A contiguous 3-Mb sequence-ready map in the S3-MX region on 21q22.2 based on high- throughput nonisotopic library screenings.

Progress in complete genomic sequencing of human chromosome 21 relies on the construction of high-quality bacterial clone maps spanning large chromosomal regions. To achieve this goal, we have applied a strategy based on nonradioactive hybridizations to contig building. A contiguous sequence-ready map was constructed in the Down syndrome congenital heart disease (DS-CHD) region in 21q22.2, as a framework for large-scale genomic sequencing and positional candidate gene approach. Contig assembly was performed essentially by high throughput nonisotopic screenings of genomic libraries, prior to clone validation by (1) restriction digest fingerprinting, (2) STS analysis, (3) Southern hybridizations, and (4) FISH analysis. The contig contains a total of 50 STSs, of which 13 were newly isolated. A minimum tiling path (MTP) was subsequently defined that consists of 20 PACs, 2 BACs, and 5 cosmids covering 3 Mb between D21S3 and MX1. Gene distribution in the region includes 9 known genes (c21-LRP, WRB, SH3BGR, HMG14, PCP4, DSCAM, MX2, MX1, and TMPRSS2) and 14 new additional gene signatures consisting of cDNA selection products and ESTs. Forthcoming genomic sequence information will unravel the structural organization of potential candidate genes involved in specific features of Down syndrome pathogenesis.

The gene encoding the alpha1A-voltage-dependent calcium channel (CACN1A4) is not a candidate for causing common subtypes of idiopathic generalized epilepsy.

Mutations in the gene encoding the alpha1A-calcium channel subunit play a causative role in the epileptogenesis of absence seizures in tottering mutant mice. The present family-based association and non-parametric linkage study tested the hypothesis that allelic variants of the homologous human gene (CACN1A4) confer susceptibility to common subtypes of idiopathic generalized epilepsy (IGE). An expressed polymorphic CAG trinucleotide repeat in the 3' end of the CACN1A4 gene was assessed in 70 families ascertained through members with either childhood (CAE) and juvenile absence epilepsy (JAE), or juvenile myoclonic epilepsy (JME). Our association analysis using the haplotype-based haplotype relative risk statistic provided no evidence for an allelic association of the CAG repeat polymorphism with either IGE, or CAE and JAE, or JME. We found no relation between the CAG repeat length and susceptibility neither to IGE, nor to CAE and JAE, nor to JME. Non-parametric linkage analysis revealed no evidence for linkage of IGE traits with the CACN1A4 locus in 42 families of patients with either CAE or JAE. A weak trend towards an excess of allele sharing (identity by descent) among family members affected by an IGE was obtained in 26 families of JME patients (Z[NPL] = 1.25 at theta = 0.000, p = 0.057). Taken together, we found no statistically significant evidence that genetic variants of the CACN1A4 gene play a causative role in the pathogenesis of common subtypes of IGE in humans.

Refined mapping of the epilepsy susceptibility locus EJM1 on chromosome 6.

Juvenile myoclonic epilepsy (JME) is a genetically determined common subtype of idiopathic generalized epilepsy. Linkage to the HLA complex on chromosome 6p21.3 and an allelic association with HLA-DR13 and -DQB1 alleles suggest that a susceptibility locus for JME, designated as "EJM1," is located within or near the HLA region. However, further studies revealed controversial results, and genetic heterogeneity has been suspected. The present study was designed to evaluate the validity of the association and linkage findings and to refine the map position of EJM1. Our association analysis showed no significant difference of the frequency of HLA-DR13 carriers in 62 German JME patients compared with that in 77 German controls (X2 = 0.98, df = 1, p = 0.161, one-tailed). Multipoint linkage analysis with use of microsatellite markers from the chromosomal region 6p25-q13 in 29 German families of JME patients provided significant evidence that an epilepsy locus (EJM1) close to the HLA locus confers susceptibility to "idiopathic" generalized seizures (Zmax = 3.27 at theta max = 0.033 centromeric to the HLA-DQ locus), assuming an autosomal dominant mode of inheritance with 70% penetrance. Haplotype analyses revealed key recombinations in five families, which locate EJM1 to the centromeric side of the HLA-DQ locus. This study confirms a causative role of EJM1 in the pathogenesis of idiopathic generalized seizures in the majority of German families of JME patients and refines a candidate region of 10.1 cM in the chromosomal region 6p21 between the flanking loci HLA-DQ and D6S1019. A possible explanation for the current controversial results in families of different populations might be ethnic variation of interfering polygenic effects that could be permissive for heterogeneous susceptibility alleles.

Allelic association of juvenile absence epilepsy with a GluR5 kainate receptor gene (GRIK1) polymorphism.

Juvenile absence epilepsy (JAE) is a common subtype of idiopathic generalized epilepsy (IGE). Hereditary factors play a major role in its etiology. The important function of glutamate receptors (GluRs) in excitatory neurotransmission, synaptic plasticity, and neurodevelopment suggests their involvement in epileptogenesis. A tetranucleotide repeat polymorphism in the non-coding region of the kainate-selective GluR5 receptor gene (GRIK1) on chromosome 21q22.1 provides the tool to investigate this candidate gene. The present association and linkage study tested the hypothesis that allelic variants of GRIK1 confer genetic susceptibility to the pathogenesis of JAE. Our family-based association analysis using the haplotype-based haplotype relative risk statistic revealed an association of JAE with the nine-repeat containing allele of the GRIK1 tetranucleotide polymorphism (chi2 = 8.31, df = 1, P = 0.004). Supportive evidence for linkage to a JAE related IGE spectrum (Zmax = 1.67 at GRIK1) under an autosomal dominant mode of inheritance and significant allele sharing (P < 0.05) among the affected family members suggest that allelic variants of GRIK1 contribute a major genetic determinant to the pathogenesis of JAE-related phenotypes.

Linkage analysis between idiopathic generalized epilepsies and the GABA(A) receptor alpha5, beta3 and gamma3 subunit gene cluster on chromosome 15.

INTRODUCTION: We tested the hypothesis that genetic variants within the GABA(A) alpha5, beta3 and gamma3 subunit gene cluster on chromosome 15q11-q13 confer genetic susceptibility to common subtypes of idiopathic generalized epilepsy (IGE). MATERIAL AND METHODS: Ninety-four families were selected from IGE patients with either juvenile myoclonic epilepsy (JME), juvenile (JAE) or childhood absence epilepsy (CAE). Cosegregation was tested between dinucleotide polymorphisms associated with the human GABA(A) alpha5, beta3 and gamma3 subunit gene cluster and three different IGE trait models. RESULTS: Evidence against linkage to the GABA(A) alpha5, beta3 and gamma3 subunit gene cluster was found in the entire family set and subsets selected from either CAE or JAE. In 61 families of JME patients, a maximum lod score (Zmax=1.40 at Theta(max)=0.00) was obtained for a broad IGE spectrum ("idiopathic" generalized seizure or generalized spike and wave discharges in the electroencephalogram) assuming genetic heterogeneity (alpha=0.37; P=0.06) and an autosomal recessive mode of inheritance. CONCLUSION: The possible hint of linkage in families of JME patients emphasizes the need for further studies to determine whether a recessively inherited gene variant within the GABA(A) alpha5, beta3 and gamma3 subunit gene cluster contributes to the pathogenesis of "idiopathic" generalized seizures and associated EEG abnormalities in a proportion of families.

Lack of linkage between idiopathic generalized epilepsies and the gene encoding the neural cell adhesion molecule.

Hereditary factors play a major role in the etiology of idiopathic generalized epilepsies (IGEs). The frequent neuropathological occurrence of microdysgeneses in the brain of IGE patients implies that genes regulating neural migration and cell adhesion might be involved in epileptogenesis of age-related generalized seizures. Our present linkage study tested the hypothesis that DNA sequence variants associated with the gene encoding the neural cell adhesion molecule (NCAM) confer genetic susceptibility to IGE traits in 57 families ascertained through patients with either juvenile myoclonic epilepsy, juvenile or childhood absence epilepsy. Our consistently negative results provide evidence against a common major effect of NCAM gene variants to the expression of IGEs with age-related onset from childhood to adolescence.

Exclusion of linkage between idiopathic generalized epilepsies and the GABAA receptor alpha 1 and gamma 2 subunit gene cluster on chromosome 5.

Hereditary factors play a major role in the etiology of idiopathic generalized epilepsies (IGEs). The pivotal function of ionotropic gamma-aminobutyric acid type A receptors (GABRs) in inhibitory neurotransmission in the mammalian central nervous system suggests that they may be involved in epileptogenesis and genetic predisposition to IGEs. Dinucleotide repeat polymorphisms associated with the human GABAA receptor alpha 1 (GABRA1) and gamma 2 subunit (GABRG2) gene cluster on chromosome 5q32-q35 offer the opportunity to test whether these candidate genes confer susceptibility to IGEs. Our linkage analyses in 63 families ascertained through IGE patients with either juvenile myoclonic epilepsy, juvenile absence epilepsy or childhood absence epilepsy do not support the hypothesis that variants within the GABRA1 and GABRG2 gene cluster contribute a frequent major gene effect to the expression of the common familial IGEs.

Common subtypes of idiopathic generalized epilepsies: lack of linkage to D20S19 close to candidate loci (EBN1, EEGV1) on chromosome 20.

Hereditary factors play a major role in the etiology of idiopathic generalized epilepsies (IGEs). A trait locus (EBN1) for a rare subtype of IGEs, the benign neonatal familial convulsions, and a susceptibility gene (EEGV1) for the common human low-voltage electroencephalogram have been mapped close together with D20S19 to the chromosomal region 20q13.2. Both loci are potential candidates for the susceptibility to IGE spectra with age-related onset beyond the neonatal period. The present study tested the hypothesis that a putative susceptibility locus linked to D20S19 predisposes to spectra of IGEs with age-related onset from childhood to adolescence. Linkage analyses were conducted in 60 families ascertained through IGE patients with juvenile myoclonic epilepsy, juvenile absence epilepsy or childhood absence epilepsy. Our results provide evidence against linkage of a putative susceptibility gene for four hierarchically broadened IGE spectra with D20S19 assuming tentative single-locus genetic models. The extent of an "exclusion region" (lod scores below-2) varied from 0.5 cM up to 22 cM on either side of D20S19 depending on the trait assumed. These results are contrary to the expectation that a susceptibility gene in vicinity to D20S19 confers a common major gene effect to the expression of IGE spectra with age-related onset from childhood to adolescence.

Refinement of map position of the human GluR6 kainate receptor gene (GRIK2) and lack of association and linkage with idiopathic generalized epilepsies.

Hereditary factors play a major role in the etiology of idiopathic generalized epilepsies (IGEs). The pivotal function of glutamate receptors (GluRs) in excitatory neurotransmission implicates their involvement in epileptogenesis and genetic susceptibility to IGEs. A trinucleotide repeat polymorphism detected in the 3' untranslated region of the kainate-selective GluR6 receptor gene (GRIK2) on chromosome 6 makes it possible to perform linkage and association studies with this high-ranking candidate gene. The present study tested the hypothesis that allelic variants of GRIK2 contribute to the genetic susceptibility to the common IGEs. Linkage and association analyses were conducted in 63 families ascertained through IGE patients with juvenile myoclonic epilepsy, juvenile absence epilepsy, or childhood absence epilepsy. Our linkage and association results suggest that allelic variants of GRIK2 are not involved in the expression of the common familial IGEs, and radiation hybrid mapping assigns GRIK2 to the chromosomal region 6q16.3-q21. This localization excludes GRIK2 as a candidate for the putative IGE susceptibility locus "EJM1" on the short arm of chromosome 6.

The phenotypic spectrum related to the human epilepsy susceptibility gene "EJM1".

Linkage studies of families ascertained through patients with juvenile myoclonic epilepsy (JME) suggest that an HLA-linked susceptibility gene on chromosome 6, designated "EJM1," predisposes to a group of idiopathic generalized epilepsies (IGEs) comprising JME, juvenile absence epilepsy (JAE), childhood absence epilepsies (CAE), and epilepsies with generalized tonic-clonic seizures (GTCS). To explore the EJM1-related phenotypic spectrum, we conducted linkage studies with HLA-DQ alpha restriction fragment length polymorphisms in 44 families ascertained through patients with CAE or JAE. Our results for the entire group of families provide evidence against a major susceptibility locus for idiopathic absence epilepsies and broader spectra of IGEs in the HLA region. Lod scores less than -2 were obtained for a region from 10 cM up to 23 cM on either side of the HLA-DQ alpha locus, depending on the assumed trait model. Suggestive evidence for linkage was found only for a subgroup of families with JME patients assuming an autosomal dominant mode of inheritance with 70% penetrance. A maximum lod score was obtained when family members with JME, JAE, CAE, and idiopathic GTCS were included into the affection status. Our results demonstrate that (1) the genetic susceptibility to idiopathic absence epilepsies and broader spectra of IGEs is heterogeneous, (2) the gene effect of EJM1 depends on the familial genetic background, and (3) EJM1 confers genetic susceptibility to idiopathic absence epilepsies and broader spectra of IGEs in the presence of family members with JME.

General roles of abscisic and jasmonic acids in gene activation as a result of mechanical wounding.

Exogenous application of abscisic acid (ABA) has been shown to induce a systemic pattern of proteinase inhibitor II (pin2) mRNA accumulation identical to that induced by mechanical wounding. Evidence is presented that the ABA-specific response is not restricted to pin2 genes but appears to be part of a general reaction to wound stress. Four other wound-induced, ABA-responsive genes that encode two additional proteinase inhibitors, the proteolytic enzyme leucine aminopeptidase, and the biosynthetic enzyme threonine deaminase were isolated from potato plants. Wounding or treatment with ABA resulted in a pattern of accumulation of these mRNAs very similar to that of pin2. ABA-deficient plants did not accumulate any of the mRNAs upon wounding, although they showed normal levels of expression upon ABA treatment. Also, application of methyl jasmonate (MeJA) induced a strong accumulation of these transcripts, both in wild-type and in ABA-deficient plants, thus supporting a role for jasmonic acid as an intermediate in the signaling pathway that leads from ABA accumulation in response to wounding to the transcriptional activation of the genes.