Biomedical subjects

Robert Lyle

Publications and source records attributed to Robert Lyle.

16 recordsLinked to original sources

PubMed2006

Frequency of genomic rearrangements involving the SHFM3 locus at chromosome 10q24 in syndromic and non-syndromic split-hand/foot malformation.

Split-hand/foot malformation (SHFM), or ectrodactyly, is characterized by underdeveloped or absent central digital rays, clefts of the hands and feet, and variable syndactyly of the remaining digits. SHFM occurs as both an isolated finding and a component of many syndromes. SHFM is a heterogeneous condition caused by multiple loci, including SHFM1 (chromosome region 7q21-q22), SHFM2 (Xq26), SHFM3 (10q24), SHFM4 (3q27), and SHFM5 (2q31). Mutations in TP63 at the SHFM4 locus are known to underlie both syndromic and non-syndromic forms SHFM, but the causes of most non-syndromic SHFM cases remain unknown. The recent identification of submicroscopic tandem chromosome duplications affecting the SHFM3 locus in seven families with non-syndromic SHFM has helped to further unravel the molecular basis of this malformation. In our ongoing studies of the SHFM3 locus in 44 additional cases of syndromic and non-syndromic SHFM, we have identified similar chromosome rearrangements in eight additional cases (18%), using pulsed-field gel electrophoresis (PFGE). We have also utilized real-time quantitative PCR (qPCR) to test for the duplications. Seven of the cases with rearrangements were non-syndromic. The current findings bring the total of SHFM3-associated cases with chromosome rearrangements to 15, which constitute 29% (15 of 51) of the cases screened to date. This includes 9 of 9 cases (100%) with known linkage to the SHFM3 locus, all of whom have non-syndromic SHFM, and 6 of 42 additional cases (14%), four of whom have non-syndromic SHFM. Thus, SHFM3 abnormalities underlie a substantial proportion of SHFM cases and appear to be a more frequent cause of non-syndromic SHFM than mutations in TP63.

Abnormalities, Multiple↗

PubMed2006

Split-hand/split-foot malformation 3 (SHFM3) at 10q24, development of rapid diagnostic methods and gene expression from the region.

Split-hand/split-foot malformation (SHFM, also called ectrodactyly) is a clinically variable and genetically heterogeneous group of limb malformations. Several SHFM loci have been mapped, including SHFM1 (7q21), SHFM2 (Xq26), SHFM3 (10q24), SHFM4 (3q27) and SHFM5 (2q31). To date, mutations in a gene (TP63) have only been identified for SHFM4. SHFM3 has been shown by pulsed-field gel electrophoresis to be caused by an approximately 500 kb DNA rearrangement at 10q24. This region contains a number of candidate genes for SHFM3, though which gene(s) is (are) involved in the pathogenesis of SHFM3 is not known. Our aim in this study was to improve the diagnosis of SHFM3, and to begin to understand which genes are involved in SHFM3. Here we show, using two different techniques, FISH and quantitative PCR that SHFM3 is caused by a minimal 325 kb duplication containing only two genes (BTRC and POLL). The data presented provide improved methods for diagnosis and begin to elucidate the pathogenic mechanism of SHFM3. Expression analysis of 13 candidate genes within and flanking the duplicated region shows that BTRC (present in three copies) and SUFU (present in two copies) are overexpressed in SHFM3 patients compared to controls. Our data suggest that SHFM3 may be caused by overexpression of BTRC and SUFU, both of which are involved in beta-catenin signalling.

Chromosomes, Human, Pair 10↗

PubMed2006

Submicroscopic deletion in patients with Williams-Beuren syndrome influences expression levels of the nonhemizygous flanking genes.

Genomic imbalance is a common cause of phenotypic abnormalities. We measured the relative expression level of genes that map within the microdeletion that causes Williams-Beuren syndrome and within its flanking regions. We found, unexpectedly, that not only hemizygous genes but also normal-copy neighboring genes show decreased relative levels of expression. Our results suggest that not only the aneuploid genes but also the flanking genes that map several megabases away from a genomic rearrangement should be considered possible contributors to the phenotypic variation in genomic disorders.

Cell Line, Transformed↗

PubMed2005

Genome-wide associations of gene expression variation in humans.

The exploration of quantitative variation in human populations has become one of the major priorities for medical genetics. The successful identification of variants that contribute to complex traits is highly dependent on reliable assays and genetic maps. We have performed a genome-wide quantitative trait analysis of 630 genes in 60 unrelated Utah residents with ancestry from Northern and Western Europe using the publicly available phase I data of the International HapMap project. The genes are located in regions of the human genome with elevated functional annotation and disease interest including the ENCODE regions spanning 1% of the genome, Chromosome 21 and Chromosome 20q12-13.2. We apply three different methods of multiple test correction, including Bonferroni, false discovery rate, and permutations. For the 374 expressed genes, we find many regions with statistically significant association of single nucleotide polymorphisms (SNPs) with expression variation in lymphoblastoid cell lines after correcting for multiple tests. Based on our analyses, the signal proximal (cis-) to the genes of interest is more abundant and more stable than distal and trans across statistical methodologies. Our results suggest that regulatory polymorphism is widespread in the human genome and show that the 5-kb (phase I) HapMap has sufficient density to enable linkage disequilibrium mapping in humans. Such studies will significantly enhance our ability to annotate the non-coding part of the genome and interpret functional variation. In addition, we demonstrate that the HapMap cell lines themselves may serve as a useful resource for quantitative measurements at the cellular level.

Chromosome Mapping↗

PubMed2005

Gene expression variation and expression quantitative trait mapping of human chromosome 21 genes.

Inter-individual differences in gene expression are likely to account for an important fraction of phenotypic differences, including susceptibility to common disorders. Recent studies have shown extensive variation in gene expression levels in humans and other organisms, and that a fraction of this variation is under genetic control. We investigated the patterns of gene expression variation in a 25 Mb region of human chromosome 21, which has been associated with many Down syndrome (DS) phenotypes. Taqman real-time PCR was used to measure expression variation of 41 genes in lymphoblastoid cells of 40 unrelated individuals. For 25 genes found to be differentially expressed, additional analysis was performed in 10 CEPH families to determine heritabilities and map loci harboring regulatory variation. Seventy-six percent of the differentially expressed genes had significant heritabilities, and genomewide linkage analysis led to the identification of significant eQTLs for nine genes. Most eQTLs were in trans, with the best result (P=7.46 x 10(-8)) obtained for TMEM1 on chromosome 12q24.33. A cis-eQTL identified for CCT8 was validated by performing an association study in 60 individuals from the HapMap project. SNP rs965951 located within CCT8 was found to be significantly associated with its expression levels (P=2.5 x 10(-5)) confirming cis-regulatory variation. The results of our study provide a representative view of expression variation of chromosome 21 genes, identify loci involved in their regulation and suggest that genes, for which expression differences are significantly larger than 1.5-fold in control samples, are unlikely to be involved in DS-phenotypes present in all affected individuals.

Chromosome Mapping↗

PubMed2005

Meiotic and epigenetic defects in Dnmt3L-knockout mouse spermatogenesis.

The production of mature germ cells capable of generating totipotent zygotes is a highly specialized and sexually dimorphic process. The transition from diploid primordial germ cell to haploid spermatozoa requires genome-wide reprogramming of DNA methylation, stage- and testis-specific gene expression, mitotic and meiotic division, and the histone-protamine transition, all requiring unique epigenetic control. Dnmt3L, a DNA methyltransferase regulator, is expressed during gametogenesis, and its deletion results in sterility. We found that during spermatogenesis, Dnmt3L contributes to the acquisition of DNA methylation at paternally imprinted regions, unique nonpericentric heterochromatic sequences, and interspersed repeats, including autonomous transposable elements. We observed retrotransposition of an LTR-ERV1 element in the DNA from Dnmt3L-/- germ cells, presumably as a result of hypomethylation. Later in development, in Dnmt3L-/- meiotic spermatocytes, we detected abnormalities in the status of biochemical markers of heterochromatin, implying aberrant chromatin packaging. Coincidentally, homologous chromosomes fail to align and form synaptonemal complexes, spermatogenesis arrests, and spermatocytes are lost by apoptosis and sloughing. Because Dnmt3L expression is restricted to gonocytes, the presence of defects in later stages reveals a mechanism whereby early genome reprogramming is linked inextricably to changes in chromatin structure required for completion of spermatogenesis.

Animals↗

PubMed2005

Different mechanisms preclude mutant CLDN14 proteins from forming tight junctions in vitro.

Mutations in claudin 14 (CLDN14) cause nonsyndromic DFNB29 deafness in humans. The analysis of a murine model indicated that this phenotype is associated with degeneration of hair cells, possibly due to cation overload. However, the mechanism linking these alterations to CLDN14 mutations is unknown. To investigate this mechanism, we compared the ability of wild-type and missense mutant CLDN14 to form tight junctions. Ectopic expression in L mouse fibroblasts (LM cells) of wild-type CLDN14 protein induced the formation of tight junctions, while both the c.254T>A (p.V85D) mutant, previously identified in a Pakistani family, and the c.301 G>A (p.G101R) mutant, identified in this study through the screen of 183 Spanish and Greek patients affected with sporadic nonsyndromic deafness, failed to form such junctions. However, the two mutant proteins differed in their ability to localize at the plasma membrane. We further identified hitherto undescribed exons of CLDN14 that are utilized in alternative spliced transcripts. We demonstrated that different mutations of CLDN14 impaired by different mechanisms the ability of the protein to form tight junctions. Our results indicate that the ability of CLDN14 to be recruited to these junctions is crucial for the hearing process.

Alternative Splicing↗

PubMed2004

Chromosome 21 and down syndrome: from genomics to pathophysiology.

The sequence of chromosome 21 was a turning point for the understanding of Down syndrome. Comparative genomics is beginning to identify the functional components of the chromosome and that in turn will set the stage for the functional characterization of the sequences. Animal models combined with genome-wide analytical methods have proved indispensable for unravelling the mysteries of gene dosage imbalance.

Animals↗

PubMed2004

Gene expression from the aneuploid chromosome in a trisomy mouse model of down syndrome.

Trisomy 21 is the prototype of human aneuploidies. Since its discovery in 1959, the hypothesis has been that overexpression of the approximately 230 human chromosome 21 (Hsa21) genes result in the complex phenotype. However, the level of overexpression of Hsa21 genes in trisomic individuals is presently unknown. We have used Taqman real-time quantitative PCR to accurately measure expression of the mouse orthologs of Hsa21 in the partial trisomy mouse model Ts65Dn. The transcript levels of 78 protein-coding genes present in three copies in Ts65Dn and 21 control genes were compared between Ts65Dn and normal mouse littermates. The mean overexpression of the aneuploid genes is very close to the expected 1.5-fold in all six tissues studied. However, only approximately a third of the genes (37%) are expressed at the theoretical value of 1.5-fold. On average, 45% of the genes are expressed at significantly lower than 1.5-fold, and 9% are not significantly different from 1.0. Interestingly, 18% of the aneuploid genes were expressed at levels significantly greater than 1.5-fold. These data provide candidate genes that might be involved in the phenotypes of Down syndrome, and reveal a complex regulation of gene expression that is not only related to gene copy number.

Aneuploidy↗

PubMed2003

The TPTE gene family: cellular expression, subcellular localization and alternative splicing.

The human TPTE (Transmembrane Phosphatase with TEnsin homology) gene family encodes a PTEN-related tyrosine phosphatase with four potential transmembrane domains. Chromosomal mapping revealed multiple copies of the TPTE gene on chromosomes 13, 15, 21, 22 and Y. Human chromosomes 13 and 21 copies encode two functional proteins, TPIP (TPTE and PTEN homologous Inositol lipid Phosphatase) and TPTE, respectively, whereas only one copy of the gene exists in the mouse genome. In the present study, we show that TPTE and TPIP proteins are expressed in secondary spermatocytes and/or prespermatids. In addition, we report the existence of several novel alternatively spliced isoforms of these two proteins with variable number of transmembrane domains. The latter has no influence on the subcellular localization of these different peptides as shown by co-immunofluorescence experiments. Finally, we identify another expressed TPTE copy, mapping to human chromosome 22, whose transcription appears to be under the control of the LTR of human endogenous retrovirus RTVL-H3.

Alternative Splicing↗

PubMed2003

A genomic rearrangement resulting in a tandem duplication is associated with split hand-split foot malformation 3 (SHFM3) at 10q24.

Split hand-split foot malformation (SHFM) is characterized by hypoplasia/aplasia of the central digits with fusion of the remaining digits. SHFM is usually an autosomal dominant condition and at least five loci have been identified in humans. Mutation analysis of the DACTYLIN gene, suspected to be responsible for SHFM3 in chromosome 10q24, was conducted in seven SHFM patients. We screened the coding region of DACTYLIN by single-strand conformation polymorphism and sequencing, and found no point mutations. However, Southern, pulsed field gel electrophoresis and dosage analyses demonstrated a complex rearrangement associated with a approximately 0.5 Mb tandem duplication in all the patients. The distal and proximal breakpoints were within an 80 and 130 kb region, respectively. This duplicated region contained a disrupted extra copy of the DACTYLIN gene and the entire LBX1 and beta-TRCP genes, known to be involved in limb development. The possible role of these genes in the SHFM3 phenotype is discussed.

Abnormalities, Multiple↗

PubMed2003

Comparison of mouse and human genomes followed by experimental verification yields an estimated 1,019 additional genes.

A primary motivation for sequencing the mouse genome was to accelerate the discovery of mammalian genes by using sequence conservation between mouse and human to identify coding exons. Achieving this goal proved challenging because of the large proportion of the mouse and human genomes that is apparently conserved but apparently does not code for protein. We developed a two-stage procedure that exploits the mouse and human genome sequences to produce a set of genes with a much higher rate of experimental verification than previously reported prediction methods. RT-PCR amplification and direct sequencing applied to an initial sample of mouse predictions that do not overlap previously known genes verified the regions flanking one intron in 139 predictions, with verification rates reaching 76%. On average, the confirmed predictions show more restricted expression patterns than the mouse orthologs of known human genes, and two-thirds lack homologs in fish genomes, demonstrating the sensitivity of this dual-genome approach to hard-to-find genes. We verified 112 previously unknown homologs of known proteins, including two homeobox proteins relevant to developmental biology, an aquaporin, and a homolog of dystrophin. We estimate that transcription and splicing can be verified for >1,000 gene predictions identified by this method that do not overlap known genes. This is likely to constitute a significant fraction of the previously unknown, multiexon mammalian genes.

Amino Acid Sequence↗

PubMed2002

Human chromosome 21 gene expression atlas in the mouse.

Genome-wide expression analyses have a crucial role in functional genomics. High resolution methods, such as RNA in situ hybridization provide an accurate description of the spatiotemporal distribution of transcripts as well as a three-dimensional 'in vivo' gene expression overview. We set out to analyse systematically the expression patterns of genes from an entire chromosome. We chose human chromosome 21 because of the medical relevance of trisomy 21 (Down's syndrome). Here we show the expression analysis of all identifiable murine orthologues of human chromosome 21 genes (161 out of 178 confirmed human genes) by RNA in situ hybridization on whole mounts and tissue sections, and by polymerase chain reaction with reverse transcription on adult tissues. We observed patterned expression in several tissues including those affected in trisomy 21 phenotypes (that is, central nervous system, heart, gastrointestinal tract, and limbs). Furthermore, statistical analysis suggests the presence of some regions of the chromosome with genes showing either lack of expression or, to a lesser extent, co-expression in specific tissues. This high resolution expression 'atlas' of an entire human chromosome is an important step towards the understanding of gene function and of the pathogenetic mechanisms in Down's syndrome.

Animals↗

PubMed2002

Numerous potentially functional but non-genic conserved sequences on human chromosome 21.

The use of comparative genomics to infer genome function relies on the understanding of how different components of the genome change over evolutionary time. The aim of such comparative analysis is to identify conserved, functionally transcribed sequences such as protein-coding genes and non-coding RNA genes, and other functional sequences such as regulatory regions, as well as other genomic features. Here, we have compared the entire human chromosome 21 with syntenic regions of the mouse genome, and have identified a large number of conserved blocks of unknown function. Although previous studies have made similar observations, it is unknown whether these conserved sequences are genes or not. Here we present an extensive experimental and computational analysis of human chromosome 21 in an effort to assign function to sequences conserved between human chromosome 21 (ref. 8) and the syntenic mouse regions. Our data support the presence of a large number of potentially functional non-genic sequences, probably regulatory and structural. The integration of the properties of the conserved components of human chromosome 21 to the rapidly accumulating functional data for this chromosome will improve considerably our understanding of the role of sequence conservation in mammalian genomes.

Animals↗

PubMed2002

Nineteen additional unpredicted transcripts from human chromosome 21.

The identification of all human chromosome 21 (HC21) genes is a necessary step in understanding the molecular pathogenesis of trisomy 21 (Down syndrome). The first analysis of the sequence of 21q included 127 previously characterized genes and predicted an additional 98 novel anonymous genes. Recently we evaluated the quality of this annotation by characterizing a set of HC21 open reading frames (C21orfs) identified by mapping spliced expressed sequence tags (ESTs) and predicted genes (PREDs), identified only in silico. This study underscored the limitations of in silico-only gene prediction, as many PREDs were incorrectly predicted. To refine the HC21 annotation, we have developed a reliable algorithm to extract and stringently map sequences that contain bona fide 3' transcript ends to the genome. We then created a specific 21q graphical display allowing an integrated view of the data that incorporates new ESTs as well as features such as CpG islands, repeats, and gene predictions. Using these tools we identified 27 new putative genes. To validate these, we sequenced previously cloned cDNAs and carried out RT-PCR, 5'- and 3'-RACE procedures, and comparative mapping. These approaches substantiated 19 new transcripts, thus increasing the HC21 gene count by 9.5%. These transcripts were likely not previously identified because they are small and encode small proteins. We also identified four transcriptional units that are spliced but contain no obvious open reading frame. The HC21 data presented here further emphasize that current gene prediction algorithms miss a substantial number of transcripts that nevertheless can be identified using a combination of experimental approaches and multiple refined algorithms.

Chromosomes, Human, Pair 21↗

PubMed2002

Chromosome 21: a small land of fascinating disorders with unknown pathophysiology.

In the year 2000 we celebrated the sequencing of the entire long arm of human chromosome 21. This achievement now provides unprecedented opportunities to understand the molecular pathophysiology of trisomy 21, elucidate the mechanisms of all monogenic disorders of chromosome 21, and discover genes and functional sequence variations that predispose to common complex disorders. All of that requires the functional analysis of gene products in model organisms, and the determination of the sequence variation of this chromosome.

Chromosome Aberrations↗