The initial step of mapping STSs to chromosome 3 has been by mapping to a reference panel of 21 somatic cell hybrids containing fragments of chromosome 3. In this study we map 638 STSs to 23 bins on chromosome 3, The bin information greatly facilitates further mapping by radiation hybrids and YAC clones.

Hyperekplexia is a rare, autosomal dominant neurological disorder characterized by hypertonia, especially in infancy, and by an exaggerated startle response. This disorder is caused by mutations in the ϵ1 subunit of the inhibitory glycine receptor (GLRA1). We previously reported two GLRA1 point mutations detected in 4 unrelated hyperekplexia families; both mutations were at nucleotide 1192 and resulted in the replacement of Arg 271 by a glutamine (R271Q) in one case and a leucine (R271L) in the other. Here, 5 additional hyperekplexia families are shown to have the most common G‐to‐A transition mutation at nucleotide 1192. Haplotype analysis using polymorphisms within and close to the GLRA1 locus suggests that this mutation has arisen at least twice (and possibly four times). In 2 additional families, a third mutation is also presented that changes a tyrosine at amino acid 279 to a cysteine (Y279C). Five patients with atypical clinical features and equivocal or absent family history of hyperekplexia and 1 patient with a classical presentation but no family history are presented in whom a mutation in the GLRA1 gene was not detected. Thus, only clinically typical hyperekplexia appears to be consistently associated with GLRA1 mutations, and these affect a specific extracellular domain of the protein.

Human chromosome 3 contains 210 million base pairs and approximately 7,000 genes. Our center is directed towards making genetic and physical maps of chromosome 3 with the goal of a YAC contig of the chromosome and 2500 STSs. Markers are first binned into 23 regions of chromosome 3 using framework somatic cell hybrids. To date, STSs for 259 microsatellite and 63 genes have been placed into bins. 125 polymorphic simple sequences repeat (SSR) markers were genetically mapped to chromosome 3 using PCR-based genotyping of the CEPH reference families. This genetic map spans 271 cM (sex averaged). A chromosome 3-specific radiation-reduced somatic cell hybrid panels has been constructed to obtain higher resolution map order information. With the map locations of these markers as a guide, 226 loci were screened using PCR-based hierarchial screening of the CEPH Mark I and Mark IV-VII yeast artificial chromosome (YAC) libraries. YAC contigs which are ordered on the chromosome consist of 33 multi-locus contigs covering 80 cM and single locus contigs accounting for 62 cM. We have isolated approximately 1000 YACs by this method covering 65% of chromosome 3. IRS-PCR screening of the CEPH YAC library has yielded several thousand more chromosome 3 YACs which aremore » being screened with the STSs to integrate them into contigs. FISH analysis on a random sampling of 60 YACs from the long arm was performed to assess the degree of chimerism. 40% of the YACs hybridized to the map locations predicted by the STSs used in their isolation. 30% were obviously chimeric while 40% hybridized uniquely to the predicted region as well as nonspecifically to centromeres and telomeres. This integrated approach to constructing a map of chromosome 3 will result in a reliably ordered YAC contig for this chromosome.« less

This chapter describes the use of radiation hybrids for constructing high-resolution maps of the human genome. Radiation hybrids have recently been implemented for mouse mapping and have been found to have a higher level of resolution compared to that of interspecies meiotic mapping panels. The chapter presents the advantages and disadvantages of the two different types of radiation hybrid panels. One advantage of the chromosome-specific panels is their reduced complexity. Because the human component in the hybrids is derived from a single chromosome or a reduced number of chromosomes, the hybrid can be used as a resource for chromosome-specific marker isolation. Using methods, such as interspersed repetitive sequence-polymerase chain reaction (IRS-PCR) to obtain the human sequences has greatly improved their usefulness. Moreover, the hybrids are easier to analyze with markers from multigenic families due to their reduced complexity. As radiation hybrid mapping has become more popular, numerous laboratories have expressed an interest in gaining access to hybrid panels. There is a renewed interest in total genome hybrid panels because they can be used without a piori knowledge of a marker's location.

Neurofibromatosis 1 maps to chromosome band 17q11.2, and the NF1 locus has been partially characterized. Even though the full-length NF1 cDNA has been sequenced, the complete genomic structure of the NF1 gene has not been elucidated. The 5′ end of NF1 is embedded in a CpG island containing a NotI restriction site, and the remainder of the gene lies in the adjacent 350-kb NotI fragment. In our efforts to develop a comprehensive screen for NF1 mutations, we have isolated genomic DNA clones that together harbor the entire NF1 cDNA sequence. We have identified all intron-exon boundaries of the coding region and established that it is composed of 59 exons. Furthermore, we have defined the 3′-untranslated region (3′- UTR) of the NF1 gene; it spans approximately 3.5 kb of genomic DNA sequence and is continuous with the stop codon. Oligonucleotide primer pairs synthesized from exon-flanking DNA sequences were used in the polymerase chain reaction with cloned, chromosome 17-specific genomic DNA as template to amplify NF1 exons 1 through 27b and the exon containing the 3′- UTR separately. This information should be useful for implementing a comprehensive NF1 mutation screen using genomic DNA as template.

Oligonucleotide primers for 125 simple sequence repeat microsatellite-based genetic markers have been assayed by polymerase chain reaction (PCR) in the CEPH reference family panel. These microsatellites include 101 dinucleotide repeats as well as 24 new tetranucleotide repeats. The average heterozygosity of this marker set was 72.4%. Genetic data were analyzed with the genetic mapping package LINKAGE. A subset of these microsatellite markers define a set of 56 uniquely ordered loci (>1000:1 against local inversion) that span 271 cM. Sixty-seven additional loci were tightly linked to markers on the uniquely ordered map, but could not be ordered with such high precision. These markers were positioned by CMAP into confidence intervals. One hundred thirteen of the microsatellite markers were also tested on a chromosome 3 framework somatic cell hybrid panel that divides this chromosome into 23 cytogenetically defined regions, integrating the genetic and physical maps of this chromosome. The high density, high heterozygosity, and PCR format of this genetically and physically mapped set of markers will accelerate the mapping and positional cloning of new chromosome 3 genes.

A locus on chromosome 17q, designated "BRCA1," has been identified as a predisposition gene for breast cancer. A panel of chromosome 17-specific radiation-reduced somatic cell hybrid clones has been assembled for high-resolution mapping of chromosome 17. A series of 35 markers, known to span the BRCA1 locus, were tested against this hybrid panel by PCR assays. Statistical analysis of these data yields a BRCA1 radiation hybrid map at a density sufficient to initiate YAC cloning and pulsed-field gel electrophoretic mapping of the candidate region. In addition, many of the markers reveal genetic polymorphisms and may be tested in breast cancer families and in loss-of-heterozygosity studies of sporadic breast cancers to better define the BRCA1 gene candidate region.

BACKGROUND:Children with type 1 neurofibromatosis (NF-1) are at increased risk for malignant myeloid disorders. Analysis of the NF-1 gene (NF1) suggests that the function of its product, neurofibromin, is reduced in affected persons and that NF1 belongs to the tumor-suppressor class of recessive cancer genes. This model is consistent with evidence that neurofibromin accelerates the intrinsic guanosine triphosphate-hydrolyzing activity of the Ras family of regulatory proteins. Loss of constitutional heterozygosity has not been reported in the benign tumors associated with NF-1, however, and has only been detected in a few malignant neural-crest tumors and in some tumor-derived cell lines.METHODS:We studied DNA extracted from the bone marrow of 11 children with NF-1 in whom malignant myeloid disorders developed and from parental leukocytes. We used a series of polymorphic markers within and near NF1 to determine whether leukemogenesis was associated with structural alterations of the gene.RESULTS:Bone marrow samples from five patients showed loss of heterozygosity. In each case, the NF1 allele was inherited from a parent with NF-1 and the normal allele was deleted.CONCLUSIONS:These data provide evidence of NF1 may function as a tumor-suppressor allele in malignant myeloid diseases in children with NF-1 and that neurofibromin is a regulator of ras in early myelopoiesis.

Polymorphic alleles at loci such as LPL (lipoprotein lipase) and MSR (macrophage scavenger receptor) in chromosome band 8p22 are frequently lost during the genesis of several types of human cancer, including colorectal, non-small cell lung, hepatocellular, and prostatic carcinomas. A physical map of 31 published or novel probes and sequence-tagged sites in this genetic region was constructed using a radiation hybrid panel and the CEPH (Centre d'Etude du Polymorphisme Humain) yeast artificial chromosome (YAC) library. Thirty-six overlapping YACs defined a physical order for the following polymorphic markers: tel-D8S26-D8S511-D8S549-MSR-D8S254-D8S233- D8S261-D8S21-LPL-D8S258-cen. These maps unify small consensus regions of allelic loss on chromosome 8p defined by restriction fragment length polymorphisms with more informative PCR-based polymorphisms and widely available YAC mapping resources.

Glucose-dependent insulinotropic polypeptide (GIP) has been regionally localized to a gene cluster on human chromosome 17q. Genetic mapping through CEPH reference families demonstrated that GIP was tightly linked to NME1 and PPY and fully linked to HOXB6 and NGFR. High-resolution radiation hybrid mapping resolved the gene order as cen-PPY-HOXB6-NGFR-GIP-NME1-tel. GIP maps distal to NGFR with an estimated distance of 250 kb.

Hereditary hyperekplexia, an autosomal dominant neurologic disorder characterized by an exaggerated startle reflex and neonatal hypertonia, can be caused by mutations in the gene encoding the alpha 1 subunit of the inhibitory glycine receptor (GLRA1). Spasmodic (spd), a recessive neurologic mouse mutant, resembles hyperekplexia phenotypically, and the two disease loci map to homologous chromosomal regions. Here we describe a Glra1 missense mutation in spd that results in reduced agonist sensitivity in glycine receptors expressed in vitro. We conclude that spd is a murine homologue of hyperekplexia and that mutations in GLRA1/Glra1 can produce syndromes with different inheritance patterns.

Our previous genetic map for chromosome 17 has been expanded to include 72 loci defined by 90 RFLP markers and four microsatellite markers assayed by the polymerase chain reaction. Forty-one of these loci were ordered with odds greater than 1000:1 against local inversion, and the other 31 were ordered within 95% confidence limits. From the set of 41 unambiguously mapped loci, 14 well-spaced "index markers" can be extracted for efficient, genetic studies. The complete map spans 173 cM (136 cM in males and 214 cM in females); average spacing between markers is 4.2 cM.

A genetic linkage map for the long arm of human chromosome 13 contains 29 loci derived from 38 probe and enzyme combinations and two protein polymorphisms. Thirteen loci form a continuous linkage map of 106 cM in males and 230 cM in females; each was placed on the map with support of at least 1000:1 against alternative orders. On a sex-combined basis, the mean distance between markers is less than 13 cM. The order of loci on the genetic map agrees with physical localization data that show that together these 13 loci cover 13q13 to 13q34. This map was used to regionally localize the 16 remaining loci. The linkage maps reported here should prove to be useful to investigators mapping disease genes and other genetic markers on human chromosome 13.