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The association of subclasses of Alu repetitive elements with various classes of trinucleotide and tetranucleotide microsatellites was characterized as a first step toward advancing our understanding of the evolution of microsatellite repeats. In addition, information regarding the association of specific classes of microsatellites with families of Alu elements was used to facilitate the development of genetic markers. Sequences containingAlu repeats were eliminated because unique primers could not be designed. Various classes of microsatellites are associated with different classes of Alu repeats. Very abundant and poly(A)-rich microsatellite classes (ATA, AATA) are frequently associated with an evolutionarily older subclass of Alurepeats, AluSx, whereas most of GATA and CA microsatellites are associated with a recent Alu subfamily, AluY. Our observations support all three possible mechanisms for the association of Alu repeats to microsatellites. Primers designed using a set of sequences from a particular microsatellite class showed higher homology with more sequences of that class than probes designed for other classes. We developed an efficient method of prescreening GGAA and ATA microsatellite clones for Alu repeats with probes designed in this study. We also showed that Alu probes labeled in a single reaction (multiplex labeling) could be used efficiently for prescreening of GGAA clones. Sequencing of these prescreened GGAA microsatellites revealed only 5% Alu repeats. Prescreening with primers designed for ATA microsatellite class resulted in the reduction of the loss of markers from ∼50% to 10%. The newAlu probes that were designed have also proved to be useful inAlu–Alu fingerprinting.

For the purpose of initial screening of the human genome in linkage mapping, two overlapping sets of high quality short tandem repeat polymorphisms (STRPs) which span the autosomes have been assembled. The higher density set contains a total of 363 markers with an average heterozygosity of 79% and an average sex-equal genetic distance between markers of 10.5 cM. The lower density set, which is a subset of the other, contains 156 markers with an average heterozygosity of 80% and an average spacing of 26.5 cM. Tri- and tetranucleotide STRPs comprised 47 and 63%, respectively, of the markers within the higher and lower density sets. Markers within the screening sets were selected to have maximum quality, where quality was defined as a blend of high informativeness, strong amplification under standard PCR conditions, low amplification background, and ease in scoring. The screening sets along with combinations of STRPs which can be amplified and electrophoresed simultaneously are available electronically through anonymous ftp.

We report a collection of tri- and tetranucleotide repeat sequence polymorphic markers used to construct genome-wide human linkage maps. Using a strategy of marker selection to create libraries highly enriched for the presence of specific tandem repeat elements, we have developed over 2000 high heterozygosity, easy-to-use tri- and tetranucleotide short tandem repeat polymorphisms (STRPs). To date, over 1300 of these markers have been genotyped on the CEPH reference families. Additional STRPs were assigned to chromosomes using human monochromosomal somatic cell hybrids. The linkage maps constructed with these markers have been integrated with other CEPH genotypes into a comprehensive high density linkage map. These STRPs have been shown to be robust for genotyping in a variety of laboratories using a variety of methods. The high quality of these STRPs makes them ideal candidates for use in genome-wide linkage searches. The integration of these markers with physical mapping reagents and other genetic markers will create a resource for moving from genome-wide linkage searches to rapid sublocalization of disease loci.

Exon trapping is a method to functionally clone expressed sequences from genomic DNA. We have previously developed the vector system pETV-SD2, which contains only a splice donor site (SD) followed by a LacZ gene, allowing trapping of internal exons of human genes by blue-white selection. We now describe the adaptation of the same system for the efficient trapping of 3'-terminal exons, by using different RT-PCR primers in a 3' RACE reaction. The addition of a T7 promoter to the RT-PCR products derived from pETV-SD2 allows their amplification in an isothermic amplification reaction called NASBA (nucleic acid sequence-based amplification reaction) and results in a strong signal from amplified 3' exons in addition to a great reduction of non-specific background. As a test for the system, 3' exon trapping was performed using a cosmid containing the alpha-globin gene cluster on chromosome 16. The 3'-terminal exons of the human alpha 1-, zeta 2-, and theta-globin genes were trapped, as well as a correctly spliced and polyadenylated sequence in the 3' flanking region of the alpha 1-globin gene. This exon appears to belong to a previously unidentified gene within the alpha-globin gene cluster. This 3' exon trapping strategy should facilitate the cloning of genes from large genomic regions.

Bardet-Biedl syndrome is an autosomal recessive disorder characterized by mental retardation, obesity, retinitis pigmentosa, polydactyly and hypogonadism. Other findings include hypertension, diabetes mellitus and renal and cardiovascular anomalies. We have performed a genome-wide search for linkage in a large inbred Bedouin family. Pairwise analysis established linkage with the locus D16S408 with no recombination and a lod score of 4.2. A multilocus lod score of 5.3 was observed. By demonstrating homozygosity, in all affected individuals, for the same allele of marker D16S408, further support for linkage is found, and the utility of homozygosity mapping using inbred families is demonstrated. In a second family, linkage was excluded at this locus, suggesting non-allelic genetic heterogeneity in this disorder.

We detected a germ-line mutation of the p53 gene in a patient with a malignant ependymoma of the posterior fossa. This mutation, which was found at codon 242, resulted in an amino acid substitution in a highly conserved site of exon 7 of the p53 gene; the same mutation was found in both the germ-line and the tumor tissue. This is the most common region of previously described somatic p53 mutations in tumor specimens and of the germ-line p53 mutations in patients with the Li-Fraumeni cancer syndrome. Evaluation of the patient's family revealed several direct maternal and paternal relatives who had died at a young age from different types of cancer. The association of a germ-line p53 mutation with an intracranial malignancy and a strong family history of cancer suggests that p53 gene mutations predispose a person to malignancy and, like retinoblastoma mutations, may be inherited.

The beige mutation is a murine autosomal recessive disorder, resulting in hypopigmentation, bleeding and immune cell dysfunction. The gene defective in beige is thought to be a homologue of the gene for the human disorder Chediak-Higashi syndrome. We have identified the murine beige gene by in vitro complementation and positional cloning, and confirmed its identification by defining mutations in two independent mutant alleles. The sequence of the beige gene message shows strong nucleotide homology to multiple human ESTs, one or more of which may be associated with the Chediak-Higashi syndrome gene. The amino acid sequence of the Beige protein revealed a novel protein with significant amino acid homology to orphan proteins identified in Saccharomyces cerevisiae, Caenorhabditis elegans and humans.

The expansion of a (CAG/CTG) n triplet repeat has been found to be associated with at least seven genetic diseases, suggesting that this mechanism of disease may be fairly common. To accelerate the discovery of new loci containing (CAG/CTG) n triplet expansions, we have isolated numerous genomic clones containing this class of repeats. We have developed 338 sequence-tagged sites (STSs) containing (CAG/CTG) n repeat sequences. Two hundred ninety-nine STSs were unambiguously assigned to chromosomes, and 89 of the total were assigned to YACs. The 141 STSs that were developed based on (CAG/CTG) n repeats of at least seven units were genotyped on four reference CEPH individuals to estimate their polymorphic quality.

The complete sequence of the polycystic kidney disease gene (PKD1) and its transcript have been described. The predicted protein is not a member of a previously described gene family, but contains several structural motifs that are present in proteins of known function. Most of these domains are present in the extracellular parts of proteins involved in interactions with other proteins and carbohydrates. The PKD1 gene product also contains potential trans- membrane sequences. The molecule is likely to be involved in cell-cell or cell-matrix interactions, which is consistent with the different manifestations of polycystic kidney disease.

Genetic markers based upon PCR amplification of short tandem repeat-containing sequence tagged sites (STSs) have become the standard for genetic mapping. We have completed a survey based on the direct isolation of representative members of each of the 10 trinucleotide repeat classes to determine their relative abundance, repeat size distribution, and general utility as genetic markers. Trinucleotide repeats, depending on the repeat class, are one to two orders of magnitude less frequent than (AC) n repeats. The average size of trinucleotide repeats sequenced was less than 15 repeat units in length, and only three of the STSs developed for this study demonstrated more than 25 repeats units. The (AAT) n class of repeats are the most abundant and also the most frequently polymorphic. Other classes of trinucleotide repeat classes observed to be frequently polymorphic include (AAC) n , (ACT) n , (ATC) n and (AAG) n ; however, the relative abundance of these classes is less than that observed for the (AAT) n class of repeats. Based upon this initial survey, we have initiated saturation cloning of the (AAT) n class of repeats. At the time of submission of this manuscript, we have developed, as part of the Cooperative Human Linkage Center (CHLC), more than 415 new high heterozygosity (AAT) n genetic markers (more than two alleles in four individuals) and 200 new low heterozygosity (AAT) n STSs from this larger screening effort combined with the initial survey.

Bardet-Biedl syndrome is an autosomal recessive disorder characterized by mental retardation, obesity, retinitis pigmentosa, polydactyly and hypogonadism. Individuals with this disorder also have an increased incidence of hypertension, diabetes mellitus, and renal and cardiac anomalies. We previously identified a locus on chromosome 16 causing this disorder, and provided evidence that Bardet-Biedl syndrome is hetero-geneous. In this study, we identify another Bardet-Biedl syndrome locus on chromosome 3 and confirm the non-allelic heterogeneity of this disorder in Bedouin populations. In addition, we demonstrate the feasibility of using pooled DNA samples from members of large kindreds as an efficient approach to homozygosity mapping

High resolution linkage maps have proven to be invaluable tools in genetic investigations. We have assembled a collection of genetic maps constructed from primary data collected from investigators performing genotyping using the Centre Etude Polymorphism Humain (CEPH) reference pedigree panel. These maps were constructed using a rigorous, semi-automated map construction algorithm that evaluates the integrity of the maps during construction. Two classes of maps were produced: a high confidence "skeletal" set composed of 544 PCR based markers, and a more highly annotated "framework" set containing maps of 1,123 markers. Genetic map locations within the framework maps are provided for an additional 1,758 loci without statistically unique interval assignments.

p53 is a tumor suppressor gene located on 17p, a region of the human genome frequently deleted in tumors. Mutation of the p53 gene is an important step leading to development of many forms of human cancer. To simplify the analysis of tumors for p53 point mutations, we describe a GC-clamped denaturing gradient gel assay for detecting single-base substitutions within highly conserved regions of the p53 gene. This assay allows for efficient screening of tumors for single-base substitutions within the p53 gene and can be used to facilitate sequence analysis of p53 point mutations.

Identification and recovery of transcribed sequences from cloned mammalian genomic DNA remains an important problem in isolating genes on the basis of their chromosomal location. We have developed a strategy that facilitates the recovery of exons from random pieces of cloned genomic DNA. The basis of this "exon trapping" strategy is that, during a retroviral life cycle, genomic sequences of nonviral origin are correctly spliced and may be recovered as a cDNA copy of the introduced segment. By using this genetic assay for cis-acting sequences required for RNA splicing, we have screened approximately 20 kilobase pairs of cloned genomic DNA and have recovered all four predicted exons.

OB-R is a high affinity receptor for leptin, an important circulating signal for the regulation of body weight. We identified an alternatively spliced transcript that encodes a form of mouse OB-R with a long intracellular domain. db/db mice also produce this alternatively spliced transcript, but with a 106 nt insertion that prematurely terminates the intracellular domain. We further identified a G→T point mutation in the genomic OB-R sequence in db/db mice. This mutation generates a donor splice site that converts the 106 nt region to a novel exon retained in the OB-R transcript. We predict that the long intracellular domain form of OB-R is crucial for initiating intracellular signal transduction, and as a corollary, the inability to produce this form of OB-R leads to the severe obese phenotype found in db/db mice.

Maturity-onset diabetes of the young (MODY) type 3 is a dominantly inherited form of diabetes, which is often misdiagnosed as non-insulin-dependent diabetes mellitus (NIDDM) or insulin-dependent diabetes mellitus (IDDM). Phenotypic analysis of members from four large Finnish MODY3 kindreds (linked to chromosome 12q with a maximum lod score of 15) revealed a severe impairment in insulin secretion, which was present also in those normoglycemic family members who had inherited the MODY3 gene. In contrast to patients with NIDDM, MODY3 patients did not show any features of the insulin resistance syndrome. They could be discriminated from patients with IDDM by lack of glutamic acid decarboxylase antibodies (GAD-Ab). Taken together with our recent findings of linkage between this region on chromosome 12 and an insulin-deficient form of NIDDM (NIDDM2), the data suggest that mutations at the MODY3/NIDDM2 gene(s) result in a reduced insulin secretory response, that subsequently progresses to diabetes and underlines the importance of subphenotypic classification in studies of diabetes.