Coexistence of the K65R/L74V and/or K65R/T215Y mutations on the same HIV-1 genome.

OBJECTIVE: To determine to which extent the mutations K65R, L74V/I and T215Y/F are linked to the same HIV-1 genome. METHODS: Retrospective analysis of the Marseille database (8866 sequences from 3720 patients). The HIV-1 pol gene from four patients' viruses harbouring the double mutant (pure species or genotypic mixtures) was amplified, cloned and sequenced. RESULTS: The four patients had a mean viral load of 5.6 log(10)copies/ml. Analysis of 73 clones (patient 1: 12 clones; patient 2: 27 clones; patient 3: 13 clones; patient 4: 21 clones) showed that 29 clones harboured K65R and L74V/I mutations. Twenty-three per cent of clones from the two bulk sequences harbouring K65K/R and T215T/Y genotypic mixtures contained K65R and T215Y on the same viral genome. CONCLUSIONS: The co-linearity of 65R and 74V or 65R and 215Y amino-acids on the same genome is rare. A high viral load (6.19 log(10)) associated with the coexistence of 65R and 74V on the same HIV-1 genome suggests possible compensatory mechanisms.     

Severe hypophosphatasia: characterization of fifteen novel mutations in the ALPL gene

Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and deficiency of serum and tissue liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. We report the characterization of ALPL gene mutations in a series of 11 families from various origins affected by perinatal and infantile hypophosphatasia. Sixteen distinct mutations were found, fifteen of them not previously reported: M45V, G46R, 388-391delGTAA, 389delT, T131I, G145S, D172E, 662delG, G203A, R255L, 876-881delAGGGGA, 962delG, E294K, E435K, and A451T. This confirms that severe hypophosphatasia is due to a large spectrum of mutations in Caucasian populations.     

Mutation analysis of the GALT gene in Czech and Slovak galactosemia populations: identification of six novel mutations, including a stop codon mutation (X380R)

A study of the galactose-1-phosphate uridyltransferase (GALT) gene from 37 unrelated galactosemia families is reported here. A total of 16 sequence variations in eleven mutated alleles was found. The two most common molecular defects were the mutations Q188R (46.0%) and K285N (25.7%). Six novel mutations in the GALT gene, X380R, Y209S, E340K, L74fsdelCT, Q169K and L256/P257delGCC, were detected. Three mutations, V151A, L195P and R204X that were previously described in other populations, were also found. The mutation X380R, which breaks the stop codon of the GALT gene, causes elongation of the GALT enzyme's protein chain. A deletion of four nucleotides in the 5' promoter region, in a position 116 - 119 nucleotides upstream from the initiate codon (5'UTR-119delGTCA), was revealed in Duarte (D2) alleles, in addition to N314D, IVS4nt-27g-->c, IVS5nt+62g-->a, and IVS5nt-24g-->a. An unusual molecular genotype was observed on 2 types of classical galactosemia alleles, with six variations from the normal nucleotide sequence presented in cis (mutation V151A or E340K plus five Duarte (D2) characteristic variations). In summary, galactosemia is a heterogeneous disorder at the molecular level, and mutation N314D, appears to be an ancient genetic variant of the GALT gene. Hum Mutat 15:206, 2000.     

Open sandwich ELISA with V(H)-/V(L)-alkaline phosphatase fusion proteins.

The Sandwich ELISA is a widely used technique to measure antigen concentration. Recently, a novel ELISA based on the interchain interaction of separated V(H) and V(L) chains from a single antibody variable region (Fv) was proposed (Open Sandwich ELISA). Since it employs a single antibody recognizing one epitope, the assay requires, in essence, only one cycle of incubation and washing steps. To demonstrate this directly, we have constructed a recombinant gene fusion encoding the V(H) chain of an anti-hen egg lysozyme (HEL) antibody HyHEL-10 and Escherichia coli alkaline phosphatase (V(H)-PhoA). The same type of gene fusion using V(L) chain instead of V(H) chain (V(L)-PhoA) was also constructed and the proteins were obtained with an E. coli expression/secretion system. Open Sandwich ELISAs were performed using microtiter plates with immobilized V(L) or V(H) fragment, and V(H)-PhoA or V(L)-PhoA, respectively, as the detection reagent which was simultaneously added to each well with samples. As a result, HEL concentrations in the samples were determined after one round of incubation and washing steps, with a signal generated in a direct relationship to the concentration of HEL added to the reaction mixture. The minimum detectable HEL concentration was approximately 10 ng/ml, which was almost equal to the value previously obtained with plate-immobilized V(L) and V(H) fragment displayed on M13 phage. When the active-site mutant V(H)-PhoA(D101S) was employed instead of V(H)-PhoA and reacted at an optimum pH of 10, a significant enhancement in signal was attained.     

Spectrum of Delta(7)-dehydrocholesterol reductase mutations in patients with the Smith-Lemli-Opitz (RSH) syndrome

The Smith-Lemli-Opitz syndrome (SLOS; also known as the RSH syndrome) is an autosomal recessive genetic disorder, leading to characteristic multi-organ developmental abnormalities, dysmorphic facies, limb malformations and mental retardation. Mutations in the gene for Delta(7)-dehydrocholesterol reductase (Delta(7)-reductase), which catalyzes the last step in cholesterol biosynthesis, cause the disease. We screened 32 patients with SLOS, 28 from the USA and four from Sweden. Twenty-two different nucleotide changes, predicted to be disease-causing mutations, were identified; 20 missense mutations, one nonsense mutation and one splice-site mutation involving the exon 9 acceptor site (IVS8 -1G-->C) were detected. All probands were heterozygous for mutations. Twelve of these mutations have not been reported previously, including missense mutations L148R, F168I, D175H, P179L, P243R, F284L, N287K, F302L, R404S, Y462H, R469P and one nonsense mutation W37X [corrected]. Coupled with previously reported mutations, these findings bring the total of different Delta(7)-reductase mutations to 36. These are distributed throughout the coding sequence of the Delta(7)-reductase gene except exons 3 and 5, with a clustering in exon 9. Three mutations account for 54% of those observed in our cohort, the splice acceptor site mutation IVS8 -1G-->C (22/64 alleles, 34%), T93M (8/64, 12.5%) and V326L (5/64, 7.8%). Severity of SLOS was negatively correlated with both plasma cholesterol and relative plasma cholesterol, but not with 7-dehydrocholesterol, the immediate precursor, confirming previous observations. However, no correlation was observed between mutations and phenotype, suggesting that the degree of severity may be affected by other factors. We estimate that between 33 and 42% of the variation in the SLOS severity score is accounted for by variation in plasma cholesterol. Thus, factors other than plasma cholesterol are additionally involved in determining severity.     

Mechanisms of cold sensitivity of paramyotonia congenita mutation R1448H and overlap syndrome mutation M1360V

Missense mutations of the human skeletal muscle voltage-gated Na⁺ channel (hSkM1) cause a variety of neuromuscular disorders. The mutation R1448H results in paramyotonia congenita and causes cold-induced myotonia with subsequent paralysis. The mutation M1360V causes an overlapping syndrome with both K⁺-induced muscle weakness and cold-induced myotonia. The molecular mechanisms of the temperature dependence of these disorders are not well understood. Therefore we investigated physiological parameters of these Na⁺ channel mutations at different temperatures. Channel proteins were recombinantly expressed in human embryonic kidney cells and studied electrophysiologically, using the whole-cell patch-clamp technique. We compared the wild-type (WT) channel with both mutants at different temperatures. Both mutations had slower inactivation and faster recovery from inactivation compared to WT channels. This effect was more pronounced at the R1448H mutation, leading to a larger depolarization of the cell membrane causing myotonia and paralysis. The voltage dependence of activation of R1448H was shifted to more negative membrane potentials at lower temperature but not at the M1360V mutation or in the WT. The window current by mutation R1448H was increased at lower temperatures. The results of this study may explain the stronger cold-induced clinical symptoms resulting from the R1448H mutation in contrast to the M1360V mutation.     

Investigation of causes of oseltamivir chemoprophylaxis failures during influenza A (H1N1-2009) outbreaks

BackgroundAntiviral post-exposure prophylaxis with oseltamivir has been used as a strategy in mitigating the Influenza A (H1N1-2009) pandemic. There have been few reports of well-documented prophylaxis failures and the reasons for failure.ObjectivesWe report herein a series of 10 cases of prophylaxis failures and explore the reasons behind their prophylaxis failure.Study designIn the early pandemic phase, the military employed oseltamivir post-exposure ring-prophylaxis of affected units. From June 22 to July 30, 2009, cases of laboratory-confirmed prophylaxis failures were identified. Nasopharyngeal swabs were collected and tested by PCR. Samples with sufficient RNA material were sent for whole genome sequencing, and screened for mutations that confer oseltamivir resistance, especially the H275Y mutation.ResultsTen cases of laboratory-confirmed prophylaxis failure were identified, with a mean age of 22.3 years. One case was asymptomatic; the remaining 9 had fever or cough but without severe complications. The mean duration of exposure before starting oseltamivir was 1.9 days (SD 0.9), while the mean duration of oseltamivir consumption before symptom onset was 1.9 days (SD 1.4). None of the samples had the H275Y mutation or other known mutations that confer resistance. From the whole genome sequencing, several mutations at the HA (T220S, E275V, T333A, D239G); PB2 (K660R, L607V, V292I); NS1 (F103S), and NP (W104G) gene segments were detected, but none of them were likely to result in anti-viral resistance.ConclusionsPrimary prophylaxis failures exhibited mild symptoms without complications; all did not have the H275Y mutation and were unlikely to result from other mutations.     

婴儿严重肌阵挛癫痫钠离子通道SCNIA基因突变分析

目的 研究婴儿严重肌阵挛癫痫(severe myoclonic epilepsy of infancy,SMEI)患儿钠离子通道a1亚单位基因(sodium channel al subunit gene,SCNIA)突变筛查及遗传特征.方法 收集SMEI患儿23例及其家系成员的临床资料及外周血DNA,采用PCR扩增和DNA直接测序的方法筛查SCNIA基因的26个外显子的突变.结果 23例SMEI患儿中17例有SCNlA基因突变.基因突变率约为73.9%(17/23),其中8例为错义突变(F90S、I91T、A239T、W952G、T1210K,V1335M、V1390M、G1433E),3例为无义突变(R612X、W768X、w1408X),3例为缺失突变(A395fsX400、L556fsX557、V1778fsX1800),1例为插入突变(Y1241fsX1270),1例为剪切部位突变(IVS10+3A>G),1例为同义突变(K1492K),截断突变约占总突变的47.1%(8/17).13个突变位点(F90S、I91T、T1210K、V1335M、G1433E、R612X、W768X,A395faX400、L556fsx557、V1778fsXl800、Y1241fsXl270、IVS10+3A>G、K1492K)经相关检索未见报道.14例突变已证实为新生突变,其余3例突变尚不能确定突变来源.结论 SCNIA基因是SMEI患儿的主要致病基因,约一半为截断突变.SMEI患儿的SCNIA基因突变无热点,且多为新生突变.     

An adenosine deaminase (ADA) allele contains two newly identified deleterious mutations (Y97C and L106V) that interact to abolish enzyme activity

Genetic deficiency of the purine salvage enzyme adenosine deaminase (ADA) results in varying degrees of immunodeficiency, ranging from neonatal onset Severe Combined Immunodeficiency (SCID) to an adult onset immunodeficiency disorder. Multiple different mutations have now been identified in these immunodeficient patients. Additional mutations, initially identified in healthy individuals, abolish ADA in erythrocytes but retain 10-80% of activity in non-erythroid cells ('partial deficiency mutations'). In general, severity of disease correlates inversely with the amount of residual ADA expressed by the mutant enzymes and directly with the accumulation of the toxic metabolites deoxyATP and deoxyadenosine. We report two newly identified mutations (Y97C and L106V), both carried on the same allele of an immunodeficient patient who was diagnosed prenatally and successfully transplanted with haploidentical bone marrow. Based on the ability of mutant cDNAs to express ADA in vitro , the L106V mutation resulted in activity similar to 'partial' mutations (30% of normal) while the Y97C mutation resulted in detectable but markedly reduced activity (1.5% of normal). However, the presence of both mutations on the same allele virtually abolished detectable enzyme activity. Analysis of the crystallographic structure of ADA to understand the marked deleterious effect of the Y97C mutation suggested a previously unappreciated role of salt bridges in the catalytic mechanism of ADA. The patient was also heteroallelic for a previously described deletion of the promoter and exon 1. Testing of additional patients in whom we had not identified a mutation on the second allele revealed presence of this deletion in three of four patients tested. This deletion is therefore relatively common, accounting for 10% of almost 100 chromosomes studied by this and other laboratories, but is easily missed by currently used methods of mutation detection. Lastly, the finding of two mutations on the same allele that interact to reduce residual enzyme function emphasizes hazards in evaluating potential genotype-phenotype correlations in individuals analyzed only for the presence of single specific mutations.      

The spectrum of Familial Mediterranean Fever gene (MEFV) mutations and genotypes in Iran,and report of a novel missense variant (R204H)

BackgroundFamilial Mediterranean Fever (FMF) is an autosomal recessive disorder, characterized by recurrent and self-limited episodes of fever, abdominal pain, synovitis and pleuritis. FMF as the most common inherited monogenic autoinflammatory disease mainly affects ethnic groups of the Mediterranean basin, Arab, Jewish, Turkish, Armenian North Africans and Arabic descent.Materials and methodsIn the present study, we selected 390 unrelated FMF patients according to the Tel-Hashomer criteria, and analyzed all patients for 12 most common mutations of MEFV gene by reverse hybridization assay (FMF strip assay). We also investigated exon 2 and 10 of MEFV gene in 78 patients by Sanger sequencing.ResultsAccording to strip assay results, at least one mutation was found in 234 patients (60%), and no mutation was found in other 156 patients (40%). The five most common mutations and allelic frequencies were M694V (13.6%), E148Q (10.4%), M694I (6.5%), V726A (4.1%), and M680I (3.8%). Moreover, we detected a novel missense variant (R204H, c.611 G > A) (SCV000297822) and following rare mutations among sequenced samples; R202Q, P115T, G304R, and E230K.ConclusionThis study describes the MEFV mutations spectrum and distribution in Iranian population, and shows different mutation patterns among Iranian ethnicities. Moreover, M694V is the most common MEFV mutation in Iran.     

The molecular basis of UDP-galactose-4-epimerase (GALE) deficiency galactosemia in Korean patients.

PURPOSE: UDP-galactose-4-epimerase (GALE) deficiency galactosemia is an autosomal recessive disorder and the prevalence of the disease varies among ethnic groups. We aimed to investigate molecular characteristics of the Korean patients with attenuated GALE activity and elevated galactose-1-phosphate levels in blood. METHODS: In order to characterize the molecular defects underlying GALE deficiency, the GALE gene of 7 patients showing severe activity decreases was sequenced. PCR-RFLP was performed to confirm the presence of the mutations identified by sequencing. RESULTS: Nine mutations were identified: 8 missense mutations (p.A25V, p.R40C, p.D69E, p.E165K, p.R169W, p.R239W, p.G302D, and p.R335H) and one nonsense mutation (p.W336X). Except for p.R335H, all of these mutations are novel. Six patients were compound heterozygotes (p.D69E/p.G302D, p.R40C/p.R169W, p.D69E/p.E165K, p.R239W/p.R335H, p.A25V/p.R169W, and p.G302D/p.R335H) and the remaining patient had only one mutation (p.W336X/not detected). Thirty patients with moderately reduced GALE activity were also tested by PCR-RFLP for the presence of the above mutation, and mutations were detected in 17 of these 30 patients. The frequency of p.G302D (9/30), p.R239W (6/30) and p.R169W (5/30) in our Korean patients with GALE deficiency galactosemia was relatively high. CONCLUSIONS: We detected 9 mutations of the GALE gene in Korean galactosemia patients, and confirmed allelic heterogeneity in this disease.     

Mutation analysis and description of sixteen RSH/Smith-Lemli-Opitz syndrome patients: polymerase chain reaction-based assays to simplify genotyping

We report the clinical and molecular data of 16 patients with RSH/Smith-Lemli-Opitz syndrome (RSH/SLOS) with varying phenotypic severity, for which we have identified mutations in both alleles. RSH/SLOS is an autosomal recessive malformation syndrome caused by mutations in the gene encoding the sterol Delta(7)-reductase. This protein catalyzes the reduction of 7-dehydrocholesterol to cholesterol in the last step of cholesterol biosynthesis via the Kandutsch-Russell pathway. In addition to previously reported mutations (T93M, L109P, G147D, W151X, T154M, R242C, A247V, T289I, IVS8-1G-->C, Y408H, and E448K), we have identified six previously undescribed mutations (321G-->C, W177R, R242H, Y318N, L341P, and C444Y). We also report rapid polymerase chain reaction (PCR)-based assays developed to detect four of the recurring mutations (T93M, W151X, V326L, and R404C) and six other RSH/SLOS mutations (321G-->C, L109P, T154M, T289I, Y318N, and L341P). The purpose of this article is to correlate detailed clinical information with molecular data in order to improve our understanding of the genotype-phenotype correlation of RSH/SLOS and to report the development of PCR-based assays that will allow more rapid mutation analysis.     

2004-2005年中国A(H1N1)亚型流感病毒抗原性及基因特性研究

目的 阐明2004-2005年中国流行的A（H1N1）亚型流感病毒血凝素抗原性及其基因变异情况.方法 对2004-2005年分离的A（H1N1）亚型毒株先进行单向血凝抑制试验;在此基础上选取不同时间、地点的A（H1N1）亚型流感毒株进行血凝素基因HA1区核苷酸序列测定并推导出其氨基酸序列,然后进行基因进化特性分析.结果 单向血凝抑制实验结果表明,2004年A（H1N1）亚型病毒株对鉴定血清的血凝抑制效价与A/Shanghai/1/1999（H1N1）毒株没有4倍差异;2005年分离的A（H1H1）亚型毒株中有62株（占6.2%）病毒与A/Shanghai/1/1999（H1N1）毒株本身的血凝抑制效价相比有4倍差异.HA1区核苷酸序列和氨基酸序列分析表明,我国2005年分离到的A（H1N1）亚型流感病毒株有以下位点发生变异,54 K＞R、90T＞K、101Y＞H、149R＞K、169V＞A、190D＞N、212R＞K、219K＞R、245W＞R、246Y＞F、258T＞N、318V＞A,其中54、190位氨基酸位于抗原决定簇.结论 我国2005年分离的A（H1N1）亚型流感毒株基因特性和抗原性已开始发生变异.     

Identification of mutations in the galactose-1-phosphate uridyltransferase (GALT) gene in 16 Turkish patients with galactosemia, including a novel mutation of F294Y. Mutation in brief no. 235. Online

Classical galactosemia caused by deficiency of galactose-1-phosphate uridyltransferase (GALT) is a severe autosomal recessive disorder. We report here molecular analysis of 16 unrelated Turkish galactosemia index cases without GALT activity. Almost 84% of all mutant alleles were identified in this study. The most common molecular defect observed in the Turkish population was Q188R (replacement of glutamine-188 by arginine) (57%). In order to facilitate the determination of unknown mutations in the entire coding region of GALT, we established an approach based on GALT cDNA synthesis and direct sequencing. We have identified one novel candidate galactosemia mutation, a T-to-A transversion at the codon 294 (F294Y) in exon 9 in addition to previously reported three missense (M142K K285N, A320T), one stop codon (E340X), and one silent (L218L) mutations in galactosemia patients which reflect considerable genetic heterogeneity in the Turkish population.     

Metachromatic leukodystrophy: Identification of the first deletion in exon 1 and nine novel point mutations in the arylsulfatase A gene

Metachromatic leukodystrophy (MLD), a lysosomal storage disease caused by the deficiency of arylsulfatase A (ASA), is inherited as an autosomal recessive trait, and its frequency is estimated to be 1 in 40,000 live births. Genomic DNA from 21 MLD patients (14 late-infantile and 7 juvenile cases) was amplified in four overlapping PCR fragments and tested by allele-specific oligonucleotide (ASO) for the two common mutations 459+1G→A and P426L. These mutations were found in only 28.6% of the alleles studied. The remaining alleles were analyzed by chemical mismatch cleavage (CMC) and automatic sequencing. In addition to five previously reported mutations (459+1G→a, A212V, R244C, R390W, P426L), 10 novel mutations were identified: 9 missense mutations (S95N, G119R, D152Y, R244H, S250Y, A314T, R384C, R496H, K367N) and one 8 bp deletion in exon 1, the first mutation reported in this exon. These methods allowed us to identify 76% of the alleles tested. Genotype-phenotype correlations could be established for some of these mutations. These results confirm the heterogeneity of mutations causing MLD and suggest that CMC is a reliable and informative screening method for point mutation detection in the arylsulfatase A gene. Hum Mutat 9:234–242, 1997. © 1997 Wiley-Liss, Inc.     

Evidence of a founder effect for the tissue-nonspecific alkaline phosphatase (TNSALP) gene E174K mutation in hypophosphatasia patients

Hypophosphatasia is a rare inborn error of metabolism characterised by defective bone mineralisation caused by a deficiency of liver-, bone- or kidney-type alkaline phosphatase due to mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. The clinical expression of the disease is highly variable, ranging from stillbirth with a poorly mineralised skeleton to pathologic skeletal fractures which develop in late adulthood only. This clinical heterogeneity is due to the strong allelic heterogeneity in the TNSALP gene. We found that mutation E174K is the most frequent in Caucasian patients, and that it was carried by 31% of our patients with mild hypophosphatasia. Because the mutation was found in patients of various geographic origins, we investigated whether it had a unique origin or rather multiple origins due to recurrence of de novo mutations. Three intragenic polymorphisms, S93S, 472+12delG and V505A, were genotyped in patients carrying E174K and in normal unrelated individuals. Our results show that all the E174K mutations are carried by a common ancestral haplotype, also found at low frequency in normal and hypophosphatasia chromosomes. We conclude that the TNSALP gene E174K mutation is the result of a relatively ancient ancestral mutation that occurred on a single chromosome in the north of Western Europe and spread throughout the rest of Europe and into the New World as a result of human migration.     

Prevalence of factor V R2 (H1299R) polymorphism in the Lebanese population

AIMS: A recently identified polymorphism in factor V gene (His1299Arg; also named HR2) has been reported to be a possible risk factor for the development of venous thromboembolism (VTE), with a high prevalence of 9.5-15.2% in patients of different ethnic groups in different parts of the world. The aim of this study is to assess the prevalence of HR2 haplotype in Lebanon. METHODS: We randomly selected 125 samples from unrelated donors logged into our HLA registry; these represent healthy Lebanese individuals originating from different provinces and religious communities of the country. Their DNA was extracted using the Pel-Freez extraction kit and stored at -80 degrees C for later use.The CVD StripAssay was used for PCR and reverse hybridisation. It screens for several gene mutations including factor V H1299R. RESULTS: A total of 125 controls were studied: 72 males and 53 females with a median age 42 years. Thirteen (10.4%) had the HR2 haplotype; 11 (8.8%) were heterozygous (R1/R2), and two (1.6%) were homozygous (R2/R2), with an allelic frequency of 0.06. CONCLUSIONS: Our study is the first report from Lebanon that describes the prevalence of HR2 haplotype and the frequency of its alleles. We are reporting a high prevalence of the HR2 in our population (10.4%). The hypothesis that A4070G polymorphism might contribute to the expression of a thrombotic phenotype deserves to be tested in our population through larger studies.     

Distribution of HFE C282Y and H63D mutations in the Balearic Islands (NE Spain)

The HFE gene contains two main missense mutations: C282Y and H63D. Individuals with these mutations carry a risk of developing hereditary haemochromatosis (HH). The common form of this disease is due to homozygosity for the C282Y mutation. Population studies have shown the variation of the prevalence of these mutations in different countries and ethnic groups. The purposes of this current study were to determine the prevalence of the C282Y and H63D mutations in the Balearic Islands and the genotypic characterization of patients diagnosed with HH, as well as those with iron overload and liver diseases. A total of 1330 Balearic chromosomes were analyzed. The results showed that the populations of the Balearic Islands were not homogeneous. No C282Y carriers were observed in a group of descendants of Majorcan Jews (Chuetas) and the frequency was very low in Minorca (1.2%) in comparison with the other islands of Majorca (4.7%) and Ibiza (6.5%). The carrier frequency of the H63D mutation was similar in the three islands and very high (43.1%) in the descendants of Majorcan Jews. The study of patients was carried out in 129 individuals. The homozygous C282Y genotype was the principal one involved in hereditary haemochromatosis (90%), whereas the other HH patients were C282Y/H63D compound heterozygous and H63D homozygous.