Towards an evidence-based process for the clinical interpretation of copy number variation.

The evidence-based review (EBR) process has been widely used to develop standards for medical decision-making and to explore complex clinical questions. This approach can be applied to genetic tests, such as chromosomal microarrays, in order to assist in the clinical interpretation of certain copy number variants (CNVs), particularly those that are rare, and guide array design for optimal clinical utility. To address these issues, the International Standards for Cytogenomic Arrays Consortium has established an EBR Work Group charged with building a framework to systematically assess the potential clinical relevance of CNVs throughout the genome. This group has developed a rating system enumerating the evidence supporting or refuting dosage sensitivity for individual genes and regions that considers the following criteria: number of causative mutations reported; patterns of inheritance; consistency of phenotype; evidence from large-scale case-control studies; mutational mechanisms; data from public genome variation databases; and expert consensus opinion. The system is designed to be dynamic in nature, with regions being reevaluated periodically to incorporate emerging evidence. The evidence collected will be displayed within a publically available database, and can be used in part to inform clinical laboratory CNV interpretations as well as to guide array design.

Spidey: a tool for mRNA-to-genomic alignments.

We have developed a computer program that aligns spliced sequences to genomic sequences, using local alignment algorithms and heuristics to put together a global spliced alignment. Spidey can produce reliable alignments quickly, even when confronted with noise from alternative splicing, polymorphisms, sequencing errors, or evolutionary divergence. We show how Spidey was used to align reference sequences to known genomic sequences and then to the draft human genome, to align mRNAs to gene clusters, and to align mouse mRNAs to human genomic sequence. We compared Spidey to two other spliced alignment programs; Spidey generally performed quite well in a very reasonable amount of time.

A radiation hybrid map of mouse genes.

A comprehensive gene-based map of a genome is a powerful tool for genetic studies and is especially useful for the positional cloning and positional candidate approaches. The availability of gene maps for multiple organisms provides the foundation for detailed conserved-orthology maps showing the correspondence between conserved genomic segments. These maps make it possible to use cross-species information in gene hunts and shed light on the evolutionary forces that shape the genome. Here we report a radiation hybrid map of mouse genes, a combined project of the Whitehead Institute/Massachusetts Institute of Technology Center for Genome Research, the Medical Research Council UK Mouse Genome Centre, and the National Center for Biotechnology Information. The map contains 11,109 genes, screened against the T31 RH panel and positioned relative to a reference map containing 2,280 mouse genetic markers. It includes 3,658 genes homologous to the human genome sequence and provides a framework for overlaying the human genome sequence to the mouse and for sequencing the mouse genome.

Database resources of the National Center for Biotechnology Information.

In addition to maintaining the GenBank nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides data analysis and retrieval resources that operate on the data in GenBank and a variety of other biological data made available through NCBI's Web site. NCBI data retrieval resources include Entrez, PubMed, LocusLink and the Taxonomy Browser. Data analysis resources include BLAST, Electronic PCR, OrfFinder, RefSeq, UniGene, HomoloGene, Database of Single Nucleotide Polymorphisms (dbSNP), Human Genome Sequencing, Human MapViewer, GeneMap'99, Human-Mouse Homology Map, Cancer Chromosome Aberration Project (CCAP), Entrez Genomes, Clusters of Orthologous Groups (COGs) database, Retroviral Genotyping Tools, Cancer Genome Anatomy Project (CGAP), SAGEmap, Gene Expression Omnibus (GEO), Online Mendelian Inheri-tance in Man (OMIM), the Molecular Modeling Database (MMDB) and the Conserved Domain Database (CDD). Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. All of the resources can be accessed through the NCBI home page at: http://www.ncbi.nlm.nih. gov.

Generation of RCAS vectors useful for functional genomic analyses.

Avian leukosis type A virus-derived retroviral vectors have been used to introduce genes into cells expressing the corresponding avian receptor tv-a. This includes the use of Replication-Competent Avian sarcoma-leukosis virus (ASLV) long terminal repeat (LTR) with Splice acceptor (RCAS) vectors in the analysis of avian development, human and murine cell cultures, murine cell lineage studies and cancer biology. Previously, cloning of genes into this virus was difficult due to the large size of the vector and sparse cloning sites. To overcome some of the disadvantages of traditional cloning using the RCASBP-Y vector, we have modified the RCASBP-Y to incorporate "Gateway" site-specific recombination cloning of genes into the construct, either with or without HA epitope tags. We have found the repetitive "att" sequences, which are the targets for site-specific recombination, do not impair the production of infectious viral particles or the expression of the gene of interest. This is the first instance of site-specific recombination being used to generate retroviral gene constructs. These viral constructs will allow for the efficient transfer and expression of cDNAs needed for functional genomic analyses.

Rapid generation of nested chromosomal deletions on mouse chromosome 2.

Nested chromosomal deletions are powerful genetic tools. They are particularly suited for identifying essential genes in development either directly or by screening induced mutations against a deletion. To apply this approach to the functional analysis of mouse chromosome 2, a strategy for the rapid generation of nested deletions with Cre recombinase was developed and tested. A loxP site was targeted to the Notch1 gene on chromosome 2. A targeted line was cotransfected with a second loxP site and a plasmid for transient expression of Cre. Independent random integrations of the second loxP site onto the targeted chromosome in direct repeat orientation created multiple nested deletions. By virtue of targeting in an F(1) hybrid embryonic stem cell line, F(1)(129S1xCast/Ei), the deletions could be verified and rapidly mapped. Ten deletions fell into seven size classes, with the largest extending six or seven centiMorgans. The cytology of the deletion chromosomes were determined by fluorescent in situ hybridization. Eight deletions were cytologically normal, but the two largest deletions had additional rearrangements. Three deletions, including the largest unrearranged deletion, have been transmitted through the germ line. Several endpoints also have been cloned by plasmid rescue. These experiments illustrate the means to rapidly create and map deletions anywhere in the mouse genome. They also demonstrate an improved method for generating nested deletions in embryonic stem cells.

Transcript mapping of the human chromosome 11q12-q13.1 gene-rich region identifies several newly described conserved genes.

Despite the localization of several human diseases to 11q13, the majority of the genes responsible for these disorders have not yet been cloned. Exon amplification and EST mapping were performed using clones derived from an approximately 1.65-Mb P1 artificial chromosome contig encompassing the region that reportedly harbors the gene mutated in the dominantly inherited eye disorder, Best disease. Fifty-eight exons isolated from the region were sequenced, resulting in 41.3% showing weak or no similarity to database sequences. Four exons had exact matches with human ESTs and 2 exons were highly similar to mouse ESTs. The sequence of 1 of these human ESTs was highly similar to that of the rat Rabin3 and mouse Pat-12 genes, which potentially encode Ras-like GTPase binding proteins. Three exon sequences were similar to those of the inner centromere proteins of Gallus gallus and Xenopus laevis, which are mitotic phosphoproteins, and 1 exon sequence had similarity to the epidermal growth factor-like repeat from several proteins. High-resolution mapping of 34 ESTs binned to the 11q12-q13 region by the Human Transcript Mapping Project identified 5 present in the PAC contig, with 1 of these ESTs identifying a human homologue of the rat synaptotagmin VII gene. Database searches identified two overlapping cDNA clones representing almost the entire open reading frame of this human gene and a sequenced cosmid indicating its partial genomic structure. Further database analyses identified another sequenced cosmid from this region that contained both exon-trap and mapped EST sequences. PowerBLAST and GRAIL analysis of this cosmid sequence identified matches with several other ESTs, the previously described FEN1 gene, and a novel evolutionarily conserved gene. These experiments identify candidate genes for disorders that map to this region and indicate that this is a gene-rich region of the human genome.

A radiation hybrid map of human chromosome 5 with integration of cytogenetic, genetic, and transcript maps.

One of the major goals of the human genome project is to establish a physical map of each human chromosome with a density of sequence-tagged site (STS) markers exceeding one every 100 kb. We report here the generation of a human chromosome 5-specific radiation hybrid (RH) map that includes 556 markers. Of these markers, 132 loci are ordered with a maximum likelihood ratio of >1000:1 compared with the next most likely order. An additional 113 loci were ordered relative to these backbone markers with a maximum likelihood ratio of >10:1 but <1000:1. Together, these 245 loci form an ordered framework map for the chromosome. Using this framework, >300 more markers were localized based on two-point analysis with the ordered set. On average, there are 50 markers in common with the RH map presented here and other chromosome 5 maps included in the current whole genome cytogenetic, genetic, and physical maps. The accuracy of all the maps is evident in that there are no more than two discrepancies between any one of them and these data. All of the maps encompassing chromosome 5 complement each other providing excellent STS coverage with >2200 loci combined. The chromosome 5-specific RH map contains 20% of these independent loci. In addition, our RH map contains STSs derived from clones suitable for fluorescent in situ hybridization, allowing alignment to the cytogenetic map. Together, these maps will assist in the assembly of sequence-ready contigs and will aid in the identification of disease loci on chromosome 5 by positional cloning and positional candidate approaches.

A high-resolution physical and transcript map of the Cri du chat region of human chromosome 5p.

A high-resolution physical and transcription map has been generated of a 3.5-Mb region of 5p15.2 that is associated with the Cri du chat (CDC) syndrome. Utilizing a variety of resources including a natural deletion panel, a chromosome specific radiation hybrid panel, and YAC, PAC, and BAC genomic clones we have ordered > 60 STSs within this region. Approximately 45% of these STSs were obtained from publicly available whole genome maps, thus allowing for integration of this map with currently available resources. Thirteen of these markers were ESTs. In addition, > 70 exon trapped products have been mapped on the natural deletion panel and bacterial clone resource. The combination of these resources has allowed for the identification of 17 transcripts within this region, all of which represent candidate genes for CDC. Further characterization of the genomic contig also revealed that this region of 5p15 contains a large number of repetitive elements.

The Ras GTPase-activating-protein-related human protein IQGAP2 harbors a potential actin binding domain and interacts with calmodulin and Rho family GTPases.

We previously described IQGAP1 as a human protein related to a putative Ras GTPase-activating protein (RasGAP) from the fission yeast Schizosaccharomyces pombe. Here we report the identification of a liver-specific human protein that is 62% identical to IQGAP1. Like IQGAP1, the novel IQGAP2 protein harbors an N-terminal calponin homology motif which functions as an F-actin binding domain in members of the spectrin, filamin, and fimbrin families. Both IQGAPs also harbor several copies of a novel 50- to 55-amino-acid repeat, a single WW domain, and four IQ motifs and have 25% sequence identity with almost the entire S. pombe sar1 RasGAP homolog. As predicted by the presence of IQ motifs, IQGAP2 binds calmodulin. However, neither full-length nor truncated IQGAP2 stimulated the GTPase activity of Ras or its close relatives. Instead, IQGAP2 binds Cdc42 and Racl but not RhoA. This interaction involves the C-terminal half of IQGAP2 and appears to be independent of the nucleotide binding status of the GTPases. Although IQGAP2 shows no GAP activity towards Cdc42 and Rac1, the protein did inhibit both the intrinsic and RhoGAP-stimulated GTP hydrolysis rates of Cdc42 and Rac1, suggesting an alternative mechanism via which IQGAPs might modulate signaling by these GTPases. Since IQGAPs harbor a potential actin binding domain, they could play roles in the Cdc42 and Rac1 controlled generation of specific actin structures.

Molecular definition of deletions of different segments of distal 5p that result in distinct phenotypic features.

Cri du chat syndrome (CDC) is a segmental aneusomy associated with deletions of chromosome 5p15. In an effort to define regions that produce the phenotypes associated with CDC, we have analyzed deletions from 17 patients. The majority of these patients had atypical CDC features or were asymptomatic. Using these patients, we have mapped several phenotypes associated with deletions of 5p, including speech delay, catlike cry, newborn facial dysmorphism, and adult facial dysmorphism. This phenotypic map should provide a framework with which to begin identification of genes associated with various phenotypic features associated with deletions of distal 5p. We have also analyzed the parental origin of the de novo deletions, to determine if genomic imprinting could be occurring in this region. In addition, we have isolated cosmids that could be useful for both prenatal and postnatal assessments of del5(p) individuals.

Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia.

Achondroplasia (ACH) is the most common genetic form of dwarfism. This disorder is inherited as an autosomal dominant trait, although the majority of cases are sporadic. A gene for ACH was recently localized to 4p16.3 by linkage analyses. The ACH candidate region includes the gene encoding fibroblast growth factor receptor 3 (FGFR3), which was originally considered as a candidate for the Huntington's disease gene. DNA studies revealed point mutations in the FGFR3 gene in ACH heterozygotes and homozygotes. The mutation on 15 of the 16 ACH-affected chromosomes was the same, a G-->A transition, at nucleotide 1138 of the cDNA. The mutation on the only ACH-affected chromosome 4 without the G-->A transition at nucleotide 1138 had a G-->C transversion at this same position. Both mutations result in the substitution of an arginine residue for a glycine at position 380 of the mature protein, which is in the transmembrane domain of FGFR3.

Isolation of genes from complex sources of mammalian genomic DNA using exon amplification.

Modifications to exon amplification have been instituted that increase its speed, efficiency and reliability. Exons were isolated from target human or mouse genomic DNA sources ranging from 30 kilobases (kb) to 3 megabases (Mb) in complexity. The efficiency was dependent upon the amount of input DNA, and ranged from isolation of an exon for every 20 kb to an exon for every 80 kb of target genomic DNA. In these studies, several novel genes and a smaller number of genes isolated previously that reside on human chromosome 9 have been identified. These results indicate that exon amplification is presently adaptable to large scale isolation of exons from complex sources of genomic DNA.

Identification of human chromosome 9 specific genes using exon amplification.

We have recently developed a method, exon amplification, that is designed for isolation of exon sequences from genomic DNA. To assess the efficacy of this method we have analyzed cosmid genomic clones derived from human chromosome 9, and have cloned several products from this analysis. Approximately 63% of cosmids produced at least one product derived from functioning splice sites within the target genomic fragment, and in many cases multiple products were isolated. In addition, an easily identifiable class of false positives was produced from 56% of cosmids analyzed; these are readily eliminated from subsequent study. Sequence analysis and database searches revealed that the majority (87%) of the putative exon clones were unique, the remainder being derived from repetitive sequences. Analysis of sequence conservation by Southern blotting in addition to cDNA screening experiments suggested that most, if not all, of these unique sequences represent true exons. The results of these studies indicate that exon amplification is a rapid and reliable approach for isolation of exon sequences from mammalian genomic DNA.