Biomedical subjects
E S Lander
Publications and source records attributed to E S Lander.
Determination of ancestral alleles for human single-nucleotide polymorphisms using high-density oligonucleotide arrays.
Here we report the application of high-density oligonucleotide array (DNA chip)-based analysis to determine the distant history of single nucleotide polymorphisms (SNPs) in current human populations. We analysed orthologues for 397 human SNP sites (identified in CEPH pedigrees from Amish, Venezuelan and Utah populations) from 23 common chimpanzee, 19 pygmy chimpanzee and 11 gorilla genomic DNA samples. From this data we determined 214 proposed ancestral alleles (the sequence found in the last common ancestor of humans and chimpanzees). In a diverse human population set, we found that SNP alleles with higher frequencies were more likely to be ancestral than less frequently occurring alleles. There were, however, exceptions. We also found three shared human/pygmy chimpanzee polymorphisms, all involving CpG dinucleotides, and two shared human/gorilla polymorphisms, one involving a CpG dinucleotide. We demonstrate that microarray-based assays allow rapid comparative sequence analysis of intra- and interspecies genetic variation.
Identification of the Finnish founder mutation for diastrophic dysplasia (DTD).
Diastrophic dysplasia (DTD) is especially prevalent in Finland and the existence of a founder mutation has been previously inferred from the fact that 95% of Finnish DTD chromosomes have a rare ancestral haplotype found in only 4% of Finnish control chromosomes. Here we report the identification of the Finnish founder mutation as a GT-> GC transition (c.-26 + 2T > C) in the splice donor site of a previously undescribed 5'-untranslated exon of the diastrophic dysplasia sulfate transporter gene (DTDST); the mutation acts by severely reducing mRNA levels. Among 84 DTD families in Finland, patients carried two copies of the mutation in 69 families, one copy in 14 families, and no copies in one family. Roughly 90% of Finnish DTD chromosomes thus carry the splice-site mutation, which we have designated DTDST(Fin). Unexpectedly, we found that nine of the DTD chromosomes having the apparently ancestral haplotype did not carry DTDST(Fin), but rather two other mutations. Eight such chromosomes had an R279W mutation and one had a V340del deletion. We consider the possible implications of presence of multiple DTD mutations on this rare haplotype.
Campomelic dysplasia translocation breakpoints are scattered over 1 Mb proximal to SOX9: evidence for an extended control region.
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.
Sequencing a genome by walking with clone-end sequences: a mathematical analysis.
One approach to sequencing a large genome is (1) to sequence a collection of nonoverlapping "seeds" chosen from a genomic library of large-insert clones [such as bacterial artificial chromosomes (BACs)] and then (2) to take successive "walking" steps by selecting and sequencing minimally overlapping clones, using information such as clone-end sequences to identify the overlaps. In this paper we analyze the strategic issues involved in using this approach. We derive formulas showing how two key factors, the initial density of seed clones and the depth of the genomic library used for walking, affect the cost and time of a sequencing project-that is, the amount of redundant sequencing and the number of steps to cover the vast majority of the genome. We also discuss a variant strategy in which a second genomic library with clones having a somewhat smaller insert size is used to close gaps. This approach can dramatically decrease the amount of redundant sequencing, without affecting the rate at which the genome is covered.
Quantitative trait locus mapping of airway responsiveness to chromosomes 6 and 7 in inbred mice.
Quantitative trait locus (QTL) mapping was used to identify chromosomal regions contributing to airway hyperresponsiveness in mice. Airway responsiveness to methacholine was measured in A/J and C3H/HeJ parental strains as well as in progeny derived from crosses between these strains. QTL mapping of backcross [(A/J x C3H/HeJ) x C3H/HeJ] progeny (n = 137-227 informative mice for markers tested) revealed two significant linkages to loci on chromosomes 6 and 7. The QTL on chromosome 6 confirms the previous report by others of a linkage in this region in the same genetic backgrounds; the second QTL, on chromosome 7, represents a novel locus. In addition, we obtained suggestive evidence for linkage (logarithm of odds ratio = 1.7) on chromosome 17, which lies in the same region previously identified in a cross between A/J and C57BL/6J mice. Airway responsiveness in a cross between A/J and C3H/HeJ mice is under the control of at least two major genetic loci, with evidence for a third locus that has been previously implicated in an A/J and C57BL/6J cross; this indicates that multiple genetic factors control the expression of this phenotype.
Dissecting the regulatory circuitry of a eukaryotic genome.
Genome-wide expression analysis was used to identify genes whose expression depends on the functions of key components of the transcription initiation machinery in yeast. Components of the RNA polymerase II holoenzyme, the general transcription factor TFIID, and the SAGA chromatin modification complex were found to have roles in expression of distinct sets of genes. The results reveal an unanticipated level of regulation which is superimposed on that due to gene-specific transcription factors, a novel mechanism for coordinate regulation of specific sets of genes when cells encounter limiting nutrients, and evidence that the ultimate targets of signal transduction pathways can be identified within the initiation apparatus.
A physical map of 30,000 human genes.
A map of 30,181 human gene-based markers was assembled and integrated with the current genetic map by radiation hybrid mapping. The new gene map contains nearly twice as many genes as the previous release, includes most genes that encode proteins of known function, and is twofold to threefold more accurate than the previous version. A redesigned, more informative and functional World Wide Web site (www.ncbi.nlm.nih.gov/genemap) provides the mapping information and associated data and annotations. This resource constitutes an important infrastructure and tool for the study of complex genetic traits, the positional cloning of disease genes, the cross-referencing of mammalian genomes, and validated human transcribed sequences for large-scale studies of gene expression.
Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome.
Single-nucleotide polymorphisms (SNPs) are the most frequent type of variation in the human genome, and they provide powerful tools for a variety of medical genetic studies. In a large-scale survey for SNPs, 2.3 megabases of human genomic DNA was examined by a combination of gel-based sequencing and high-density variation-detection DNA chips. A total of 3241 candidate SNPs were identified. A genetic map was constructed showing the location of 2227 of these SNPs. Prototype genotyping chips were developed that allow simultaneous genotyping of 500 SNPs. The results provide a characterization of human diversity at the nucleotide level and demonstrate the feasibility of large-scale identification of human SNPs.
Zebrafish genomic library in yeast artificial chromosomes.
We have constructed a zebrafish yeast artificial chromosome (YAC) library using genomic DNA isolated from the inbred AB zebrafish strain. The average insert size is 470 kb, estimated from analysis of 155 random selected YACs. The library consists of 17,000 clones, providing about a 4.7-fold coverage of zebrafish genome. The YAC clones have been arrayed in individual wells of 96-well microplates and also pooled to permit rapid polymerase chain reaction screening of the entire library. We have also found that the YAC ends can be easily rescued and sequenced from pRML1/pRML2-based mini-YAC clones.
Sib-pair collection strategies for complex diseases.
When planning an affected sib pair collection for use in a genomewide search for complex trait loci, researchers must ask: (a) Which family structures will yield the most informative pairs? and (b) Should recruitment extend beyond the index sib pair? The optimal collection strategy will depend on the trait's genetic architecture, but this is rarely known for non-Mendelian diseases. In the present report, we study the consequences of collecting only those sib pairs arising from pedigrees with a precisely specified structure as opposed to a strategy that collects all affected sib pairs at random (i.e., blind to the affection status of first-degree relatives). The former approach turns out to be risky because the power of specific pedigree structures can vary dramatically even among models producing identical observable parameters (such as population prevalence and sibling recurrence rate). In contrast, the latter approach typically involves only a modest loss of power as compared with the optimal (but unknowable) design. Further, we compare the strategy of collecting all affected sib pairs at random with the alternative of imposing some modest limitations on family structure (e.g., presence of at least one unaffected sib or parent). The latter approach generally provides some increase in power but entails additional effort to contact and phenotype relatives: the overall merit of imposing such requirements needs to be evaluated in the context of the specific disease to be studied and of the clinical and analytical resources available. In addition, these findings suggest that a further explanation for failure to replicate positive complex trait linkages lies in differences in ascertainment strategy between data sets.
Genetic isolation of iddm 1 on chromosome 4 in the biobreeding (BB) rat.
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An integrated genetic linkage map of the laboratory rat.
The laboratory rat, Rattus novegicus, is a major model system for physiological and pathophysiological studies, and since 1966 more than 422,000 publications describe biological studies on the rat (NCBI/Medline). The rat is becoming an increasingly important genetic model for the study of specific diseases, as well as retaining its role as a major preclinical model system for pharmaceutical development. The initial genetic linkage map of the rat contained 432 genetic markers (Jacob et al. 1995) out of 1171 developed due to the relatively low polymorphism rate of the mapping cross used (SHR x BN) when compared to the interspecific crosses in the mouse. While the rat genome project continues to localize additional markers on the linkage map, and as of 11/97 more than 3,200 loci have been mapped. Current map construction is using two different crosses (SHRSP x BN and FHH x ACI) rather than the initial mapping cross. Consequently there is a need to provide integration among the different maps. We set out to develop an integrated map, as well as increase the number of markers on the rat genetic map. The crosses available for this analysis included the original mapping cross SHR x BN reciprocal F2 intercross (448 markers), a GH x BN intercross (205 markers), a SS/Mcw x BN intercross (235 markers), and a FHH/Eur x ACI/Hsd intercross (276 markers), which is also one of the new mapping crosses. Forty-six animals from each cross were genotyped with markers polymorphic for that cross. The maps appear to cover the vast majority of the rat genome. The availability of these additional markers should facilitate more complete whole genome scans in a greater number of strains and provide additional markers in specific genomic regions of interest.
Gene-based anchoring of the rat genetic linkage and cytogenetic maps: new regional localizations, orientation of the linkage groups, and insights into mammalian chromosome evolution.
In order to generate anchor points connecting the rat cytogenetic and genetic maps, the cytogenetic position of 62 rat markers (including 55 genes) already localized genetically was determined by fluorescence in situ hybridization. Whenever possible, markers located near one end of the linkage groups were included. These new localizations allowed us to unambiguously orient the 20 autosomal and the X chromosome linkage groups. The position of the centromere in the linkage map could also be determined in the case of several metacentric chromosomes. In addition, the regional localization of 15 other rat genes was determined. These new data bring useful information with respect to comparative mapping with the mouse and the human and to mammalian evolution. They illustrate, for instance, that groups of genes can remain syntenic during mammalian evolution while being subjected to intrachromosomal rearrangements in some lineages (synteny is conserved while gene order is not). This analysis also disclosed cases of synteny conservation in one the two rodent species and the human, while the synteny is split in the other rodent species: such configurations are likely examples of lineage-specific interchromosomal rearrangements associated with speciation.
Absence of linkage between inflammatory bowel disease and selected loci on chromosomes 3, 7, 12, and 16.
BACKGROUND & AIMS: Linkage data derived from genome-wide scans of inflammatory bowel disease (IBD) sibling-pair families have identified 4 loci on chromosomes 3, 7, 12, and 16 as potential sites for IBD susceptibility genes. The aim of this study was to investigate whether linkage analysis of another independently collected set of sibling pairs with IBD would provide further evidence of linkage between these previously reported loci and IBD. METHODS: Using the MAPMAKER/SIBS program, the segregation of 21 microsatellite marker loci spanning the 4 putative IBD gene loci was analyzed in a study population comprising 161 families with 114 Crohn's disease, 36 ulcerative colitis, and 50 mixed IBD sibling pairs from the Greater Toronto area. RESULTS: The results of multipoint linkage analysis showed no evidence for linkage between IBD and each of the 21 marker loci studied; the logarithm of odds scores in all instances were less than 0.8. These linkage data were found, by exclusion mapping analysis, to exclude values of lambdas ranging from 1.5 to 3.0, depending on the locus evaluated. CONCLUSIONS: The loci previously suggested as representing IBD susceptibility loci are not linked to IBD in the Toronto population examined in this analysis.
The mouse pudgy mutation disrupts Delta homologue Dll3 and initiation of early somite boundaries.
Pudgy (pu) homozygous mice exhibit clear patterning defects at the earliest stages of somitogenesis, resulting in adult mice with severe vertebral and rib deformities. By positional cloning and complementation, we have determined that the pu phenotype is caused by a mutation in the delta-like 3 gene (Dll3), which is homologous to the Notch-ligand Delta in Drosophila. Histological and molecular marker analyses show that the pu mutation disrupts the proper formation of morphological borders in early somite formation and of rostral-caudal compartment boundaries within somites. Viability analysis also indicates an important role in early development. The results point to a key role for a Notch-signalling pathway in the initiation of patterning of vertebrate paraxial mesoderm.
CLN5, a novel gene encoding a putative transmembrane protein mutated in Finnish variant late infantile neuronal ceroid lipofuscinosis.
The neuronal ceroid lipofuscinoses (NCLs) represent a group of common recessive inherited neurodegenerative disorders of childhood, with an incidence of 1:12,500 live births. They are characterized by accumulation of autofluorescent lipopigments in various tissues. Several forms of NCLs have been identified, based on age at onset, progression of disease, neurophysiological and histopathological findings and separate genetic loci. All types of NCL cause progressive visual and mental decline, motor disturbance, epilepsy and behavioral changes, and lead to premature death. One of the subtypes, Finnish variant late infantile neuronal ceroid lipofuscinosis (vLINCL; MIM256731) affects children at 4-7 years of age. The first symptom is motor clumsiness, followed by progressive visual failure, mental and motor deterioration and later by myoclonia and seizures. We have previously reported linkage for vLINCL on chromosome 13 (ref. 5) and constructed a long-range physical map over the region. Here, we report the positional cloning of a novel gene, CLN5, underlying this severe neurological disorder. The gene encodes a putative transmembrane protein which shows no homology to previously reported proteins. Sequence analysis of DNA samples from patients with three different haplotypes revealed three mutations; one deletion, one nonsense and one missense mutation, suggesting that mutations in this gene are responsible for vLINCL.