Paul C. Watkins

Linkage of a gene causing familial amyotrophic lateral sclerosis to chromosome 21 and evidence of genetic-locus heterogeneity

BACKGROUND Amyotrophic lateral sclerosis is a progressive neurologic disorder that commonly results in paralysis and death. Despite more than a century of research, no cause of, cure for, or means of preventing this disorder has been found. In a minority of cases, it is familial and inherited as an autosomal dominant trait with age-dependent penetrance. In contrast to the sporadic form of amyotrophic lateral sclerosis, the familial form provides the opportunity to use molecular genetic techniques to localize an inherited defect. Furthermore, such studies have the potential to discover the basic molecular defect causing motor-neuron degeneration. METHODS AND RESULTS We evaluated 23 families with familial amyotrophic lateral sclerosis for linkage of the gene causing this disease to four DNA markers on the long arm of chromosome 21. Multipoint linkage analyses demonstrated linkage between the gene and these markers. The maximum lod score--5.03--was obtained 10 centimorgans distal (telomeric) to the DNA marker D21S58. There was a significant probability (P less than 0.0001) of genetic-locus heterogeneity in the families. CONCLUSIONS The localization of a gene causing familial amyotrophic lateral sclerosis provides a means of isolating this gene and studying its function. Insight gained from understanding the function of this gene may be applicable to the design of rational therapy for both the familial and sporadic forms of the disease.

Regional assignment of the erythropoietin gene to human chromosome region 7pter→q22

The chromosomal location of the human gene for erythropoietin (EPO) was determined by Southern blot hybridization analysis of a panel of human-mouse somatic hybrid cell DNAs. DNAs from cell hybrids containing reduced numbers of human chromosomes were treated with the restriction enzyme PstI and screened with a cloned human EPO cDNA probe. EPO is assigned to human chromosome 7 based on the complete cosegregation of EPO with this chromosome in all 45 cell hybrids tested. A cell hybrid containing a translocated derivative of chromosome 7 localizes EPO to 7pter----q22. A HindIII restriction fragment length polymorphism is detected by hybridization of the EPO cDNA probe to human genomic DNA.

DNA sequences of chromosome 21-specific YAC detect the t(8;21) breakpoint of acute myelogenous leukemia

The t(8;21)(q22;q22) is a nonrandom translocation specifically marking blasts of acute myelogenous leukemia (AML) with undifferentiated phenotype. The breakpoint on chromosome 21 involved by this rearrangement has been precisely localized relative to cloned DNA markers by physical and genetic linkage analysis enabling the use of positional cloning for its isolation. Yeast artificial chromosome (YAC) clones for loci proximal (D21S65) and distal (ERG) to the (21q22) breakpoint have been developed and their chromosome 21 origin and location relative to the breakpoint has been established. By using in situ hybridization analysis, a 240 kb YAC clone for the D21S65 locus clearly identified both derivative chromosomes of the (8;21) translocation in metaphase spreads of leukemia blasts with the rearrangement. The characterization of the DNA sequences contained in this 240 kb YAC can reveal the functional consequences of their derangement in leukemia with abnormalities of the (21q22) region.

Assignment of the gene for β-spectrin (SPTB) to chromosome 14q23→q24.2 by in situ hybridization

Type I hereditary spherocytosis results from a molecular defect in the beta-polypeptide of the erythrocyte cytoskeletal protein spectrin. Using a cDNA probe, we had previously assigned the gene for human erythrocyte beta-spectrin (SPTB) to chromosome 14 based upon analysis of its segregation in panels of human x rodent somatic cell hybrids (Winkelmann et al., 1988). Here we report the regional localization of this gene by in situ hybridization to 14q23----q24.2.

Molecular genetics of human chromosome 21.

Chromosome 21 is the smallest autosome, comprising only about 1.9% of human DNA, but represents one of the most intensively studied regions of the genome. Much of the interest in chromosome 21 can be attributed to its association with Downs syndrome, a genetic disorder that afflicts one in every 700 to 1000 newborns. Although only 17 genes have been assigned to chromosome 21, a very large number of cloned DNA segments of unknown function have been isolated and regionally mapped. The majority of these segments detect restriction fragment length polymorphisms (RFLPs) and therefore represent useful genetic markers. Continued molecular genetic investigation of chromosome 21 will be central to elucidating molecular events leading to meiotic non-disjunction and consequent trisomy, the contribution of specific genes to the pathology of Downs syndrome, and the possible role of chromosome 21 in Alzheimers disease and other as yet unmapped genetic defects.

A linkage map of three anonymous human DNA fragments and SOD-1 on chromosome 21.

Using DNA polymorphisms adjacent to single‐copy genomic fragments derived from human chromosome 21, we initiated the construction of a linkage map of human chromosome 21. The probes were genomic EcoRI fragments pW228C, pW236B, pW231C and a portion of the superoxide dismutase gene (SOD‐1). DNA polymorphisms adjacent to each of the probes were used as markers in informative families to perform classical linkage analysis. No crossing‐over was observed between the polymorphic sites adjacent to genomic fragments pW228C and pW236B in 31 chances for recombination. Therefore, these fragments are closely linked to one another (theta = 0.00, lod score = 6.91, 95% confidence limits = 0‐10 cM) and can be treated as one ‘locus’ with four high‐frequency markers. There is a high degree of non‐random association of markers adjacent to each of these two probes which suggests that they are physically very close to one another in the genome. The pW228C ‐ pW236B ‘locus’ was also linked to the SOD‐1 gene (theta = 0.07, lod score = 4.33, 95% confidence limits = 1‐20 cM). On the other hand, no evidence for linkage was found between the pW228C‐pW236B ‘locus’ and the genomic fragment pW231C (theta = 0.5, lod score = 0.00). Based on the fact that pW231C maps to 21q22.3 and SOD‐1 to 21q22.1, we suggest that the pW228C‐pW236B ‘locus’ lies in the proximal long arm of chromosome 21. These data provide the outline of a linkage map for the long arm of chromosome 21, and indicate that the pW228C‐pW236B ‘locus’ is a useful marker system to differentiate various chromosome 21s in a population.

Generation of 19 STS markers that can be anchored at specific sites on human chromosome 21

Sequence-tagged sites (STSs) are short stretches of DNA that can be specifically detected by the polymerase chain reaction (PCR) and can be used to construct long-range physical maps of chromosomal DNA. These STSs can be detected by PCR assays developed by reference to data obtained from the sequencing of restriction fragment length polymorphism-DNA markers for chromosome 21, which were derived from recombinant lamba-phage and plasmid clones made from DNA of a human-hamster hybrid cell line. In this report, we describe the generation of 19 new STSs that are specific for human chromosome 21.

Owl monkey gene map: Evidence for a homologous human chromosome 7q region near the cystic fibrosis locus

We have demonstrated the assignments of two gene loci (COLIA2, MET) and two noncoding DNA markers (D7S13, D7S8) to owl monkey chromosome 14 (K-VI) by hybridizing DNA probes from the cystic fibrosis (CF) region of human chromosome 7q21-32 to panels of rodent-owl monkey somatic cell hybrids. The assignments are substantiated by in situ chromosome hybridization of markers COLIA2, MET, and D7S13 to the distal long arm of chromosome 14 (K-VI). These results support genomic conservation of the human CF region, at least in the higher primates.

Yeast artificil chromosome (YAC) clones and sequence tagged site (STS) markers anchored at human chromosome 21

Human chromosome 21 has been well-characterized genetically because it includes several potential genes involved in numerous inherited disorders, particularly Down syndrome (Scoggin and Patterson, 1982) and familial Alzheimers disease (St. George-Hyslop et al., 1987). Several known genes or restriction fragment length polymorphic (RFLP) DNA markers have been mapped regionally on chromosome 21 (Watkins et al., 1985, 1987; Tanzi et al., 1988). In order to clarify genome structure and to define molecular bases of these inherited disorders, great efforts to make a physical map have been made during the past decades. Yeast artificial chromosome (YAC) vector developed recently provides a powerful tool for cloning several hundred kilobases of exogenous DNA in yeast cells and has allowed us to analyze a large region of human genome (Burke et al., 1987; Hieter et al., 1990; Imai and Olson, 1990a; Kai et al., 1990; Yokoyama et al., 1990). We have constructed a YAC library from a human lymphoblastoid cell line, CGM-1 according to the Burkes method (Burke and Olson, 1990) with a slight modification (Imai and Olson, 1990a). The library contains 14,000 clones with an average insert DNA size of 360 kb and the total length of human insert DNA was estimated approximately to be two equivalents of human haploid genome. We have chosen several known genes and RFLP markers localized in chromosome 21 in order to isolate YAC clones by using the polymerase chain reaction

Molecular Genetic Strategies in Familial Alzheimer’s Disease: Theoretical and Practical Considerations

Genetic linkage studies have provided evidence to indicate that there is a defective gene on chromosome 21 which causes the autosomal dominant form of Alzheimer’s disease (AD), at least in the four large pedigrees examined. Further studies have indicated that the β-amyloid gene and the superoxide dismutase-1 gene are not the site of the familial AD (FAD) mutation, and that duplication of large regions of chromosome 21 is not the pathogenetic mechanism in either FAD or sporadic AD. Additional studies are currently under way to more precisely map the location of the FAD gene in order to expedite the ultimate goal of isolating and characterizing the actual FAD gene.