Greg Lennon

Mutations in the Cacnl1a4 calcium channel gene are associated with seizures, cerebellar degeneration, and ataxia in tottering and leaner mutant mice

Tottering and leaner, two mutations of the mouse tottering locus, have been studied extensively as models for human epilepsy. Here we describe the isolation, mapping, and expression analysis of Cacnl1a4, a gene encoding the alpha subunit of a proposed P-type calcium channel, and also report the physical mapping and expression patterns of the orthologous human gene. DNA sequencing and gene expression data demonstrate that Cacnl1a4 mutations are the primary cause of seizures and ataxia in tottering and leaner mutant mice, and suggest that tottering locus mutations and human diseases, episodic ataxia 2 and familial hemiplegic migraine, represent mutations in mouse and human versions of the same channel-encoding gene.

An encyclopedia of mouse genes

The laboratory mouse is the premier model system for studies of mammalian development due to the powerful classical genetic analysis possible (see also the Jackson Laboratory web site, http://www.jax.org/) and the ever–expanding collection of molecular tools. To enhance the utility of the mouse system, we initiated a program to generate a large database of expressed sequence tags (ESTs) that can provide rapid access to genes. Of particular significance was the possibility that cDNA libraries could be prepared from very early stages of development, a situation unrealized in human EST projects. We report here the development of a comprehensive database of ESTs for the mouse. The project, initiated in March 1996, has focused on 5´ end sequences from directionally cloned, oligo–dT primed cDNA libraries. As of 23 October 1998, 352,040 sequences had been generated, annotated and deposited in dbEST, where they comprised 93% of the total ESTs available for mouse. EST data are versatile and have been applied to gene identification, comparative sequence analysis, comparative gene mapping and candidate disease gene identification, genome sequence annotation, microarray development and the development of gene–based map resources.

Molecular profiling of clinical tissue specimens: feasibility and applications.

The relationship between gene expression profiles and cellular behavior in humans is largely unknown. Expression patterns of individual cell types have yet to be precisely measured, and, at present, we know or can predict the function of a relatively small percentage of genes. However, biomedical research is in the midst of an informational and technological revolution with the potential to increase dramatically our understanding of how expression modulates cellular phenotype and response to the environment. The entire sequence of the human genome will be known by the year 2003 or earlier. 1,2 In concert, the pace of efforts to complete identification and full-length cDNA sequencing of all genes has accelerated, and these goals will be attained within the next few years. 3-7 Accompanying the expanding base of genetic information are several new technologies capable of global gene expression measurements. 8-16 Taken together, the expanding genetic database and developing expression technologies are leading to an exciting new paradigm in biomedical research known as molecular profiling.

SNPedia: a wiki supporting personal genome annotation, interpretation and analysis

SNPedia (http://www.SNPedia.com) is a wiki resource of the functional consequences of human genetic variation as published in peer-reviewed studies. Online since 2006 and freely available for personal use, SNPedia has focused on the medical, phenotypic and genealogical associations of single nucleotide polymorphisms. Entries are formatted to allow associations to be assigned to single genotypes as well as sets of genotypes (genosets). In this article, we discuss the growth of this resource and its use by affiliated software to create personal genome reports.

Molecular Profiling of Clinical Tissue Specimens : Feasibility and Applications

The relationship between gene expression profiles and cellular behavior in humans is largely unknown. Expression patterns of individual cell types have yet to be precisely measured, and, at present, we know or can predict the function of a relatively small percentage of genes. However, biomedical research is in the midst of an informational and technological revolution with the potential to increase dramatically our understanding of how expression modulates cellular phenotype and response to the environment. The entire sequence of the human genome will be known by the year 2003 or earlier. 1, 2 In concert, the pace of efforts to complete identification and full-length cDNA sequencing of all genes has accelerated, and these goals will be attained within the next few years. 3, 4, 5, 6, 7 Accompanying the expanding base of genetic information are several new technologies capable of global gene expression measurements. 8, 9, 10, 11, 12, 13, 14, 15, 16 Taken together, the expanding genetic database and developing expression technologies are leading to an exciting new paradigm in biomedical research known as molecular profiling.

IMAGEne I: clustering and ranking of I.M.A.G.E. cDNA clones corresponding to known genes

MOTIVATION To enhance the usefulness of the I.M.A.G.E. Consortium (Lennon et al., 1996, Genomics, 33, 151-152) cDNA clone collection by directed analysis and organization of their associated Expressed Sequence Tags (ESTs), thus enabling effective mining of the immense amounts of public cDNA information. RESULTS This paper introduces the IMAGEne suite of tools, which clusters ESTs around known genes, then ranks each clone within a cluster. IMAGEne filters data from known gene sequence databases and the GenBanks EST database (Boguski and Shuler, 1995, Nature Genet., 10, 369-371). It applies biological criteria in connection with judicious use of the BLAST (Altschul et al., 1990, J. Mol. Biol., 215), FASTA (Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA, 85, 2444-2448; Pearson, 1990, Methods Enzymol., 183, 63-98; Gusfield, 1997, Algorithms on Strings, Trees, and Sequences, Cambridge University Press), and SIM (Huang et al., 1990, Comput. Appl. Biosci., 6, 373-381) tools to form known gene clusters. It then applies criteria derived from experienced biologists to select the best representative I.M.A.G.E. clone for a gene. The tool provides an intuitive Java interface for query and display of the gene and its associated clones, thus directing researchers in selecting a clone that will best enhance their research. An important product is a listing of clones that best represent all known genes. The listing will be used for re-arraying clones into minimally redundant Master Arrays. Both the listings and Master Arrays will be made available to the public, which will be a valuable resource to the genomic community in furthering discovery in the area of gene function.

SIX TRANSCRIPTS MAP WITHIN 200 KILOBASES OF THE MYOTONIC DYSTROPHY EXPANDED REPEAT

Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disorder characterized by a highly variable clinical phenotype. Symptoms include myotonia, progressive weakness and wasting of muscles, cardiac conduction defects, cataracts, and diabetes (Harper 1989). The underlying genetic mutation in this disease is an expanded triplet repeat sequence (CTG) located within the 38 untranslated region of the myotonic dystrophy protein kinase gene (DMPK) (Brook et al. 1992a). Normal alleles have between 5 and 34 copies of the repeat, whereas affected individuals have up to several thousand copies. There is a general correlation between severity of the disease and length of the repeat (Harley et al. 1993), certainly for expansions less than 1.2 kb (Hamshere et al. 1997). The mechanism by which the expanded repeat causes the underlying pathology in DM is not clear, and three different mechanisms have been suggested. First, the repeat may exert an effect directly on the expression of DMPK, altering the levels of RNA and protein. Second, the repeat may cause an effect in trans at either the DNA or RNA level via interactions with other nuclear proteins or nucleic acids. Third, the expanded repeat may affect the expression of several genes through a field effect, possibly on chromatin structure. To test this third possibility it is necessary to identify all of the transcripts in the vicinity of the DM triplet repeat. In order to construct a detailed transcript map around the DMassociated repeat, we performed exon trap analysis of a 200-kb contig for which the closest flanking markers are D19S219 and D19S412 (Fig. 1).BamHI and BglI double-digested phage DNA was shot-gun cloned into the pSPL3 vector and transfected into COS-7 cells. Following established protocols (Church et al. 1994; Buckler et al. 1991; Li et al. 1996), exons were isolated through the selection of functional 5 8 and 38 splice sites in COS-7 cells. Several exons and cDNAs have been identified, sequenced, and searched against available DNA databases. In addition to exons and cDNAs corresponding to DMPK (Brook et al. 1992b), 59 (Shaw et al. 1993), and DMAHP (Boucher et al. 1995) which are known to map to this interval, seven other exons have been identified including pSV23B, pSV20D7, and pSV5C. Each of these exons has been hybridized to zoo blots and screened against cDNA libraries. Figure 2 shows the results of the zoo blot analysis with pSV23B (size 105 bp), pSV20D7 (size 354 bp), pSV5C (size 238 bp) and pSV9T9 (size 369 bp) which corresponds to gene 59. All four exons demonstrated cross species sequence conservation. Two cDNAs have been identified with exon pSV20D7 when screened against brain cDNA libraries. No clones were obtained from a fetal muscle cDNA library. Clones p20D7-FC4 and p20D7SN10 were obtained from adult frontal cortex and substantia nigra libraries respectively. Both cDNAs map to overlapping genomic clonesl36 andl20 (Fig. 1). Sequence analysis of p20D7-FC4 (1.6 kb) and p20D7-SN10 (0.8 kb) indicates that the latter clone is contained entirely within the former, and p20D7-FC4 contains a long poly A stretch indicative of the 3 8 end of the transcript. This gene has been designated 20D7. There is a strong Kozak consensus sequence (Kozak 1987) at nucleotides 29 to 37 (accATGACC) of p20D7-FC4 followed by an open reading frame which would encode a putative protein of 202 amino acids (Fig. 3a). This is the longest open reading frame for this sequence. The gene is transcribed in the same orientation as DMPK. DNA database searches failed to reveal significant similarities to known genes, though complete sequences from expressed sequence tags (ESTs) T73173, T11851, T24520, and AA035309 correspond to parts of 20D7. AA035309 (268 bp) has been assigned as a Unigene (Schuler et al. 1996). Comparison of the sequence from exon pSV20D7 to the 20D7 cDNA sequences revealed that the exon trapped product was spliced in the opposite orientation to which the cDNAs are transcribed. Similar findings of exons spliced in the opposite orientation to the cDNAs they identify have been reported previously (Church et al. 1994). cDNAs transcribed in the same orientation as pSV20D7 have yet to be identified. Northern blot analysis of poly (A) RNA from six rat tissues (muscle, lung, heart, testis, brain, and liver) probed with a gel-purified restriction fragment from p20D7-FC4 revealed expression of a 2.3-kb transcript in testis (Fig. 3b). cDNAs p5C-K7 and p5C-FC6 were identified by screening fetal muscle and adult brain libraries respectively, with exon pSV5C (Buckler et al. 1991). The cDNA clones were mapped back to genomic phage clones lM5C, lM13J, lM11F, andlM12D (Fig. 1). A previously isolated cDNA clone, pK2C-17 (Brook et al. 1992b) also hybridized to genomic clones lM11F andlM12D. The sequences of cDNAs p5C-K7 (1.64 kb), p5C-FC6 (2.18 kb), and pK2C-17 (2.2 kb) were compared and found to overlap. A polyadenylation addition site and long poly A tail were detected within the sequence of pK2C-17, indicating that this clone contains the 38 end of the transcript. A composite cDNA of 4011bp, termed 5C2C, was assembled with p5C-K7, p5C-FC6, and pK2C-17, and confirmed by RT-PCR analysis of poly (A) + RNA from adult human brain, testis, and muscle with primer sequences derived from p5C-FC6 and pK2C-17. DNA sequence database searches with 5C2C revealed almost identical sequence to human SYMPLEKIN, a tight junction plaque protein. This protein was reported to be the product of a ∼3.7-kb transcript encoding a polypeptide of 1142 amino acids (Keon et al. 1996). The composite 5C2C cDNA encodes a predicted protein of the same size as SYMPLEKIN but with a longer 58UTR and covers approximately 60 kb of genomic DNA. Northern blot analysis with pK2C-17 (Fig. 3c) and p5C-K7 (data not shown) indicated that these clones show the same extent of expression in heart and brain of an mRNA of around 4.5 kb. Correspondence to: J.D. Brook Mammalian Genome 9, 485–487 (1998).

Localization and genomic structure of human deoxyhypusine synthase gene on chromosome 19p13.2-distal 19p13.1.

The amino acid hypusine is formed post-translationally in a single cellular protein, the eukaryotic translation initiation factor 5A, by two enzymes, namely deoxyhypusine synthase and deoxyhypusine hydroxylase. Hypusine is found in all eukaryotes and in some archaebacteria, but not in eubacteria. The deoxyhypusine synthase cDNA was cloned and mapped by fluorescence in situ hybridization on chromosome 19p13.11-p13.12. Rare cDNAs containing internal deletions were also found. We localized the deoxyhypusine synthase gene on a high resolution cosmid/BAC contig map of chromosome 19 to a region in 19p13.2-distal 19p13.1 between MANB and JUNB. Analysis of the genomic exon/intron structure of the gene coding region showed that it consists of nine exons and spans a length of 6.6kb. From observation of the genomic structure, it seems likely that the internally deleted forms of mature RNA are the result of alternative splicing, rather than of artifacts.

Genetic and Physical Localisation of the Gene Causing Cone-Rod Dystrophy (CORD2)

Choroidoretinal dystrophies are incurable and essentially untreatable, representing the most common cause of genetic visual loss in childhood (1). They are a clinically and genetically heterogeneous group of disorders. Dystrophies which primarily affect rod function such as retinitis pigmentosa have been extensively studied and a number of genes have been implicated in the disease pathogenesis (2). In contrast dystrophies which primarily affect cone photoreceptors have been less well studied and examples of this group of diseases includes cone dystrophies and cone-rod dystrophies. Cone dystrophies are characterised by photophobia, loss of visual acuity and colour vision defects associated with reduced cone photoreceptor ERG responses. Abnormal pigmentation with atrophy is often seen at the macula. Cone-rod dystrophies are distinct from cone dystrophies in that abnormalities of cone dysfunction is seen with progressive peripheral retinal disease. Diminished visual acuity and loss of colour discrimination is followed by nyctalopia, progressive peripheral visual field deficit and decreasing rod photoreceptor ERG amplitudes from an early age. Advancing chorioretinal atrophy of the central and peripheral retina is characteristic (3). Autosomal dominant, recessive and X-linked patterns of inheritance for cone dystrophies and cone-rod dystrophy (CRD) have been described and studies have implicated a number of loci for the disease-causing genes. Loci associated with cone dystrophies include a balanced translocation on chromosome 6q which was reported in a patient with mental retardation and cone dystrophy (4), an X-linked cone dystrophy mapping to Xp21.1–p11.3 (5) and two independent studies have used linkage analysis to identify an autosomal dominant cone dystrophy locus on chromosome 17p (6–7). Loci implicated in CRD include two case reports that have suggested localisation of CRD genes on chromosome 18q (8) and 17q (9), an autosomal dominant form of CRD mapping to chromosome 19q (10) and a transverse mutation in the peripherin/RDS gene (Asn244His) has been found in one Japanese CRD family (11).

Challenges and opportunities in constructing comprehensive gene expression databases

MAExplorer is a Java applet that runs in a user’s Web browser. It allows the exploratory data analysis of quantitative cDNA expression profiles across multiple microarrays. Data may be viewed and directly manipulated in images, scatter plots, histograms, expression profile plots, cluster analysis and so on. A key feature is the clone “Filter” for constraining a working set of clones passing a variety of user specified tests. Reports may be generated with Web access to UniGene, GeneBank and other Internet databases for sets of clones found to be of interest. Reports may also be exported to MS-Excel. MAExplorer is being developed in a collaboration between the Laboratory of Genetics and Physiology (LGP, NIDDK) and the Laboratory of Experimental and Computational Biology (LECB,NCI). LGP has established a program designed to identify and understand genetic pathways operative during normal mammary gland development and tumorigenesis. One arm of this program focuses on the use of cDNA microarrays to profile gene expression patterns. For this purpose, cDNA (EST) libraries are generated, sequenced and clone inserts are spotted on nylon membranes. These membranes are used to monitor expression profiles under various physiological conditions. At this point, expression profiles have been obtained from several stages of normal mammary gland development and different tumour models. With this program, you may: (i) analyse the expression of individual genes; (ii) analyse the expression of gene families and clusters; (iii) compare expression patterns. Data is downloaded as required to the user’s Web browser to perform real-time analyses on the user’s computer. However, the user has access to the entire database and may save and share their explorations in a groupware environment. The MAExplorer may be accessed through the MGAP web site at http://mammary.nih.gov/mgap