Integrating clinical and laboratory data in genetic studies of complex phenotypes: a network-based data management system.

The identification of genes underlying a complex phenotype can be a massive undertaking, and may require a much larger sample size than thought previously. The integration of such large volumes of clinical and laboratory data has become a major challenge. In this paper we describe a network-based data management system designed to address this challenge. Our system offers several advantages. Since the system uses commercial software, it obviates the acquisition, installation, and debugging of privately-available software, and is fully compatible with Windows and other commercial software. The system uses relational database architecture, which offers exceptional flexibility, facilitates complex data queries, and expedites extensive data quality control. The system is particularly designed to integrate clinical and laboratory data efficiently, producing summary reports, pedigrees, and exported files containing both phenotype and genotype data in a virtually unlimited range of formats. We describe a comprehensive system that manages clinical, DNA, cell line, and genotype data, but since the system is modular, researchers can set up only those elements which they need immediately, expanding later as needed.

A novel, heritable, expanding CTG repeat in an intron of the SEF2-1 gene on chromosome 18q21.1.

There are currently 13 diseases known to be caused by unstable triplet repeat mutations; however, there are some instances (as with FRAXF and FRA16) when these mutations appear to be asymptomatic. In a search for polymorphic CTG repeats as candidate genes for bipolar disorder, we screened a genomic human chromosome 18-specific library and identified a 1.6 kb clone (7,6A) with a CTG24 repeat that maps to 18q21.1. The CTG repeat locus, termed CTG18.1, is located within an intron of human SEF2-1, a gene encoding a basic hellx-loop-hellx DNA binding protein involved in transcriptional regulation. The CTGn repeat is highly polymorphic and very enlarged alleles, consistent with expansions of up to CTG2100, were identified. PCR and Southern blot analysis in pedigrees ascertained for a Johns Hopkins University bipolar disorder linkage study and in CEPH reference pedigrees revealed a tripartite distribution of CTG18.1 alleles with stable alleles (CTG10-CTG37), moderately enlarged and unstable alleles (CTG53-CTG250), and very enlarged, unstable alleles (CTG800-CTG2100). Moderately enlarged alleles were not associated with an abnormal phenotype and have a combined enlarged allele frequency of 3% in the CEPH and bipolar populations. Very enlarged alleles, detectable only by Southern blot analysis of genomic digests, have thus far been found in only three individuals from our bipolar pedigrees, and to date, have not been found in any of the CEPH reference pedigrees. These enlarged alleles may arise, at least in part, via somatic mutation.

cDNAs with long CAG trinucleotide repeats from human brain.

Twelve diseases, most with neuropsychiatric features, arise from trinucleotide repeat expansion mutations. Expansion mutations may also cause a number of other disorders, including several additional forms of spinocerebellar ataxia, bipolar affective disorder, schizophrenia, and autism. To obtain candiate genes for these disorders, cDNA libraries from adult and fetal human brain were screened at high stringency for clones containing CAG repeats. Nineteen cDNAs were isolated and mapped to chromosomes 1, 2, 4, 6, 7, 8, 9, 12, 16, 19, 20, and X. The clones contain between 4 and 17 consecutive CAG, CTG, TCG, or GCA triplets. Clone H44 encodes 40 consecutive glutamines, more than any other entry in the nonredundant GenBank protein database and well within the range that causes neuronal degeneration in several of the glutamine expansion diseases. Eight cDNAs encode 15 or more consecutive glutamine residues, suggesting that the gene products may function as transcription factors, with a potential role in the regulation of neurodevelopment or neuroplasticity. In particular, the conceptual translation of clone CTG3a contains 18 consecutive glutamines and is 45% identical to the C-terminal 306 residues of the mouse numb gene product. These genes are therefore candidates for diseases featuring anticipation, neurodegeneration, or abnormalities of neurodevelopment.

Linkage disequilibrium analysis of G-olf alpha (GNAL) in bipolar affective disorder.

This study examines G-olf alpha as a possible candidate gene for susceptibility to bipolar affective disorder (BPAD) using the Transmission Disequilibrium Test (TDT). G-olf alpha, which encodes a subunit of a G-protein involved in intracellular signaling, maps within a region of chromosome 18 that has been implicated by two different linkage studies as a potential site of BPAD susceptibility loci. The expression pattern of G-olf alpha in the brain, its coupling to dopamine receptors, and the effects of lithium salts on G-proteins all support G-olf alpha as a candidate gene for BPAD. Our study population consisted of 106 probands and sibs with bipolar I disorder, with a median age-at-onset of 21.5 years ascertained from the United States. There was no evidence of linkage disequilibrium between BPAD and any of the observed G-olf alpha alleles in our sample. Division of families based on sex of the transmitting parent did not significantly change the results. This sample had good power (78%) to detect linkage disequilibrium with alleles conferring a relative risk equal to that estimated for the putative 18p locus (2.58). Our results do not support a major role for G-olf alpha as a susceptibility locus for BPAD in a substantial portion of our sample. Other genes lying near G-olf alpha within the linked region on chromosome 18 cannot be excluded by our data. This study illustrates the use of the TDT in evaluating candidate genes within linked chromosome regions.

cDNA cloning of a human homologue of the Caenorhabditis elegans cell fate-determining gene mab-21: expression, chromosomal localization and analysis of a highly polymorphic (CAG)n trinucleotide repeat.

The two most consistent features of the diseases caused by trinucleotide repeat expansion-neuropsychiatric symptoms and the phenomenon of genetic anticipation-may be present in forms of dementia, hereditary ataxia, Parkinsonism, bipolar affective disorder, schizophrenia and autism. To identify candidate genes for these disorders, we have screened human brain cDNA libraries for the presence of gene fragments containing polymorphic trinucleotide repeats. Here we report the cDNA cloning of CAGR1, originally detected in a retinal cDNA library. The 2743 bp cDNA contains a 1077 bp open reading frame encoding 359 amino acids. This amino acid sequence is homologous (56% amino acid identify and 81% amino acid conservation) to the Caenorhabditis elegans cell fate-determining protein mab-21. CAGR1 is expressed in several human tissues, most prominently in the cerebellum, as a message of approximately 3.0 kb. The gene was mapped to 13q13, just telomeric to D13S220. A 5'-untranslated CAG trinucleotide repeat is highly polymorphic, with repeat length ranging from six to 31 triplets and a heterozygosity of 87-88% in 684 chromosomes from several human populations. One allele from an individual with an atypical movement disorder and bipolar affective disorder type II contains 46 triplets, 15 triplets longer than any other allele detected. Though insufficient data are available to link the long repeat to this clinical phenotype, an expansion mutation of the CAGR1 repeat can be considered a candidate for the etiology of disorders with anticipation or developmental abnormalities, and particularly any such disorders linked to chromosome 13.

Evidence for linkage of bipolar disorder to chromosome 18 with a parent-of-origin effect.

A susceptibility gene on chromosome 18 and a parent-of-origin effect have been suggested for bipolar affective disorder (BPAD). We have studied 28 nuclear families selected for apparent unilineal transmission of the BPAD phenotype, by using 31 polymorphic markers spanning chromosome 18. Evidence for linkage was tested with affected-sib-pair and LOD score methods under two definitions of the affected phenotype. The affected-sibpair analyses indicated excess allele sharing for markers on 18p within the region reported previously. The greatest sharing was at D18S37: 64% in bipolar and recurrent unipolar (RUP) sib pairs (P = .0006). In addition, excess sharing of the paternally, but not maternally, transmitted alleles was observed at three markers on 18q: at D18S41, 51 bipolar and RUP sib pairs were concordant for paternally transmitted alleles, and 21 pairs were discordant (P = 0004). The evidence for linkage to loci on both 18p and 18q was strongest in the 11 paternal pedigrees, i.e., those in which the father or one of the father's sibs is affected. In these pedigrees, the greatest allele sharing (81%; P = .00002) and the highest LOD score (3.51; phi = 0.0) were observed at D18S41. Our results provide further support for linkage of BPAD to chromosome 18 and the first molecular evidence for a parent-of-origin effect operating in this disorder. The number of loci involved, and their precise location, require further study..

Polymorphic (AAT) in trinucleotide repeats derived from a human brain cDNA library.

Seven cDNA fragments containing polymorphic (AAT)n trinucleotide repeats were isolated from a human brain cDNA library and mapped by linkage to specific loci. These repeats may serve as gene markers or as candidates for diseases caused by expansion mutation.

Characterization of cDNA clones containing CCA trinucleotide repeats derived from human brain.

Expansion mutation is the cause of eight neuropsychiatric disorders. Thus far each disease is the result of expansion of a C-G rich trinucleotide repeat that is polymorphic for length in the general population. We now report the identification of seven novel cDNA clones with CCA or equivalent trinucleotide repeats obtained by screening a human frontal cortex cDNA library. The repeat lengths of two clones, CCA11 (linked to D20S101, expressed in human brain as a 3.2 kb message) and CCA38 (linked to D5S404), are highly polymorphic in a normal human population. CCA54, mapped to chromosome 19, appears to correspond to a portion of the human gene encoding the alpha 1 subunit of a P-type calcium channel. Expansion mutations at these loci should be considered as possible candidates in evaluating the genetic etiologies of diseases linked to chromosomes 5, 19, and 20.

Use of the API rapid NFT system for identifying nonfermentative and fermentative marine bacteria.

Thirty-five American Type Culture Collection type strains of marine bacteria were used to evaluate the Rapid NFT system (API Analab Products, Plainview, N.Y.) for use in identifying heterotrophic marine bacteria. The 21 biochemical and assimilation tests on the Rapid NFT test strips were treated according to the manufacturer's protocol, which included use of AUX medium (provided with the Rapid NFT system) for preparing assimilation tests, and by substituting phenol red broth base (BBL Microbiology Systems, Cockeysville, Md.) with and without an oil overlay for the AUX medium. A seven-digit numerical profile was obtained for each NFT test strip from each of the three procedures and matched to its corresponding number in the Rapid NFT identification codebook. Also, all biochemical and assimilation test results were analyzed with SASTAXAN and SAS/GRAPH programs (SAS Institute, Inc., Cary, N.C.); similarity matrices were computed for all 35 strains. For comparison purposes, bacterial strains were grouped at a similarity level of 70%. The results indicated a low efficacy of identification for all three procedures. In addition, similarity matrix analysis showed more cohesive grouping based on results of phenol red broth base-treated strains than for the AUX medium provided by the manufacturer. However, none of the three treatments provided exclusive grouping of type strains at the genus level. Thus, the reliability of the data obtained from the NFT system and modifications thereof should be evaluated carefully when environmental isolates are characterized.