Identification of WASP mutations in 14 Spanish families with Wiskott‐Aldrich syndrome

The Wiskott‐Aldrich syndrome (WAS) is an X‐linked immunodeficiency caused by mutations in the WASP gene. The disease is known to be associated with extensive clinical variability, and mutation studies indicate that genotypes are also highly variant among WAS patients. In this study, we performed mutation analysis of the WASP gene in 14 unrelated Spanish families by single strand conformation analysis (SSCA) and sequencing, resulting in the identification of a novel mutation and nine known mutations. No mutation was identified in one family. The ten different mutations include point mutations resulting in amino acid substitutions, stop codons, and small deletions and insertions causing frameshifts. Missense mutations were preferentially located in the amino‐terminal part of the protein, exons 2 and 4, whereas stop and frameshift mutations were located in the carboxyl‐terminal region, exons 10 and 11. However, in two families, two missense mutations in exon 11 were identified. Our study demonstrates that WASP genotypes have some concordance with the patients' phenotypes, although mutation 1019delC, identified in a family with several affected members, resulted in high intrafamilial clinical variability. © 2001 Wiley‐Liss, Inc.     

Re‐evaluation of the big blue® mouse assay of propiconazole suggests lack of mutagenicity

Propiconazole (PPZ) is a conazole fungicide that is not mutagenic, clastogenic, or DNA damaging in standard in vitro and in vivo genetic toxicity tests for gene mutations, chromosome aberrations, DNA damage, and cell transformation. However, it was demonstrated to be a male mouse liver carcinogen when administered in food for 24 months only at a concentration of 2,500 ppm that exceeded the maximum tolerated dose based on increased mortality, decreased body weight gain, and the presence of liver necrosis. PPZ was subsequently tested for mutagenicity in the Big Blue® transgenic mouse assay at the 2,500 ppm dose, and the result was reported as positive by Ross et al. ([2009]: Mutagenesis 24:149‐152). Subsets of the mutants from the control and PPZ‐exposed groups were sequenced to determine the mutation spectra and a multivariate clustering analysis method purportedly substantiated the increase in mutant frequency with PPZ (Ross and Leavitt. [2010]: Mutagenesis 25:231‐234). However, as reported here, the results of the analysis of the mutation spectra using a conventional method indicated no treatment‐related differences in the spectra. In this article, we re‐examine the Big Blue® mouse findings with PPZ and conclude that the compound does not act as a mutagen in vivo. © Environ. Mol. Mutagen. 2012. Published 2011 Wiley Periodicals, Inc.     

Severe lactic acidosis caused by a novel frame‐shift mutation in mitochondrial‐encoded cytochrome c oxidase subunit II

We report the first frame‐shift truncation mutation in a mitochondrial DNA (mtDNA)‐encoded subunit II of cytochrome c oxidase (COXII). The mutation was identified by temporal temperature gradient gel electrophoresis (TTGE) followed by direct DNA sequencing in an infant who died at 12 days of age following a course of apnea, bradycardia, and severe lactic acidosis. The patient had a twin brother who died at two days of age of similar course. The mutation, 8042delAT, produced a truncated protein that was 72 amino acids shorter than the wild type protein. The mutant protein, missing one third of the amino acid residues at the C‐terminal essential for hydrophilic interaction with cytochrome c, ligand binding to Cu_A and Mg, and the formation of proton and water channels, apparently has devastating effects on mitochondrial respiratory function. © 2001 Wiley‐Liss, Inc.     

PCR‐ and Restriction Endonuclease‐Based Detection of IDH1 Mutations

Hotspot mutations in codon 132 of the gene encoding isocitrate dehydrogenase 1 (IDH1) have emerged as the most frequent DNA alteration in astrocytomas, oligodendrogliomas and oligoastrocytomas. These mutations have been shown to be of significant diagnostic and prognostic value. So far, assessment of IDH1 mutation relied on DNA sequencing techniques.We generated a set of primers suitable for endonuclease‐based detection of hotspot mutations in codon 132 of IDH1. This primer set will allow determining these mutations without the need of DNA sequencing. One set of primer sets is designed to detect the presence or absence of IDH1 mutations in codon 132, while the other primer sets individually recognize the R132H, R132C, R132S, R132G and R132L mutations.     

Classical galactosemia and mutations at the galactose‐1‐phosphate uridyl transferase (GALT) gene

Classical galactosemia is caused by a deficiency in activity of the enzyme galactose‐1‐phosphate uridyl transferase (GALT), which, in turn, is caused by mutations at the GALT gene. The disorder exhibits considerable allelic heterogeneity and, at the end of 1998, more than 150 different base changes were recorded in 24 different populations and ethnic groups in 15 countries worldwide. The mutations most frequently cited are Q188R, K285N, S135L, and N314D. Q188R is the most common mutation in European populations or in those predominantly of European descent. Overall, it accounts for 60–70% of mutant chromosomes, but there are significant differences in its relative frequency in individual populations. Individuals homoallelic for Q188R tend to have a severe phenotype and this is in keeping with the virtually complete loss of enzyme activity observed in in vitro expression systems. Globally, K285N is rarer, but in many European populations it can be found on 25–40% of mutant chromosomes. It is invariably associated with a severe phenotype. S135L is found almost exclusively in African Americans. In vitro expression results are discrepant, but some individuals carrying S135L appear to exhibit GALT activity in some tissues. Duarte 1 (or Los Angeles) and Duarte 2 (or Duarte) variants carry the same amino acid substitution, N314D, even though D1 is associated with increased erythrocyte GALT activity and D2 with reduced activity. N314D is in linkage disequilibrium with other base changes that differ on the D1 and D2 alleles. N314D does not impair GALT activity in in vitro expression systems. However, there are differences in the abundance of GALT protein in lymphoblastoid cells lines from D2 and D1 individuals. It is unclear whether the specific molecular changes that distinguish the D1 and D2 alleles account for the different activities. The considerable genetic heterogeneity documented to date undoubtedly contributes to the phenotypic heterogeneity that is observed in galactosemia. The additional effects of nonallelic variation and other constitutional factors on phenotypic variability remain to be elucidated. Hum Mutat 13:417–430, 1999. © 1999 Wiley‐Liss, Inc.     

Human CD36 deficiency is associated with elevation in low‐density lipoprotein‐cholesterol

To find out whether CD36 plays a role in the human lipoprotein metabolism, we studied lipoprotein profiles in subjects with CD36 deficiency. Apparently healthy Japanese volunteers (n = 790) were classified by flow cytometry into three groups of normal (platelet and monocyte CD36+, n = 741, 93.8%), type‐II deficiency (platelet CD36− and monocyte CD36+, n = 45, 5.7%), and type‐I deficiency (platelet and monocyte CD36−, n = 4, 0.5%). At least one of reported mutations in the CD36 gene was found in all four subjects with type‐I deficiency and in 23 of the 45 subjects with type II. Among 779 subjects (731 normals, 44 type II, and four type I) with serum triglyceride levels of <400 mg/dL, serum total cholesterol and low‐density lipoprotein (LDL) cholesterol were significantly elevated in type‐II deficiency (P = 0.0095 and 0.0382 versus normal, respectively, Scheffe's F‐test), while differences were not significant in triglyceride and high‐density lipoprotein‐cholesterol. Similar tendency was observed in type‐I deficiency, although the differences were not statistically significant because of small sample size. We conclude that CD36 deficiency elevates LDL cholesterol, indicating a contribution of CD36 to LDL metabolism. Am. J. Med. Genet. 93:299–304, 2000. © 2000 Wiley‐Liss, Inc.     

Electrophysiological characteristics of a population of mushroom body neurons in <Emphasis Type="Italic">Apis Mellifera</Emphasis> honeybees in kynurenine deficiency

Electrophysiological methods were used to study the neurophysiological characteristics of mushroom body neurons in snow ^laranja mutant and wild-type bees. The snow ^laranja mutation, which produces a sharp reduction in the activity of the enzyme tryptophan oxygenase, thus creating a deficiency of all kynurenines in the body, modifies the bioelectrical properties of mushroom body neurons. The parameters most dependent on the snow ^laranja mutation and, thus, the level of endogenous kynurenines, were the duration of action potential afterdepolarization recorded from neurons in the calyx and the amplitude of the postsynaptic potential of neurons in the calyx evoked by focal stimulation of the antennal lobes. There was also a tendency to an increase in the spontaneous spike frequency. These data lead to the conclusion that the effect of the mutation is recessive in nature and appears only in homozygotes (bearing two mutant alleles). __________ Translated from Zhurnal Vysshei Nervnoi Deyatel’nosti imeni I. P. Pavlova, Vol. 56, No. 6, pp. 796–800, November–December, 2006.     

Mutational and haplotype analysis of <Emphasis Type="Italic">AGL</Emphasis> in patients with glycogen storage disease type III

Glycogen storage disease type III (GSD III) is a rare autosomal recessive inherited disorder caused by a deficiency of the glycogen-debranching enzyme (AGL). We investigated two GSD III patients and identified four different mutations. Nucleotide sequence analysis revealed patient 1 of Chinese descent to be a compound heterozygote for a novel nonsense mutation, R34X, and the splicing mutation (IVS32−12A > G) reported in a Japanese patient. Patient 2 of Japanese origin was found to be compound heterozygous for a novel nonsense mutation, Y1148X, and the splicing mutation (IVS14+1G > T) that we had described previously. To determine whether splicing mutations occurred independently, we performed intense AGL haplotype analysis using 21 intragenic polymorphic markers plus a novel polymorphism IVS32−97 A/G in the vicinity of the IVS32 splicing mutation. Patient 1 of Chinese origin and the Japanese patient homozygous for the IVS32−12A > G were found to have different haplotypes, indicating the IVS32−12A > G mutation to be a recurrent mutation. This is the first recurrent mutation established by intense haplotyping in the AGL gene. Received: October 3, 2001 / Accepted: November 12, 2001     

dbNSFP v3.0: A One‐Stop Database of Functional Predictions and Annotations for Human Nonsynonymous and Splice‐Site SNVs

The purpose of the dbNSFP is to provide a one‐stop resource for functional predictions and annotations for human nonsynonymous single‐nucleotide variants (nsSNVs) and splice‐site variants (ssSNVs), and to facilitate the steps of filtering and prioritizing SNVs from a large list of SNVs discovered in an exome‐sequencing study. A list of all potential nsSNVs and ssSNVs based on the human reference sequence were created and functional predictions and annotations were curated and compiled for each SNV. Here, we report a recent major update of the database to version 3.0. The SNV list has been rebuilt based on GENCODE 22 and currently the database includes 82,832,027 nsSNVs and ssSNVs. An attached database dbscSNV, which compiled all potential human SNVs within splicing consensus regions and their deleteriousness predictions, add another 15,030,459 potentially functional SNVs. Eleven prediction scores (MetaSVM, MetaLR, CADD, VEST3, PROVEAN, 4× fitCons, fathmm‐MKL, and DANN) and allele frequencies from the UK10K cohorts and the Exome Aggregation Consortium (ExAC), among others, have been added. The original seven prediction scores in v2.0 (SIFT, 2× Polyphen2, LRT, MutationTaster, MutationAssessor, and FATHMM) as well as many SNV and gene functional annotations have been updated. dbNSFP v3.0 is freely available at http://sites.google.com/site/jpopgen/dbNSFP .     

Mutational specificity: Mutation frequencies but not mutant frequencies in Big Blue® mice fit a Poisson distribution

Transgenic mutation assays generally use mutant frequencies to estimate mutation frequencies but the degree to which clonal expansion inflates mutant frequencies is largely unknown. Mutant frequency is defined as the fraction of cells carrying mutations in the gene of interest and, according to the standard Big Blue® protocol, is determined by dividing the number of mutant plaques by the total number of plaques screened. Mutation frequency is determined as the fraction of cells carrying definitely independent mutations and therefore requires correction for clonal expansion. Mutant and mutation frequencies were determined for brain, thymus and male germ cells of four mice from two age groups (3- versus 10-month old). The mutant frequency in thymus differed significantly between 3- and 10-month old mice (P < 0.05). By sequencing all mutants, the mutation frequency (i.e., corrected for jackpot mutations) in thymus was determined and was not significantly different between 3- and 10-month old mice. Mutant frequency does not fit a Poisson distribution, but mutation frequency corrected for jackpot mutations is substantially less variable and does fit a Poisson distribution. © 1996 Wiley-Liss, Inc.     

Free the Data: One Laboratory's Approach to Knowledge‐Based Genomic Variant Classification and Preparation for EMR Integration of Genomic Data

Current technology allows clinical laboratories to rapidly translate research discoveries from small patient cohorts into clinical genetic tests; therefore, a potentially large proportion of sequence variants identified in individuals with clinical features of a genetic disorder remain unpublished. Without a mechanism for clinical laboratories to share data, interpretation of sequence variants may be inconsistent. We describe here the two components of Emory Genetics Laboratory's (EGL) in‐house developed data management system. The first is a highly curated variant database with a data structure designed to facilitate sharing of information about variants identified at EGL with curated databases. This system also tracks changes in variant classifications, creating a record of previous cases in need of updated reports when a classification is changed. The second component, EmVClass, is a Web‐based interface that allows any user to view the inventory of variants classified at EGL. These software tools provide a solution to two pressing issues faced by clinical genetics laboratories: how to manage a large variant inventory with evolving variant classifications that need to be communicated to healthcare providers and how to make that inventory of variants freely available to the community.