-(Syp, DXMit55, DXMit26)-Tfe3-Gata1-EB-Xk-Cybb-telomere. In the proximal region of the human X chromosome short arm, the position of evolutionary breakpoints with respect to key loci has been established as DMD-EB-XK-PFC-EB-GATA1-C1CN5-EB-DXS1272E-ALAS2-E B-DXF34-centromere. These data have enabled us to construct a high-resolution genetic map for the approximately 3-cM interval between DXWas70 and Cybb on the mouse X chromosome, which encompasses 10 loci. This detailed map demonstrates the power of high-resolution genetic mapping in the mouse as a means of determining locus order in a small chromosomal region and of providing an accurate framework for the construction of physical maps.

Abstract

We have constructed two YAC contigs in the Xp11.23-p11.22 interval of the human X chromosome, a region that was previously poorly characterized. One contig, of at least 1.4 Mb, links the pseudogene OATL1 to the genes GATA1, TFE3, and SYP and also contains loci implicated in Wiskott-Aldrich syndrome and synovial sarcoma. A second contig, mapping proximal to the first, is estimated to be over 2.1 Mb and links the hypervariable locus DXS255 to DXS146, and also contains a chloride channel gene that is responsible for hereditary nephrolithiasis. We have used plasmid rescue, inverse PCR, and Alu-PCR to generate 20 novel markers from this region, 1 of which is polymorphic, and have positioned these relative to one another on the basis of YAC analysis. The order of previously known markers within our contigs, Xpter-OATL1-GATA-TFE3-SYP-DXS255146- Xcen, agrees with genomic pulsed-field maps of the region. In addition, we have constructed a rare-cutter restriction map for a 710-kb region of the DXS255-DXS146 contig and have identified three CPG islands. These contigs and new markers will provide a useful resource for more detailed analysis of Xp11.23-p11.22, a region implicated in several genetic diseases.

Abstract

Dent disease, an X-linked familial renal tubular disorder, is a form of Fanconi syndrome associated with proteinuria, hypercalciuria, nephrocalcinosis, kidney stones, and eventual renal failure. We have previously used positional cloning to identify the 3' part of a novel kidney-specific gene (initially termed hClC-K2, but now referred to as CLCN5), which is deleted in patients from one pedigree segregating Dent disease. Mutations that disrupt this gene have been identified in other patients with this disorder. Here we describe the isolation and characterization of the complete open reading frame of the human CLCN5 gene, which is predicted to encode a protein of 746 amino acids, with significant homology to all known members of the ClC family of voltage-gated chloride channels. CLCN5 belongs to a distinct branch of this family, which also includes the recently identified genes CLCN3 and CLCN4. We have shown that the coding region of CLCN5 is organized into 12 exons, spanning 25-30 kb of genomic DNA, and have determined the sequence of each exon-intron boundary. The elucidation of the coding sequence and exon-intron organization of CLCN5 will both expedite the evaluation of structure/function relationships of these ion channels and facilitate the screening of other patients with renal tubular dysfunction for mutations at this locus.

Abstract

A t(X;1)(p11.2;q21.2) has been reported in cases of papillary renal cell tumors arising in males. In this study two cell lines derived from this tumor type have been used to indicate the breakpoint region on the X chromosome. Both cell lines have the translocation in addition to other rearrangements and one is derived from the first female case to be reported with the t(X;1)(p11.2;q21.2). Fluorescence in situ hybridization (FISH) has been used to position YACs belonging to contigs in the Xp11.2 region relative to the breakpoint. When considered together with detailed mapping information from the Xp11.2 region the position of the breakpoint in both cell lines was suggested as follows: Xpter-->Xp11.23-OATL1-GATA1-WAS-TFE3-SY P-t(X;1)-DXS255-CLCN5-DXS146-OATL2- Xp11.22-->Xcen. The breakpoint was determined to lie in an uncloned region between SYP and a YAC called FTDM/1 which extends 1 Mb distal to DXS255. These results are contrary to the conclusion from previous FISH studies that the breakpoint was near the OATL2 locus, but are consistent with, and considerably refine, the position that had been established by molecular analysis.