Showing papers by "Bruce W. Birren published in 1999"

An azoospermic man with a de novo point mutation in the Y-chromosomal gene USP9Y

Abstract: In humans, deletion of any one of three Y-chromosomal regions—AZFa, AZFb or AZFc—disrupts spermatogenesis, causing infertility in otherwise healthy men1,2,3,4,5. Although candidate genes have been identified in all three regions3,6,7,8, no case of spermatogenic failure has been traced to a point mutation in a Y-linked gene, or to a deletion of a single Y-linked gene. We sequenced the AZFa region of the Y chromosome and identified two functional genes previously described: USP9Y (also known as DFFRY) and DBY (refs 7,8). Screening of the two genes in 576 infertile and 96 fertile men revealed several sequence variants, most of which appear to be heritable and of little functional consequence. We found one de novo mutation in USP9Y: a 4-bp deletion in a splice-donor site, causing an exon to be skipped and protein truncation. This mutation was present in a man with nonobstructive azoospermia (that is, no sperm was detected in semen), but absent in his fertile brother, suggesting that the USP9Y mutation caused spermatogenic failure. We also identified a single-gene deletion associated with spermatogenic failure, again involving USP9Y, by re-analysing a published study.

Campomelic Dysplasia Translocation Breakpoints Are Scattered over 1 Mb Proximal to SOX9: Evidence for an Extended Control Region

Abstract: Campomelic dysplasia (CD), a skeletal malformation syndrome with or without XY sex reversal, is usually caused by mutations within the SOX9 gene on distal 17q. Several CD translocation and inversion cases have been described with breakpoints outside the coding region, mapping to locations >130 kb proximal to SOX9. Such cases are generally less severely affected than cases with SOX9 coding-region mutations, as is borne out by three new translocation cases that we present. We have cloned the region extending 1.2 Mb upstream of the SOX9 gene in overlapping bacterial-artificial-chromosome and P1-artificial-chromosome clones and have established a restriction map with rare-cutter enzymes. With sequence-tagged-site-content mapping in somatic-cell hybrids, as well as with FISH, we have precisely mapped the breakpoints of the three new and of three previously described CD cases. The six CD breakpoints map to an interval that is 140-950 kb proximal to the SOX9 gene. With exon trapping, we could isolate five potential exons from the YAC 946E12 that spans the region, four of which could be placed in the contig in the vicinity of the breakpoints. They show the same transcriptional orientation, but only two have an open reading frame (ORF). We failed to detect expression of these fragments in several human and mouse cDNA libraries, as well as on northern blots. Genomic sequence totaling 1,063 kb from the SOX9 5'-flanking region was determined and was analyzed by the gene-prediction program GENSCAN and by a search of dbEST and other databases. No genes or transcripts could be identified. Together, these data suggest that the chromosomal rearrangements most likely remove one or more cis-regulatory elements from an extended SOX9 control region.

A novel member of the F-box/WD40 gene family, encoding dactylin, is disrupted in the mouse dactylaplasia mutant.

Abstract: Early outgrowth of the vertebrate embryonic limb requires signalling by the apical ectodermal ridge (AER) to the progress zone (PZ), which in response proliferates and lays down the pattern of the presumptive limb in a proximal to distal progression1. Signals from the PZ maintain the AER until the anlagen for the distal phalanges have been formed2. The semidominant mouse mutant dactylaplasia (Dac) disrupts the maintenance of the AER, leading to truncation of distal structures of the developing footplate, or autopod3,4,5. Adult Dac homozygotes thus lack hands and feet except for malformed single digits, whereas heterozygotes lack phalanges of the three middle digits. Dac resembles the human autosomal dominant split hand/foot malformation (SHFM) diseases. One of these, SHFM3, maps to chromosome 10q24 (Refs 6,7), which is syntenic to the Dac region on chromosome 19, and may disrupt the orthologue of Dac. We report here the positional cloning of Dac and show that it belongs to the F-box/WD40 gene family, which encodes adapters that target specific proteins for destruction by presenting them to the ubiquitination machinery8. In conjuction with recent biochemical studies9,10,11,12, this report demonstrates the importance of this gene family in vertebrate embryonic development.

A YAC-based physical map of the mouse genome.

Abstract: A physical map of the mouse genome is an essential tool for both positional cloning and genomic sequencing in this key model system for biomedical research. Indeed, the construction of a mouse physical map with markers spaced at an average interval of 300 kb is one of the stated goals of the Human Genome Project. Here we report the results of a project at the Whitehead Institute/MIT Center for Genome Research to construct such a physical map of the mouse. We built the map by screening sequenced-tagged sites (STSs) against a large-insert yeast artificial chromosome (YAC) library and then integrating the STS-content information with a dense genetic map. The integrated map shows the location of 9,787 loci, providing landmarks with an average spacing of approximately 300 kb and affording YAC coverage of approximately 92% of the mouse genome. We also report the results of a project at the MRC UK Mouse Genome Centre targeted at chromosome X. The project produced a YAC-based map containing 619 loci (with 121 loci in common with the Whitehead map and 498 additional loci), providing especially dense coverage of this sex chromosome. The YAC-based physical map directly facilitates positional cloning of mouse mutations by providing ready access to most of the genome. More generally, use of this map in addition to a newly constructed radiation hybrid (RH) map provides a comprehensive framework for mouse genomic studies.

Human genome anatomy: BACs integrating the genetic and cytogenetic maps for bridging genome and biomedicine.

Abstract: Translating problems of human disease into the language of the human genome requires a unified resource that bridges DNA sequence through chromosome bands. Such a resource must link the three types of linear arrays that represent the human genome: database arrays (genetic and physical maps and ultimately DNA sequence), chromosome bands visible in single cells, and ordered clone arrays. Genome maps have been previously either STS-based, with marker order obtained using a combination of STS-content of large insert yeast artificial chromosome (YAC) clones, radiation hybrid (RH) mapping, and genetic mapping (Hudson et al. 1995; Deloukas et al. 1998), or BAC-based, with order obtained at 2–6 Mb through high resolution mapping by fluorescence in situ hybridization (FISH) with respect to human chromosome landmarks (Korenberg et al. 1992). During the course of these efforts, a strategy to integrate these maps was established. BACs are well suited for a permanent FISH-mapped and integrated clone resource in that they represent a stable and easily manipulated form of cloned DNA and produce bright, well defined signals on metaphase and interphase chromosome preparations (Korenberg and Chen 1995). We now report the construction of a genome-wide array of bacterial artificial chromosomes (BACs) that is integrated with the cytogenetic, genetic, and STS maps and characterized for homology to the remainder of the human genome by FISH.

Mouse Molecular Cytogenetic Resource: 157 BACs Link the Chromosomal and Genetic Maps

Abstract: The achievements of the human and mouse genome projects provide increasing numbers of genes and phenotype-associated mutations (DeBry and Seldin 1996; Schuler et al. 1996). Linking the normal and mutant forms of the genes with their functions remains one of biology’s great challenges. To do this the DNA sequence must be defined ultimately with respect to the chromosomal location of the genes, a task that is facilitated by the many disease models available for study in the mouse. Although the genetic map of the mouse provides a powerful tool to link mutations and diseases with the genes (Dietrich et al. 1996), cytogenetic analysis is essential to define genes associated with chromosomal rearrangements. Molecular cytogenetic analysis, which has been exploited widely for localizing cloned genes, genomic sequences, and disease-related chromosomal rearrangements in the human, has not proven as useful in murine genetics. Whereas human genes and breakpoints can be rapidly and accurately mapped cytogenetically (X.-N. Chen, S. Mitchell, Z.-G. Sun, D. Noya, S. Ma, G.S. Sekhon, K. Thompson, W.T. Hsu, P. Wong, N. Wang et al., unpubl.), the common morphology and less distinct landmarks of mouse chromosomes have combined with the lack of molecular cytogenetic markers to hamper similar analyses in the mouse. To bridge the gap between molecular and cytological methods, previous studies have developed a number of reagents for mouse chromosome identification. These include whole-chromosome reagents (Breneman et al. 1993; Weier et al. 1994; Rabbitts et al. 1995; Liyanage et al. 1996; Xiao et al. 1996) and cosmid or P1 clones used with repeat-sequence-based alternatives to dye-based banding techniques (Boyle et al. 1990). Additionally, mouse yeast artificial chromosome (YAC) and P1 clones have been used to generate chromosome-specific probes through FISH and DAPI counterstaining (Mongelard et al. 1996; Shi et al. 1997). This report combines the products of the mouse genome project with molecular cytogenetic techniques to generate a new set of reagents, bacterial artificial chromosome (BAC) clones with a subset linked to centromeric and telomeric genetic markers. These serve to identify mouse chromosomes 1–19 and X, to fluorescently tag the ends of their genetic maps, and to define multiple band landmarks on each chromosome with unique BACs. The stability of BAC clones, the ease of BAC DNA purification, and the strong fluorescence in situ hybridization (FISH) signals resulting from the large insert size, make them superior molecular cytogenetic reagents (Korenberg and Chen 1995; X-.N. Chen, S. Mitchell, Z.-G. Sun, D. Noya, S. Ma, G.S. Sekhon, K. Thompson, W.T. Hsu, P. Wong, N. Wang et al., unpubl.). By including BAC clones that span each chromosome and contain markers from the ends of the genetic maps, the collection serves to integrate cytogenetic, genetic, and physical maps. The availability of such probes extends mouse analysis to interphase, and facilitates the rapid definition of chromosomal rearrangements and their associated candidate genes.

Perfect Conserved Linkage Across the Entire Mouse Chromosome 10 Region Homologous to Human Chromosome 21

Abstract: The distal end of human Chromosome (HSA) 21 from PDXK to the telomere shows perfect conserved linkage with mouse Chromosome (MMU) 10. This region is bounded on the proximal side by a segment of homology to HSA22q11.2, and on the distal side by a region of homology with HSA19p13.1. A high-resolution PAC-based physical map is described that spans 2.8 Mb, including the entire 2.1 Mb from Pdxk to Prmt2 corresponding to HSA21. Thirty-four expressed sequences are mapped, three of which were not mapped previously in any species and nine more that are mapped in mouse for the first time. These genes confirm and extend the conserved linkage between MMU10 and HSA21. The ordered PACs and dense STS map provide a clone resource for biological experiments, for rapid and accurate mapping, and for genomic sequencing. The new genes identified here may be involved in Down syndrome (DS) or in several genetic diseases that map to this conserved region of HSA21.