Michael R. Altherr

A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes

The Huntingtons disease (HD) gene has been mapped in 4p16.3 but has eluded identification. We have used haplotype analysis of linkage disequilibrium to spotlight a small segment of 4p16.3 as the likely location of the defect. A new gene, IT15, isolated using cloned trapped exons from the target area contains a polymorphic trinucleotide repeat that is expanded and unstable on HD chromosomes. A (CAG)n repeat longer than the normal range was observed on HD chromosomes from all 75 disease families examined, comprising a variety of ethnic backgrounds and 4p16.3 haplotypes. The (CAG)n repeat appears to be located within the coding sequence of a predicted approximately 348 kd protein that is widely expressed but unrelated to any known gene. Thus, the HD mutation involves an unstable DNA segment, similar to those described in fragile X syndrome, spino-bulbar muscular atrophy, and myotonic dystrophy, acting in the context of a novel 4p16.3 gene to produce a dominant phenotype.

Structure and expression of the Huntington's disease gene: Evidence against simple inactivation due to an expanded CAG repeat

Huntingtons disease, a neurodegenerative disorder characterized by loss of striatal neurons, is caused by an expanded, unstable trinucleotide repeat in a novel 4p16.3 gene. To lay the foundation for exploring the pathogenic mechanism in HD, we have determined the structure of the disease gene and examined its expression. TheHD locus spans 180 kb and consists of 67 exons ranging in size from 48 bp to 341 bp with an average of 138 bp. Scanning of theHD transcript failed to reveal any additional sequence alterations characteristic of HD chromosomes. A codon loss polymorphism in linkage disequilibrium with the disorder revealed that both normal and HD alleles are represented in the mRNA population in HD heterozygotes, indicating that the defect does not eliminate transcription. The gene is ubiquitously expressed as two alternatively polyadenylated forms displaying different relative abundance in various fetal and adult tissues, suggesting the operation of interacting factors in determining specificity of cell loss. TheHD gene was disrupted in a female carrying a balanced translocation with a breakpoint between exons 40 and 41. The absence of any abnormal phenotype in this individual argues against simple inactivation of the gene as the mechanism by which the expanded trinucleotide repeat causes HD. Taken together, these observations suggest that the dominant HD mutation either confers a new property on the mRNA or, more likely, alters an interaction at the protein level.

Pathogenomic Sequence Analysis of Bacillus cereus and Bacillus thuringiensis Isolates Closely Related to Bacillus anthracis

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are closely related gram-positive, spore-forming bacteria of the B. cereus sensu lato group. While independently derived strains of B. anthracis reveal conspicuous sequence homogeneity, environmental isolates of B. cereus and B. thuringiensis exhibit extensive genetic diversity. Here we report the sequencing and comparative analysis of the genomes of two members of the B. cereus group, B. thuringiensis 97-27 subsp. konkukian serotype H34, isolated from a necrotic human wound, and B. cereus E33L, which was isolated from a swab of a zebra carcass in Namibia. These two strains, when analyzed by amplified fragment length polymorphism within a collection of over 300 of B. cereus, B. thuringiensis, and B. anthracis isolates, appear closely related to B. anthracis. The B. cereus E33L isolate appears to be the nearest relative to B. anthracis identified thus far. Whole-genome sequencing of B. thuringiensis 97-27and B. cereus E33L was undertaken to identify shared and unique genes among these isolates in comparison to the genomes of pathogenic strains B. anthracis Ames and B. cereus G9241 and nonpathogenic strains B. cereus ATCC 10987 and B. cereus ATCC 14579. Comparison of these genomes revealed differences in terms of virulence, metabolic competence, structural components, and regulatory mechanisms.

The DNA sequence and biology of human chromosome 19

Chromosome 19 has the highest gene density of all human chromosomes, more than double the genome-wide average. The large clustered gene families, corresponding high G + C content, CpG islands and density of repetitive DNA indicate a chromosome rich in biological and evolutionary significance. Here we describe 55.8 million base pairs of highly accurate finished sequence representing 99.9% of the euchromatin portion of the chromosome. Manual curation of gene loci reveals 1,461 protein-coding genes and 321 pseudogenes. Among these are genes directly implicated in mendelian disorders, including familial hypercholesterolaemia and insulin-resistant diabetes. Nearly one-quarter of these genes belong to tandemly arranged families, encompassing more than 25% of the chromosome. Comparative analyses show a fascinating picture of conservation and divergence, revealing large blocks of gene orthology with rodents, scattered regions with more recent gene family expansions and deletions, and segments of coding and non-coding conservation with the distant fish species Takifugu.

The DNA rearrangement associated with facioscapulohumeral muscular dystrophy involves a heterochromatin-associated repetitive element: Implications for a role of chromatin structure in the pathogenesis of the disease

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant form of muscular dystrophy. The FSHD locus has been linked to the most distal genetic markers on the long arm of chromosome 4. Recently, a probe was identified that detects anEcoRI fragment length polymorphism which segregates with the disease in most FSHD families. Within theEcoRI fragment lies a tandem array of 3.2 kb repeats. In several familial cases and four independent sporadic FSHD mutations, the variation in size of theEcoRI fragment was due to a decrease in copy number of the 3.2 kb repeats. To gain further insight into the relationship between the tandem array and FSHD, a single 3.2 kb repeat unit was characterized. Fluorescencein situ hybridization (FISH) demonstrates that the 3.2 kb repeat cross-hybridizes to several regions of heterochromatin in the human genome. In addition, DNA sequence analysis of the repeat reveals a region which is highly homologous to a previously identified family of heterochromatic repeats, LSau. FISH on interphase chromosomes demonstrates that the tandem array of 3.2 kb repeats lies within 215 kb of the 4q telomere. Together, these results suggest that the tandem array of 3.2 kb repeats, tightly linked to the FSHD locus, is contained in heterochromatin adjacent to the telomere. In addition, they are consistent with the hypothesis that the gene responsible for FSHD may be subjected to position effect variegation because of its proximity to telomeric heterochromatin.

A gene encoding a fibroblast growth factor receptor isolated from the Huntington disease gene region of human chromosome 4

The gene responsible for Huntington disease (HD), an autosomal dominant neurodegenerative disorder, is located near the terminus of the short arm of chromosome 4. Detailed genetic linkage and physical mapping studies have defined a region of approximately 2.5 million basepairs where the disease gene is likely to be located. Efforts to identify the disease gene are now focused on the identification and characterization of expressed genes in this region. Nucleotide sequence analysis of a cDNA clone derived from the HD gene region has revealed that it encodes a member of the fibroblast growth factor subfamily of tyrosine kinase receptors, some members of which are known to be involved in the differentiation and survival of certain cell types within the central nervous system. Histochemical analysis using in situ hybridization revealed its expression in many areas of the brain, among them being the caudate and putamen. The nature of this gene, FGFR3, and its map location make it a possible candidate for the HD gene.

Bone morphogenetic protein: chromosomal localization of human genes for BMP1, BMP2A, and BMP3.

Bone morphogenetic protein (BMP) induces endochondral bone formation in vivo. The human genes have been cloned for a group of proteins containing BMP activity (BMP1, BMP2A, and BMP3). Two of the proteins are members of the transforming growth factor-beta supergene family (BMP2A and BMP3), while BMP1 is a novel regulatory protein. Using somatic cell hybrid lines, cDNA probes were used to map BMP1 to chromosome 8, BMP2A to chromosome 20, and BMP3 to the p14-q21 region of chromosome 4. This analysis reveals that the BMP2A and BMP3 genes map to conserved regions between mouse and human, while the BMP1 gene does not. The locations of the BMP genes were found to overlap with the loci for several disorders of cartilage and bone formation.

The complete sequence of human chromosome 5

Chromosome 5 is one of the largest human chromosomes and contains numerous intrachromosomal duplications, yet it has one of the lowest gene densities. This is partially explained by numerous gene-poor regions that display a remarkable degree of noncoding conservation with non-mammalian vertebrates, suggesting that they are functionally constrained. In total, we compiled 177.7 million base pairs of highly accurate finished sequence containing 923 manually curated protein-coding genes including the protocadherin and interleukin gene families. We also completely sequenced versions of the large chromosome-5-specific internal duplications. These duplications are very recent evolutionary events and probably have a mechanistic role in human physiological variation, as deletions in these regions are the cause of debilitating disorders including spinal muscular atrophy.

Genetic and physical maps of human chromosome 4 based on dinucleotide repeats

Characterization of inherited variations within tandem arrays of dinucleotide repeats has substantially advanced the construction of genetic maps using linkage approaches over the last several years. Using a backbone of 10 newly identified microsatellite repeats on human chromosome 4 and 6 previously identified short tandem repeat element polymorphisms, we have constructed several genetic maps and a physical map of human chromosome 4. The genetic and physical maps are in complete concordance with each other. The genetic maps include a 15-locus microsatellite-based linkage map, a framework map of high support incorporating a total of 39 independent loci, a 25-locus high-heterozygosity, easily used index map, and a gene-based comprehensive map that provides the best genetic location for 35 genes mapped to chromosome 4. The 16 microsatellite markers are each localized to one of nine regions of chromosome 4, delineated by a panel of somatic cell hybrids. These results demonstrate the utility of PCR-based repeat elements for the construction of genetic maps and provide a valuable resource for continued high-resolution mapping of chromosome 4 and of genetic disorders to this chromosome.

Mapping of cosmid clones in Huntington's disease region of chromosome 4

Huntingtons disease (HD) is tightly linked to genetic markers in 4p16.3. We have used a regional somatic cell hybrid mapping panel to isolate and map 25 cosmids to the proximal portion of 4p16.3 and 17 cosmids to the distal portion. The latter were positioned by long-range restriction mapping relative to previously mapped markers. One cosmid, L6 (D4S166), spans the critical breakpoint in the mapping panel that distinguishes proximal and distal 4p16.3. Four of the cosmids mapped distal toD4S90, the previous terminal marker on 4p, and stretched to within 75 kb of the telomere. Several of the cosmids that mapped between L6 andD4S90 were clustered near a number of previously isolated clones in a region with many NotI sites. Cosmid E4 (D4S168) was localized immediately proximal to the one remaining gap in the long-range restriction map of distal 4p16.3. Although pulsed field gel mapping with E4 failed to link the two segments of the map, the intervening gap was excluded as a potential site for theHD gene by genetic analysis.