Chromosomal microaberrations in patients with epilepsy, intellectual disability, and congenital anomalies.

Epilepsy is a common finding in patients with chromosomal macro- and micro-rearrangements but only few aberrations show a constant pattern of seizures. DNA array-based studies have reported causative copy number variations (CNVs) in 5-30% of patients with epilepsy with or without co-morbidities. The interpretation of many of the detected CNVs remains challenging. In order to identify CNVs carrying epilepsy-related genes we investigated 43 children with various patterns of epileptic seizures, intellectual disability (ID), and minor dysmorphism, using the Illumina® Infinium Human1M-DuoV1 array. In three patients we found likely causative de novo CNVs, i.e. deletions in 1q41q42.12 (3.4 Mb) and 19p13.2 (834 kb), and a mosaic two-segment duplication in 17p13.2 (218 kb) and 17p13.1 (422 kb). In six additional patients there were aberrations (a deletion in one and duplications in five patients) with uncertain clinical consequences. In total, the finding of causative chromosomal micro-rearrangements in 3 out of 43 patients (7%) and potentially causative CNVs in 6 additional patients (14%) with epilepsy and ID but without major malformations confirms the power of DNA arrays for the detection of new disease-related genetic regions.

Lipoprotein(a): resurrected by genetics.

Plasma lipoprotein(a) [Lp(a)] is a quantitative genetic trait with a very broad and skewed distribution, which is largely controlled by genetic variants at the LPA locus on chromosome 6q27. Based on genetic evidence provided by studies conducted over the last two decades, Lp(a) is currently considered to be the strongest genetic risk factor for coronary heart disease (CHD). The copy number variation of kringle IV in the LPA gene has been strongly associated with both Lp(a) levels in plasma and risk of CHD, thereby fulfilling the main criterion for causality in a Mendelian randomization approach. Alleles with a low kringle IV copy number that together have a population frequency of 25-35% are associated with a doubling of the relative risk for outcomes, which is exceptional in the field of complex genetic phenotypes. The recently identified binding of oxidized phospholipids to Lp(a) is considered as one of the possible mechanisms that may explain the pathogenicity of Lp(a). Drugs that have been shown to lower Lp(a) have pleiotropic effects on other CHD risk factors, and an improvement of cardiovascular endpoints is up to now lacking. However, it has been established in a proof of principle study that lowering of very high Lp(a) by apheresis in high-risk patients with already maximally reduced low-density lipoprotein cholesterol levels can dramatically reduce major coronary events.

Parental origin of de novo cytogenetically balanced reciprocal non-Robertsonian translocations.

De novo cytogenetically balanced reciprocal non-Robertsonian translocations are rare findings in clinical cytogenetics and might be associated with an abnormal phenotype. Knowledge of the parental origin and mechanisms of formation is still limited. By microdissection of the derivative chromosomes and their normal homologs from metaphases followed by microsatellite-mediated marker analysis we identified 7 cases of paternal and 3 cases of maternal origin in a cohort of 10 patients with de novo cytogenetically balanced reciprocal non-Robertsonian translocations. Neither in the maternal nor in the paternal group of our study parental age seems to be increased. Together with the data from the literature our results confirm that the majority of de novo cytogenetically balanced reciprocal translocations are of paternal origin, but the preponderance does not appear to be as distinct as previously thought and the paternal age does not seem to be necessarily a major contributing factor.

Parental origin of apparently balanced de novo complex chromosomal rearrangements investigated by microdissection, whole genome amplification, and microsatellite-mediated haplotype analysis.

Complex chromosomal rearrangements (CCRs) are rare findings in clinical cytogenetics. As a result of the high risk of unbalanced segregation, familial cases are even rarer and maternal transmission occurs more frequently than paternal transmission. Analogous to Drosophila and mice, as well as to CCRs involving the Y chromosome or a clinically relevant associated deletion, a preferential origin in spermatogenesis has been assumed but not proven directly and systematically thus far. Here, we investigated three healthy adults, one healthy child, and one child with multiple congenital anomalies and various balanced de novo CCRs. The analyses were performed in each case on 10 copies of a derivative chromosome and their normal homologs by glass-needle microdissection, whole genome amplification (WGA), and microsatellite-mediated haplotype analysis. With respect to the number of chromosomes involved in each case and in all cases together, the number of chromosomal segments in each case and in all cases together, and the number of breakpoints in each case and in all cases together, the conformity for paternal origin of all derivative chromosomes and maternal origin of their normal homologs makes formation in paternal germline more likely than a postzygotic formation with an accidental uniformity. In conclusion, our results confirm the preferential formation of de novo balanced CCRs in the paternal germline.

Whole genome amplification from microdissected chromosomes.

Over the last years various whole genome amplification (WGA) methods have been established for genetic investigations from a limited number of cells or small quantities of DNA but not for molecular analysis of isolated chromosomes, which is important for the direct investigation of haplotypes or molecular rearrangements of derivative chromosomes in clinical cytogenetics and oncology. Here, the results of a pilot study in which the GenomePlex Single Cell Kit linker adapter PCR approach (Sigma-Aldrich, Vienna, Austria) was modified for WGA of glass needle based microdissected chromosomes are presented. Compared with two other WGA strategies (Improved-Primer Extension Preamplification PCR and Multiple Displacement Amplification) the GenomePlex Single Cell Kit shows a higher rate of successfully amplified markers, a lower WGA drop out rate and faster feasibility.

Age and origin of major Smith-Lemli-Opitz syndrome (SLOS) mutations in European populations.

BACKGROUND: Smith-Lemli-Opitz syndrome (SLOS) (MIM 270 400) is an autosomal recessive multiple congenital anomalies/mental retardation syndrome caused by mutations in the Delta7-sterol reductase (DHCR7, E.C.1.3.1.21) gene. The prevalence of SLOS has been estimated to range between 1:15000 and 1:60000 in populations of European origin. METHODS AND RESULTS: We have analysed the frequency, origin, and age of DHCR7 mutations in European populations. In 263 SLOS patients 10 common alleles (c.964-1G>C, p.Trp151X, p.Thr93Met, p.Val326Leu, p.Arg352Trp, p.Arg404Cys, p.Phe302Leu, p.Leu157Pro, p.Gly410Ser, p.Arg445Gln) were found to constitute approximately 80% of disease-causing mutations. As reported before, the mutational spectra differed significantly between populations, and frequency peaks of common mutations were observed in North-West (c.964-1G>C), North-East (p.Trp151X, p.Val326Leu) and Southern Europe (p.Thr93Met). SLOS was virtually absent from Finland. The analysis of nearly 8000 alleles from 10 different European populations confirmed a geographical distribution of DHCR7 mutations as reported in previous studies. The common Null mutations in Northern Europe (combined ca. 1:70) occurred at a much higher frequency than expected from the reported prevalence of SLOS. In contrast the most common mutation in Mediterranean SLOS patients (p.Thr93Met) had a low population frequency. Haplotypes were constructed for SLOS chromosomes, and for wild-type chromosomes of African and European origins using eight cSNPs in the DHCR7 gene. The DHCR7 orthologue was sequenced in eight chimpanzees (Pan troglodytes) and three microsatellites were analysed in 50 of the SLOS families in order to estimate the age of the three major SLOS-causing mutations. CONCLUSIONS: The results indicate a time of first appearance of c.964-1G>C and p.Trp151X some 3000 years ago in North-West and North-East Europe, respectively. The p.Thr93Met mutations on the J haplotype has probably first arisen approximately 6000 years ago in the Eastern Mediterranean. Together, it appears that a combination of founder effects, recurrent mutations, and drift have shaped the present frequency distribution of DHCR7 mutations in Europe.

Identification of 14 novel mutations in DHCR7 causing the Smith-Lemli-Opitz syndrome and delineation of the DHCR7 mutational spectra in Spain and Italy.

The Smith-Lemli-Opitz syndrome (SLOS) is a phenotypically variable metabolic malformation and mental retardation syndrome for which more than 80 mutations in the DHCR7 disease-causing gene have been described. The DHCR7 mutational spectra differ significantly in different areas of Europe, and several common putative founder mutations account for a substantial fraction of all mutations in some ethnic groups. Here we have analysed 47 SLOS patients and describe 14 newly identified mutations in 18 SLOS patients of Ashkenazi Jewish, Austrian, British, German, Italian, Irish, Polish, Portuguese, and Spanish origins. Half of the new mutations are in the transmembrane domains of the protein. In addition, there were two null mutations, one mutation in the 4th cytoplasmic loop, two mutations in the first and last codons, and three mutations in other regions such as the second cytoplasmic loop and the first endoplasmic loop. The analysis included 20 Spanish and 12 Italian SLOS patients and revealed very different mutation spectra in these patients compared to previously described patients from Czechoslovakia, Germany, Poland, and the UK and implicated p.Thr93Met on the J haplotype as the most frequent Mediterranean founder mutation.

Mutation spectrum of type I glycogen storage disease in Hungary.

We performed mutation analysis in 12 Hungarian type I glycogen storage disease (GSD I) patients in order to determine the mutation spectrum. All patients were clinically classified as GSD Ia. Nine patients carried biallelic G6PC mutations (p.Q27fsX35, p.D38V, p.W70X, p.K76N, p.W77R, p.R83C, p.E110Q, p.G222R), with E110Q reported only in Hungary. However, three patients displayed two common G6PT1 (SLC37A4) mutations (p.L348fsX400, p.C183R) which were originally described in association with GSD Inon-a. Review of the literature and our data show that G6PT1 mutations are not associated with neutropenia and related clinical findings in approximately 10% of these cases. Homozygosity for the truncating G6PT1 mutation p.L348fsX400 can be observed with and without neutropenia, indicating that one or more modifiers of the action of G6PT1 exist. Our data are suitable to provide DNA-based and thus noninvasive confirmation of diagnosis in Hungarian patients with this disorder.

DHCR7 mutations and genotype-phenotype correlation in 37 Polish patients with Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder of cholesterol biosynthesis caused by mutations in the DHCR7 gene. Thirty-seven ethnic Polish patients with SLOS underwent mutation analysis. The mutation frequencies in Polish patients were significantly different from those observed in Western European populations. Two mutations, W151X (22/68 alleles, 32%) and V326L (19/68 alleles, 28%), accounted for 60% of all observed in our cohort. Two missense mutations L68P and L360P have not been reported previously. In total, we report 15 DHCR7 mutations identified in Polish patients. By comparing clinical severity scores and the biochemical and molecular data, a genotype-phenotype correlation was attempted. In compound heterozygotes with one null mutation, the phenotype severity depends on the localization and type of the second mutation: mild phenotypes are correlated with mutations affecting the putative transmembrane domains TM1-TM6 or CT regions and severe phenotypes with mutations localized in TM7 and 4L region. The phenotypic differences of patients with the same genotype suggest that severity of the disease may be affected by other factors.

Maternal apo E genotype is a modifier of the Smith-Lemli-Opitz syndrome.

BACKGROUND: Smith-Lemli-Opitz syndrome (MIM 270400) is an autosomal recessive malformation and mental retardation syndrome that ranges in clinical severity from minimal dysmorphism and mild mental retardation to severe congenital anomalies and intrauterine death. Smith-Lemli-Opitz syndrome is caused by mutations in the Delta7 sterol-reductase gene (DHCR7; EC 1.3.1.21), which impair endogenous cholesterol biosynthesis and make the growing embryo dependent on exogenous (maternal) sources of cholesterol. We have investigated whether apolipoprotein E, a major component of the cholesterol transport system in human beings, is a modifier of the clinical severity of Smith-Lemli-Opitz syndrome. METHOD: Common apo E, DHCR7, and LDLR genotypes were determined in 137 biochemically characterised patients with Smith-Lemli-Opitz syndrome and 59 of their parents. RESULTS: There was a significant correlation between patients' clinical severity scores and maternal apo E genotypes (p = 0.028) but not between severity scores and patients' or paternal apo E genotypes. In line with their effects on serum cholesterol levels, the maternal apo epsilon2 genotypes were associated with a severe Smith-Lemli-Opitz syndrome phenotype, whereas apo E genotypes without the epsilon2 allele were associated with a milder phenotype. The correlation of maternal apo E genotype with disease severity persisted after stratification for DHCR7 genotype. There was no association of Smith-Lemli-Opitz syndrome severity with LDLR gene variation. CONCLUSIONS: These results suggest that the efficiency of cholesterol transport from the mother to the embryo is affected by the maternal apo E genotype and extend the role of apo E and its disease associations to modulation of embryonic development and malformations.

Gabon black population data on the ten short tandem repeat loci D3S1358, VWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D19S433, TH01 and FGA.

Allele frequencies for ten short tandem repeat (STR) loci D3S1358, VWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D19S433, TH01 and FGA were determined in a Black African sample population from Gabon. All loci were highly polymorphic and except for TH01, D21S11 and D16S539, all met Hardy-Weinberg expectations. There was little evidence of association of alleles between the loci in this database. The combined power of exclusion for the ten STR loci was 0.999981. While significant differences between the Gabon population and the Austrian Caucasian population were found at all loci, significant differences were found between the Gabon population and Zimbabweans only for D3S1358 and between the Gabon population and African Americans only for TH01 and D8S1179.

Direct interaction of the extracellular matrix protein DANCE with apolipoprotein(a) mediated by the kringle IV-type 2 domain.

Lipoprotein(a) [Lp(a)] consists of LDL and apolipoprotein(a), and has been shown to be a major, independent, risk factor for arteriosclerosis and thrombosis in humans. To further elucidate the (patho)physiological function(s) of Lp(a)/apo(a), we searched for new protein ligands, using the yeast two-hybrid system and employing the highly repetitive kringle IV type 2 (KIV-2) sequence from apo(a) as bait. The extracellular matrix protein DANCE [developmental arteries and neural crest epidermal growth factor (EGF)-like] recently implicated in atherogenesis was identified as an interactor. A direct physical interaction between DANCE and apo(a) was confirmed by co-purification of both recombinant proteins derived from culture supernatants of transiently transfected COS-1 cells. Furthermore, binding of human plasma-derived Lp(a) to recombinant DANCE was also observed. Finally, when monoclonal anti-apo(a) and polyclonal anti-DANCE antibodies were applied to tissue slices of atherosclerotic carotid artery, the two proteins were found to be co-localized in endothelial and smooth muscle cells, suggesting that they occur together in the arterial wall. However, as yet, the in vivo relevance and the possible functional role of this newly identified DANCE:Lp(a)/apo(a) interaction remains speculative.

Combined study of prostatic carcinoma by classical cytogenetic analysis and comparative genomic hybridization.

Conventional cytogenetic analysis of prostatic carcinoma (PC) is characterized by inefficient growth of tumor cells during in vitro culture, leading to a lack of aberrant karyotypes in many of investigated tumors. In this study we have combined a modified short-term tissue culture method for conventional banding analysis and comparative genomic hybridization (CGH) to examine genetic changes in PC, and to evaluate the effect of the in vitro culture on chromosomal changes by comparing results of the two methods. Cytogenetic analysis was performed on 34 PCs using both, conventional and molecular methods. Tumor tissues were obtained predominantly from untreated primary tumors from 48 patients. For karyotyping all tumor samples were short-term cultured using a feeder layer technique. Additionally DNA from uncultured tumor material from 17 of those patients was isolated and screened for copy number changes using CGH. Conventional banding analysis: clonal aberrations were detected in 65% of the tumor samples. Most of the chromosomal findings were numerical changes, including loss of chromosomes Y (32%), 18, 19 and 21 (each 12%). Less frequent, trisomy of chromosome 7 and monosomy of chromosomes 9, 12 and 22 (each 9%) was found. Additionally an inversion of chromosome 9p and a deletion at chromosome 7q was found in two cases. In 35% no clonal aberrations could be detected. CGH: DNA copy number changes were detected in 65% of the analyzed tumors. Predominantly losses of DNA sequences were found. The most common losses were found at chromosome regions 13q21q33 (29%), 6q11q23 (24%), 16q, and 18 (each 18%), and the most common gains at 19 (18%). In six tumors no copy number changes were found. Both methods showed a similar aneuploidy rate, suggesting that the feeder layer technique is quite a suitable method for in vitro culture of PC cells. However, the two techniques produced substantially differing results for most of the tumor samples, and in some cases the discrepancies are quite striking. Therefore eventual culture effects need to be taken into account when comparing results from conventional cytogenetics and CGH. Some contrary findings from the two methods are discussed.

High-resolution mapping of the human 4q21 and the mouse 5E3 SCYB chemokine cluster by fiber-fluorescence in situ hybridization.

The CXC chemokine or small inducible cytokine B (SCYB) subfamily includes the T-cell chemoattractants MIG (CXCL9, SCYB9), IP-10 (CXCL10, SCYB10), and I-TAC (CXCL11, SCYB11). These three highly homologous chemokines lack the glutamic acid-leucine-arginine (ELR) motif and signal via the CXCR3 receptor. Previous work showed that the genes encoding these chemokines are localized in an individual mini-cluster on human Chromosome (Chr) 4 at position 4q21.2. Recently, we identified mouse Scyb11 and mapped this gene by fluorescence in situ hybridization (FISH) to mouse Chr 5E3, the orthologous locus to human 4q21 where the other two homologous mouse genes, Scyb9 and Scyb10, have also been localized. Since SCYB10 and SCYB11 are not represented in the recently published draft sequence of the human genome, we wanted to clarify exactly the order and distances of the three chemokine genes using two-color FISH on stretched DNA fiber preparations. Here, we report the simultaneous localization of all three genes and provide high-resolution visual maps of this chemokine cluster from both mouse and human. The three chemokine genes were found within a range of 32 kb on mouse and 29 kb on human DNA fiber targets. The precise physical distances were defined, and an almost identical arrangement of the human and mouse homologues was identified, indicating that this CXC chemokine mini-cluster has been completely conserved evolutionarily since the divergence of mouse and human. Our results refine previous maps of the three genes, support the hypothesis that they resulted from gene duplication that took place in a common ancestor of mouse and human, and provide complementary information on a region of the draft sequence of human Chr 4 that is not yet covered.

Intracellular apolipoprotein E affects Amyloid Precursor Protein processing and amyloid Abeta production in COS-1 cells.

The apoE gene has been identified as a major susceptibility locus for late-onset Alzheimer's disease (LOAD). The epsilon4 allele greatly reduces age of onset of LOAD as compared to the wild-type 3 allele. The molecular mechanism(s) underlying the association has not yet been fully elucidated. The apoE protein has been shown to physically interact with the Abeta region of the Amyloid Precursor Protein (APP), but also with the ectodomain of the APP holoprotein itself. In this study we have used apoE fusion proteins containing either the ER retention sequence KDEL or trans-Golgi network (TGN) signal sequence in order to define potential apoE-mediated alterations in APP protein processing. Co-expression and pulse-chase experiments showed that a functional apoE:APP interaction occurs intracellularly which directly affects maturation and subsequently the secretion kinetics of APP. In addition, an epsilon4 allele-specific induction of Abeta production has been demonstrated. apoE3 resulted in increased Abeta production only when targeted to the ER, as observed in cells transfected with an apoE3KDEL fusion protein as well as following treatment with brefeldin A. The findings suggest that in cells that express both apoE and APP, such as astrocytes and microglia, a functional apoE:APP interaction may occur which modulates APP processing and Abeta production.

Molecular genetics of type 1 glycogen storage disease.

Glycogen storage disease type 1 (GSD 1) comprises a group of autosomal recessive inherited metabolic disorders caused by deficiency of the microsomal multicomponent glucose-6-phosphatase system. Of the two known transmembrane proteins of the system, malfunction of the catalytic subunit (G6Pase) characterizes GSD 1a. GSD 1 non-a is characterized by defective microsomal glucose-6-phosphate or pyrophosphate/phosphate transport due to mutations in G6PT (glucose-6-phosphate translocase gene) encoding a microsomal transporter protein. Mutations in G6Pase and G6PT account for approximately 80 and approximately 20% of GSD 1 cases, respectively. G6Pase and G6PT work in concert to maintain glucose homeostasis in gluconeogenic organs. Whereas G6Pase is exclusively expressed in gluconeogenic cells, G6PT is ubiquitously expressed and its deficiency generally causes a more severe phenotype. Rapid confirmation of clinically suspected diagnosis of GSD 1, reliable carrier testing, and prenatal diagnosis are facilitated by mutation analyses of the chromosome 11-bound G6PT gene as well as the chromosome 17-bound G6Pase gene.

De novo mutation of the connexin 26 gene associated with dominant non-syndromic sensorineural hearing loss.

Mutations of the connexin 26 (Cx26) gene cause isolated recessive or dominant hearing loss or both sensorineural hearing impairment and keratoderma. We have identified the first de novo mutation of the Cx26 gene, R75 W, in a sporadic case of isolated profound hearing loss. R75 W has been previously observed in association with hearing impairment and keratoderma in one family and is thus thought to cause both syndromic and non-syndromic hearing loss. This case illustrates the risk of a possible erroneous diagnosis of autosomal recessive hearing loss in a sporadic case.

Protein kinase C isoenzyme: selective expression pattern of protein kinase C-θ during mouse development.

Protein kinase C (PKC)-θ, a serine/threonine protein kinase and novel PKC subfamily member, has been recently identified as an essential component of the T cell synapse which activates the NF-kB signaling cascade leading to expression of the IL-2 gene during T cell activation. By RNA in situ hybridization to whole-body embryo sections it is shown that the murine PKCθ is specifically expressed in tissues with hematopoietic and lymphopoietic activity. Expression is also evident in skeletal muscle. A further highly specific expression was observed in the peripheral and central nervous system which is described in detail. Expression in the brain persists up to adult stages.