Febrile seizures and generalized epilepsy associated with a mutation in the Na+-channel beta1 subunit gene SCN1B.

Febrile seizures affect approximately 3% of all children under six years of age and are by far the most common seizure disorder. A small proportion of children with febrile seizures later develop ongoing epilepsy with afebrile seizures. Segregation analysis suggests the majority of cases have complex inheritance but rare families show apparent autosomal dominant inheritance. Two putative loci have been mapped (FEB1 and FEB2), but specific genes have not yet been identified. We recently described a clinical subset, termed generalized epilepsy with febrile seizures plus (GEFS+), in which many family members have seizures with fever that may persist beyond six years of age or be associated with afebrile generalized seizures. We now report linkage, in another large GEFS+ family, to chromosome region 19q13.1 and identification of a mutation in the voltage-gated sodium (Na+)-channel beta1 subunit gene (SCN1B). The mutation changes a conserved cysteine residue disrupting a putative disulfide bridge which normally maintains an extracellular immunoglobulin-like fold. Co-expression of the mutant beta1 subunit with a brain Na+-channel alpha subunit in Xenopus laevis oocytes demonstrates that the mutation interferes with the ability of the subunit to modulate channel-gating kinetics consistent with a loss-of-function allele. This observation develops the theme that idiopathic epilepsies are a family of channelopathies and raises the possibility of involvement of other Na+-channel subunit genes in febrile seizures and generalized epilepsies with complex inheritance patterns.

Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo.

Peptide nucleic acids (PNAs) form stable and tight complexes with complementary DNA and/or RNA and would be promising antisense reagents if their cellular delivery could be improved. We show that a 21-mer PNA, complementary to the human galanin receptor type 1 mRNA, coupled to the cellular transporter peptides, transportan or pAntennapedia(43-58), is efficiently taken up into Bowes cells where they block the expression of galanin receptors. In rat, the intrathecal administration of the peptide-PNA construct results in a decrease in galanin binding in the dorsal horn. The decrease in binding results in the inability of galanin to inhibit the C fibers stimulation-induced facilitation of the rat flexor reflex, demonstrating that peptide-PNA constructs act in vivo to suppress expression of functional galanin receptors.

Localisation of a gene for an autosomal recessive syndrome of macrocephaly, multiple epiphyseal dysplasia, and distinctive facies to chromosome 15q26.

BACKGROUND: We have previously described an autosomal recessive syndrome of macrocephaly, multiple epiphyseal dysplasia (MED), and distinctive facies in a large, extended Omani family. The MED observed seems to be part of a larger malformation syndrome, since both craniofacial and central nervous system changes were present in the family. We performed a whole genome scan in this family in order to identify the gene locus for this malformation syndrome. METHODS AND RESULTS: Using homozygosity mapping, we show linkage to the telomeric region of the long arm of chromosome 15. The position of both the disease gene and the principal glycoprotein, chondroitin sulphate proteoglycan (aggrecan, AGC1) on chromosome 15q26, suggested that the aggrecan gene is a candidate for the disease in this family. However, three of the four affected children were heterozygous for a polymorphism at position 831 of the coding sequence of AGC1, providing strong evidence against its involvement. CONCLUSION: We have identified a gene locus for a recessive syndrome of macrocephaly, MED, and distinctive facies in a large Omani family. Aggrecan located on chromosome 15q26, within the critical region determined for this syndrome in this family, was excluded as a candidate gene.

Fine mapping and single nucleotide polymorphism association results of candidate genes for asthma and related phenotypes.

Several genome-wide screens for asthma and related phenotypes have been published to date but data on fine-mapping are scarce. For higher resolution we performed a fine-mapping study with 2 cM average spacing in often discussed asthma candidate regions (2p, 5q, 6p, 7p, 9q, 11p, and 12q) to narrow down the regions of interest. All participants of a Caucasian family study (97 families with at least two affected sib pairs) were genotyped for 49 supplementary polymorphic dinucleotide markers. Our results indicate increased evidence for linkage on chromosome 6p, 9q, and 12q. These candidate regions were further analyzed with SNP polymorphisms in the endothelin 1 (EDN1), lymphotoxin alpha (LTA), and neuronal nitric oxide synthase (NOS1) genes. In addition, IL4 -590C>T and IL10 -592C>A, localized on chromosomes 5q and 1q, respectively, have been analyzed for SNP association. Of the six SNPs tested, four revealed weak association with the examined phenotypes. These are the IL10 -592C>A SNP in the interleukin 10 gene (p=0.036 for eosinophil cell counts), the 4124T>C SNP in EDN1 (p=0.044 for asthma), the 3391C>T SNP in NOS1 with eosinophil cell counts (p=0.0086), and the 5266C>T polymorphism, also in the NOS1 gene, for high IgE levels (p=0.022). In summary, fine mapping data enable us to confine asthma candidate regions, while variants of EDN1 and NOS1, or nearby genes, may play an important role in this context.

Low incidence of UPD in spontaneous abortions beyond the 5th gestational week.

Approximately 15-20% of all clinically recognised pregnancies abort, most commonly between 8-12 gestational weeks. While the majority of early pregnancy losses is attributed to cytogenetic abnormalities, the aetiology of approximately 40% of early abortions remains unclear. To determine additional factors causing spontaneous abortions we retrospectively searched for uniparental disomies (UPD) in 77 cytogenetically normal diploid spontaneous abortions. In all cases an unbalanced chromosome anomaly was ruled out by cytogenetic investigation of chorionic/amniotic membranes and/or chorionic villi. For UPD screening microsatellite analyses were performed on DNA of abortion specimens and parental blood using highly polymorphic markers showing UPD in two cases. The distribution of markers analysed indicated maternal heterodisomy for chromosome 9 (UPhD(9)mat) in case 1 and paternal isodisomy for chromosome 21 (UPiD(21)pat) in case 2. The originating mechanism suggested was monosomy complementation in UPiD(21)pat and trisomy rescue in UPhD(9)mat. In the case of UPhD(9)mat purulent chorioamnionitis was noted and a distinctly growth retarded embryo of 3 cm crown-rump length showing no gross external malformations. Histological analysis in the case of UPiD(21)pat suggested a primary anlage defect. Our results indicate that less than 3% of genetically unexplained pregnancy wastage is associated with total chromosome UPD. UPD may contribute to anlage defects of human conception. Chromosome aneuploidy correction can occur in very early cleavage stages. More research, however, ought to be performed into placental mosaicism to further clarify timing and mechanisms involved in foetal UPD.

Localisation of a Fanconi anaemia gene to chromosome 9p.

Using homozygosity mapping in a large consanguineous family, we have localised to chromosome 9p a further gene for the autosomal recessive, genetically heterogeneous disease Fanconi anaemia (FA). This is the fourth of at least eight FA genes to be localised to a discrete chromosomal region. Previously localised genes are FAA, FAC and FAD. By analysis of assigned families we show that the gene localised to chromosome 9p is FAF, FAG or FAH, or a new FA gene, and refine the localisation to the 21 cM region between markers D9S1678 and D9S175.

Familial hypomagnesaemia with hypercalciuria and nephrocalcinosis maps to chromosome 3q27 and is associated with mutations in the PCLN-1 gene.

Familial hypomagnesaemia with hypercalciuria and nephrocalcinosis (FHHNC, MIM 248250) is a complex renal tubular disorder characterised by hypomagnesaemia, hypercalciuria, advanced nephrocalcinosis, hyposthenuria and progressive renal failure. The mode of inheritance is autosomal recessive. A primary defect in the reabsorption of magnesium in the medullary thick ascending limb of the loop of Henle (mTAL) has been proposed to be essential in FHHNC pathophysiology. To identify the underlying genetic defect we performed linkage analysis in eight families, including three with consanguineous marriages. We found linkage to microsatellite markers on chromosome 3q27 with a maximum two-point lod score (Zmax) of 5.208 for D3S3530 without evidence for genetic heterogeneity. Haplotype analysis revealed crucial recombination events reducing the critical interval to 6.6cM. Recently, mutations in the gene PCLN-1, mapping to 3q27 and coding for paracellin-1, were identified by Simon et al (1999) as the underlying genetic defect in FHHNC. Paracellin-1 represents a renal tight junction protein predominantly expressed in the TAL. Mutational analysis in our patient cohort revealed eight different mutations in the PCLN-1 gene, within six novel mutations. In seven of 13 mutant alleles we detected a Leu151 substitution without evidence for a founder effect. Leu151 is a residue of the first extracellular loop of paracellin-1, the part of the protein expected to bridge the intercellular space and to be important for paracellular conductance. This study confirms the implication of paracellin-1 defects in FHHNC and points to a predominant role of this protein in the paracellular reabsorption of divalent cations in the TAL.

Linkage and family-based association study of schizophrenia and the synapsin III locus that maps to chromosome 22q13.

The human synapsin III gene (synapsin III) is a member of a neuron-specific phosphoprotein gene family involved in short-term neurotransmitter release. We mapped synapsin III to chromosomal region 22q13 (13.1-13.31) by fluorescence in situ hybridization, a region that has been identified as a potential schizophrenia susceptibility locus. The dinucleotide repeat marker D22S280 located in intron 5 of synapsin III was genotyped in a linkage and family-based association study to assess the role of the synapsin III locus in the etiology of schizophrenia. In 12 pedigrees with periodic catatonia comprising 135 individuals, we found exclusion of linkage of marker D22S280 using lod score analysis with autosomal dominant/recessive models as well as affected only LOD score methods with dominant/recessive models. In a family-based association study of 61 unrelated parent-offspring trios with schizophrenia (according to the the Diagnostic and Statistical Manual of Mental Disorders, fourth edition [DSM-IV, American Psychiatric Association, 1994]), we found no association of individual D22S280 alleles to disease. Results of a multiallelic transmission/disequilibrium test (TDT(max) = 3.00; P = 0.55) challenged the possibility that D22S280 alleles appear with DSM-IV schizophrenia more frequently than expected. In addition, no evidence for gender differences or parent-of-origin effects were found. Thus, the synapsin III locus at chromosome 22q13 is not likely to contain a schizophrenia susceptibility gene.

Evaluation of a putative major susceptibility locus for juvenile myoclonic epilepsy on chromosome 15q14.

Juvenile myoclonic epilepsy (JME) is a genetically determined common subtype of idiopathic generalized epilepsy (IGE). Significant evidence for linkage has been reported for a susceptibility locus for JME in the chromosomal region 15q14 that harbors the gene encoding the alpha7 subunit of the neuronal nicotinic acetylcholine receptor (CHRNA7). The present study was designed to test the earlier linkage finding and to explore whether this susceptibility locus is involved in the epileptogenesis of a broader spectrum of IGE syndromes. Multipoint parametric and nonparametric linkage analyses with seven microsatellite polymorphisms encompassing the region of the CHRNA7 gene were performed using two diagnostic schemes of JME-related traits in two groups of multiplex families ascertained through probands with either JME (n = 27) or idiopathic absence epilepsy (n = 30). The present linkage study failed to replicate evidence for a major susceptibility locus for JME in the region encompassing the CHRNA7 gene. In addition, we found no hint in favor of linkage to 15q14 under the broad diagnostic scheme in any of the sets of families. If genetic variation in this region confers susceptibility to JME, then its effect size might be too small or its occurrence too rare to be detected in the investigated families.

Characterisation of a new chimeric ligand for galanin receptors: galanin(1-13)-[D-Trp(32)]-neuropeptide Y(25-36)amide.

In this work, we studied a novel chimeric peptide, M242, galanin(1-13)-[D-Trp(32)]-neuropeptide Y(25-36)amide, and examined its properties in comparison with its parent peptide, M32, galanin(1-13)-neuropeptide Y(25-36)amide, a previously known high-affinity ligand for galanin receptors, and galanin itself. Binding assays performed in Bowes cells known to express human galanin receptor type 1 (hGalR1) and in Chinese hamster ovary cells overexpressing human galanin receptor type 2 (hGalR2) revealed that all three ligands had comparable affinities: at hGalR1<1 nM and at hGalR2<10 nM. However, in rat hippocampal membranes M242 had a 24-fold lower affinity than galanin (9.4 vs. 0.4 nM) and 134-fold lower affinity than M32 (9.4 vs. 0.07 nM). In the same tissue, we also examined the effects of these peptides on adenylate cyclase activity. M32 showed a weak antagonistic behaviour but M242 acted as a potent biphasic regulator of adenylate cyclase. In conclusion, we present and characterise a new peptide M242, which could be a useful tool in studies of galaninergic signalling.

Effects of vasopressin-mastoparan chimeric peptides on insulin release and G-protein activity.

Two chimeric peptides, consisting of the linear vasopressin receptor V1 antagonist PhAc-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr, in the N-terminus and mastoparan in the C-terminus connected directly (M375) or via 6-aminohexanoic acid (M391), have been synthesised. At 10 microM concentration, these novel peptides increased insulin secretion from isolated rat pancreatic islet cells 18-26-fold at 3.3 mM glucose and 3.5-5-fold at 16.7 mM glucose. PTX pretreatment of the islets decreased the peptide-induced insulin release. M375 and M391 bind to V1a vasopressin receptors with affinities lower than the unmodified vasopressin antagonist, but with K(D) values of 3.76 nM and 9.02 nM, respectively, both chimeras are high affinity ligands. The GTPase activity and GTPgammaS binding in the presence of these peptides has been characterised in Rin m5F cells. Comparison of the influence of the peptides M375 and M391 on GTPase activity in native and pertussis toxin-treated cells suggests a selective activation of G alpha(i)/G alpha(o) subunits, combined with a suppression of other GTPases, primarily G alpha(s). However, the GTPgammaS binding data show that the peptides retain some of the activating property even in PTX-treated cell membranes. In conclusion, the conjugation of mastoparan with the V1a receptor antagonists produce peptides with properties different from the parent peptides that could be used to elucidate the role of different G proteins in insulin release.

Binding of chimeric NPY/galanin peptides M32 and M242 to cloned neuropeptide Y receptor subtypes Y1, Y2, Y4, and Y5.

Ligand binding to neuropeptide Y (NPY) receptors Y1, Y2, Y4, and Y5 from guinea-pig was investigated using the two NPY-galanin hybrids M32 (galanin1-13-NPY25-36-amide) and M242 ([D-Trp(32)]M32). The affinity of M32 for Y1, Y2, and Y4 receptors was 13, 4, and 30nM, respectively, similar to that of NPY18-36 and NPY22-36 but 40-fold to 300-fold lower than the affinity of intact porcine NPY. M242 bound to the Y1, Y2, and Y4 receptors with 9-fold to 20-fold lower affinity than did M32. The affinities of M32 and M242 for Y5 were 400 and 800 nM, respectively. Thus, M32 seems to gain affinity relative to both of its constituent peptide portions although the NPY25-36 part may be sufficient for NPY-receptor recognition, especially at the Y2 receptor. This suggests that the galanin portion of M32 influences and/or stabilizes the conformation of the NPY portion, similar to the effect seen for the NPY portion of M32 in binding to galanin receptors.

Role of third intracellular loop of galanin receptor type 1 in signal transduction.

To determine the domains essential for G-protein coupling of the human galanin receptor type 1 (GalR1), we have used both GalR1 mutants and synthetic receptor-derived peptides in(125)I-galanin and [(35)S]-GTPgammaS binding studies. Replacement of potential phosphorylation sites by Leu in the third intracellular loop (IC3) of GalR1 did not affect K(D)values for the receptor. Peptides derived form the IC3 loop, and especially the N-terminal part of it were able to increase the rate of [(35)S]-GTPgammaS binding to the trimeric Gialpha1beta1gamma2, but not to Gsalphabeta1gamma2, whereas the peptides corresponding to the IC1 and IC2 loops had no such effect. IC3 loop peptides also inhibited the binding of(125)I-galanin to GalR1 in membranes from Rin m5F cells. Our results suggest that the IC3 loop of GalR1, especially its N-terminal part, defines the coupling of the receptor to the Gialpha1beta1gamma2 protein and consequently, to the signal transduction cascade.

No evidence for a susceptibility locus for idiopathic generalized epilepsy on chromosome 5 in families with typical absence seizures.

A recent genome-wide scan revealed suggestive evidence for two susceptibility loci for idiopathic generalized epilepsy (IGE) in the chromosomal regions 5p15 and 5q14-q22 in families with typical absence seizures. The present replication study tested the validity of the tentative IGE loci on chromosome 5. Our study included 99 multiplex families in which at least one family member had typical absence seizures. Parametric and non-parametric multipoint linkage analyses were carried out between the IGE trait and 23 microsatellite polymorphisms covering the entire region of chromosome 5. Multipoint parametric heterogeneity lod scores < -2 were obtained along chromosome 5 when a proportion of linked families greater than 50% was assumed under recessive inheritance and > 60% under dominant inheritance. Furthermore, non-parametric multipoint linkage analyses revealed no hint of linkage throughout the candidate region (P > 0.05). Accordingly, we failed to support previous evidence for common IGE loci on chromosome 5. If there is a susceptibility locus for IGE on chromosome 5 then the size of the effect or the proportion of linked families is too small to detect linkage in the investigated family sample.

Impaired p53-mediated DNA damage response, cell-cycle disturbance and chromosome aberrations in Nijmegen breakage syndrome lymphoblastoid cell lines.

PURPOSE: To investigate the p53 ionizing radiation-induced response, G1/S cell-cycle block and cytogenetic damage in Nijmegen breakage syndrome (NBS) cells characterized by different haplotypes. MATERIAL AND METHODS: Lymphoblastoid cell lines derived from three normals, five NBS and two ataxia telangiectasia (AT) individuals were treated with moderate doses of X-rays and changes in the p53 response were studied by dose-response and time-course experiments. Multiparametric flow cytometry analysis of bromodesoxyuridine-incorporated cells was carried out to analyse G1/S checkpoint alterations. Cytogenetic damage induced by 2 Gy radiation was assessed in cells harvested 28 h later. RESULTS: Comparison of mean values of p53 accumulation in NBS, AT and control cells indicated that protein induction in NBS cells was between normal and AT cells. Cell-cycle experiments showed a markedly reduced S-phase fraction in irradiated samples of normal cell lines, while NBS, and particularly AT cells, showed less reduction in S-phase fraction. Irrespective of differences in p53 induction and G1/S block, chromatid-type aberrations were induced at a comparable level in both syndromes, while being almost absent in normal cells. CONCLUSIONS: The data suggested that failure of NBS cells to initiate cell-cycle delay cannot account alone for their extreme sensitivity to radiation.

Family-based association study of serotonergic candidate genes and attention-deficit/hyperactivity disorder in a German sample.

Alterations in the serotonergic pathway have been implicated in the pathogenesis of attention-deficit/hyperactivity disorder (ADHD). The aim of this study was to investigate seven genetic variants in three genes (serotonin transporter (5-HTT), serotonin receptor 1B (5-HTR1B) and serotonin receptor 2A (5-HTR2A)), which have previously been shown to be associated with ADHD. The polymorphisms under investigation were the 5-HTTLPR, the VNTR in intron 2 and the 3'UTR SNP in 5-HTT, the 5-HTR1B variations 861G>C and 102T>C, and the 5-HTR2A variations His452Tyr and 1438G>A. We genotyped these variants in a sample of 102 families with 229 children with ADHD according to DSM-IV criteria. Among the affected children, 69% fulfilled criteria for the combined type, 27% for the predominantly inattentive type, and 4% for the predominantly hyperactive-impulsive type. Associations were tested by the pedigree transmission disequilibrium test (PDT). All investigated polymorphisms in serotonergic candidate genes showed no association to ADHD in our sample. Earlier studies of these polymorphisms had also shown inconsistent results, with some studies reporting significant associations and others demonstrating no association. This discordance between studies may reflect variation in patient ascertainment criteria, genetic heterogeneity, too low statistical power for the expected effects or false positive results in the initial reports. We cannot rule out the possibility that other variations in the investigated genes contribute to the etiology of ADHD.

Association and linkage of allelic variants of the dopamine transporter gene in ADHD.

Previously, we had reported a genome-wide scan for attention-deficit/hyperactivity disorder (ADHD) in 102 families with affected sibs of German ancestry; the highest multipoint LOD score of 4.75 was obtained on chromosome 5p13 (parametric HLOD analysis under a dominant model) near the dopamine transporter gene (DAT1). We genotyped 30 single nucleotide polymorphisms (SNPs) in this candidate gene and its 5' region in 329 families (including the 102 initial families) with 523 affected offspring. We found that (1) SNP rs463379 was significantly associated with ADHD upon correction for multiple testing (P=0.0046); (2) the global P-value for association of haplotypes was significant for block two upon correction for all (n=3) tested blocks (P=0.0048); (3) within block two we detected a nominal P=0.000034 for one specific marker combination. This CGC haplotype showed relative risks of 1.95 and 2.43 for heterozygous and homozygous carriers, respectively; and (4) finally, our linkage data and the genotype-IBD sharing test (GIST) suggest that genetic variation at the DAT1 locus explains our linkage peak and that rs463379 (P<0.05) is the only SNP of the above haplotype that contributed to the linkage signal. In sum, we have accumulated evidence that genetic variation at the DAT1 locus underlies our ADHD linkage peak on chromosome 5; additionally solid association for a single SNP and a haplotype were shown. Future studies are required to assess if variation at this locus also explains other positive linkage results obtained for chromosome 5p.

A genome-wide scan for attention-deficit/hyperactivity disorder in 155 German sib-pairs.

Three groups have previously performed genome scans in attention-deficit/hyperactivity disorder (ADHD); linkage to chromosome 5p13 was detected in all of the respective studies. In the current study, we performed a whole-genome scan with 102 German families with two or more offspring who currently fulfilled the diagnostic criteria for ADHD. Including subsequent fine mapping on chromosome 5p, a total of 523 markers were genotyped. The highest nonparametric multipoint LOD score of 2.59 (empirical genome-wide significance 0.1) was obtained for chromosome 5p at 17 cM (according to the Marshfield map). Subsequent analyses revealed (a) a higher LOD score of 3.37 at 39 cM for a quantitative severity score based on symptoms of inattention than for hyperactivity/impulsivity (LOD score of 1.11 at 59 cM), and (b) an HLOD of 4.75 (empirical genome-wide significance 0.001) based on a parametric model assuming dominant inheritance. The locus of the solute carrier 6A3 (SLC6A3; dopamine transporter 1; DAT1) localizes to 5p15.33; the gene has repeatedly been implicated in the etiology of ADHD. However, in our sample the DAT1 VNTR did not show association with ADHD. We additionally identified nominal evidence for linkage to chromosomes 6q, 7p, 9q, 11 q, 12q and 17p, which had also been identified in previous scans. Despite differences in ethnicity, ascertainment and phenotyping schemes, linkage results in ADHD appear remarkably consistent.

CD4(+) T-cell responses against the VP1-unique region in individuals with recent and persistent parvovirus B19 infection.

To date cellular immune responses against parvovirus B19 (B19) have not been studied extensively. The aim of this study was to examine the T-cell response against the VP1-unique region as the immunodominant part of the viral structural protein VP1 in individuals with different courses of B19 infection. Therefore, a group of 13 parvovirus-positive probands was separated into subgroups characterized for recent or acute, past or persistent infection by means of the presence of specific immunoglobulin (Ig)M and IgG isotypes and of viral DNA in blood and tissue. Transiently transfected B-cells expressing VP1-unique region were used in ELISpot assays to investigate T-cell responses directed against the VP1-unique region in peripheral blood mononuclear cells (PBMC) of individual donors. Significant numbers of interferon-gamma (IFN-gamma) secreting lymphocytes were detectable in PBMC of all individuals with recent, acute or persistent B19 infection, but not in PBMC of donors with past B19 infection and seronegative individuals. A more detailed analysis of IFN-gamma producing cells by intracellular cytokine staining by flow cytometry revealed, that CD4(+) T cells but not CD8(+) cytotoxic lymphocytes (CTL) were the major subpopulation of IFN-gamma producing cells. These data strongly suggest the need of virus protein production for the maintenance of VP1-unique region-specific CD4(+) T-helper cell responses in B19-infected individuals.

Total luminescent spectroscopy for remote laser diagnostics of natural water conditions.

In undertaking the rapid diagnostics of water conditions (for organic impurities, eutrophication etc.), it is expedient to confine the effort to establishing the statistically normal state and departures from it rather than to seek the full chemical composition of the medium. What others have termed spectral signatures obtained by such methods as total luminescent spectroscopy can serve as representative indicators of both background conditions and those deviating from the normal. In this paper we show how, with the aid of laboratory catalogs of spectral signatures, it is possible to detect oil pollution at levels substantially below those of slicks (of a few microliters per liter) and to identify groups of pollutants (by remote sensing). And we describe a spectroscopic lidar we developed for marine studies.