Mutational analysis of the cardiac actin gene in familial and sporadic dilated cardiomyopathy

Dilated cardiomyopathy (DCM) results in part from genetic disorders. Recently, missense mutations of the cardiac actin gene have been reported to cause DCM. We studied 136 Japanese DCM cases to elucidate how frequently the gene mutations are involved in its pathogenesis. Genomic DNA samples were obtained from 136 DCM cases (107 males, 29 females), containing 30 familial DCM (5 confirmed and 25 suspected). All six exons of the cardiac actin gene were analyzed by polymerase chain reaction, single-strand conformation polymorphism, and sequencing. We detected no mutations of the disease causation previously reported (G867A or A1014G) but two silent mutations (G979C and C1018T) in exon 6 and one point mutation (T1080A) in the 3′-untranslated region. As a result of screening 128 healthy subjects, these novel silent mutations were found to be mere genetic polymorphisms, not responsible for the disease. Although some genetic polymorphisms exist in the cardiac actin gene, mutations of the gene are rarely responsible for DCM, at least in the Japanese patients. Am. J. Med. Genet. 86:325–327, 1999. © 1999 Wiley-Liss, Inc.     

Mutational analysis of the cardiac actin gene in familial and sporadic dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) results in part from genetic disorders. Recently, missense mutations of the cardiac actin gene have been reported to cause DCM. We studied 136 Japanese DCM cases to elucidate how frequently the gene mutations are involved in its pathogenesis. Genomic DNA samples were obtained from 136 DCM cases (107 males, 29 females), containing 30 familial DCM (5 confirmed and 25 suspected). All six exons of the cardiac actin gene were analyzed by polymerase chain reaction, single-strand conformation polymorphism, and sequencing. We detected no mutations of the disease causation previously reported (G867A or A1014G) but two silent mutations (G979C and C1018T) in exon 6 and one point mutation (T1080A) in the 3'-untranslated region. As a result of screening 128 healthy subjects, these novel silent mutations were found to be mere genetic polymorphisms, not responsible for the disease. Although some genetic polymorphisms exist in the cardiac actin gene, mutations of the gene are rarely responsible for DCM, at least in the Japanese patients.     

Identification of four novel PMM2 mutations in congenital disorders of glycosylation (CDG) Ia French patients

We screened 11 unrelated French patients with congenital disorders of glycosylation (CDG) Ia for PMM2 mutations. Twenty one missense mutations on the 22 chromosomes (95%) including four novel mutations were identified: C9Y (G26A) in exon 1, L32R (TA95GC) in exon 2, and T226S (C677G) and C241S (G722C) in exon 8. We studied the PMM activity of these four novel mutant proteins and of the R141H mutant protein in an E coli expression system. The T226S, C9Y, L32R, and C241S mutant proteins have decreased specific activity (23 to 41% of normal), are all more or less thermolabile, and R141H has no detectable activity. Our results indicate that the new mutations identified here are less severe than the inactive R141H mutant protein, conferring residual PMM activity compatible with life.


Keywords: CDG; phosphomannomutase; PMM2 mutations     

一个少见的β地中海贫血突变家系

目的　鉴定中国人中 1个少见的 β地中海贫血 (β-地贫 )家系成员的基因转录突变 (- 90C→ T)。　方法　表型分析采用常规的红细胞指数和血红蛋白电泳分析技术 ;反向点杂交技术用于检测中国人 18种已知类型的β-地贫突变 ;β珠蛋白基因全长 DNA测序技术用于确定样品的基因突变及其基因型 ;RT- PCR半定量法分析该突变对 β-珠蛋白基因转录水平的影响。　结果　家系分析发现 :先证者、先证者之兄和其母亲为有典型 β-地贫特征的个体 ,反向点杂交筛查未发现已知的 β-地贫突变 ,DNA直接测序分析发现该家系的这 3个成员均携带有 1种中国人中未见报道的 β地贫等位基因—— β-珠蛋白基因启动子区近侧 CACCC盒中 - 90位的 C→ T转录突变。RT- PCR分析显示该突变引起β-珠蛋白基因转录水平下降 (突变 :2 .2 33± 0 .0 1vs正常 :3.779± 1.19;95 % CI:3.0 6 0 ,4 .4 99) ,与其他类型的β-地贫杂合子β-基因的表达水平相当 (2 .110± 0 .5 3,95 % CI:1.732 ,2 .4 88)。　结论　 - 90位的 C→ T转录突变是中国人中未见报道的稀少类型的 β-地贫转录突变。     

Identification and in silico analyses of novel TGFBR1 and TGFBR2 mutations in Marfan syndrome-related disorders

Very recently, heterozygous mutations in the genes encoding transforming growth factor beta receptors I (TGFBR1) and II (TGFBR2) have been reported in Loeys-Dietz aortic aneurysm syndrome (LDS). In addition, dominant TGFBR2 mutations have been identified in Marfan syndrome type 2 (MFS2) and familial thoracic aortic aneurysms and dissections (TAAD). In the past, mutations of these genes were associated with atherosclerosis and several human cancers. Here, we report a total of nine novel and one known heterozygous sequence variants in the TGFBR1 and TGFBR2 genes in nine of 70 unrelated individuals with MFS-like phenotypes who previously tested negative for mutations in the gene encoding the extracellular matrix protein fibrillin-1 (FBN1). To assess the pathogenic impact of these sequence variants, in silico analyses were performed by the PolyPhen, SIFT, and Fold-X algorithms and by means of a 3D homology model of the TGFBR2 kinase domain. Our results showed that in all but one of the patients the pathogenic effect of at least one sequence variant is highly probable (c.722C > T, c.799A > C, and c.1460G > A in TGFBR1 and c.773T > G, c.1106G > T, c.1159G > A, c.1181G > A, and c.1561T > C in TGFBR2). These deleterious alleles occurred de novo or segregated with the disease in the families, indicating a causative association between the sequence variants and clinical phenotypes. Since TGFBR2 mutations found in patients with MFS-related disorders cannot be distinguished from heterozygous TGFBR2 mutations reported in tumor samples, we emphasize the importance of segregation analysis in affected families. In order to be able to find the mutation that is indeed responsible for a MFS-related phenotype, we also propose that genetic testing for sequence alterations in TGFBR1 and TGFBR2 should be complemented by mutation screening of the FBN1 gene.     

Novel fibroblast growth factor receptor 3 (FGFR3) mutations in bladder cancer previously identified in non-lethal skeletal disorders

Activating mutations in the fibroblast growth factor receptor 3 (FGFR3) gene are responsible for several autosomal dominant craniosynostosis syndromes and chondrodysplasias i.e. hypochondroplasia, achondroplasia, SADDAN and thanatophoric dysplasia--a neonatal lethal dwarfism syndrome. Recently, activating FGFR3 mutations have also been found to be present in cancer, i.e. at high frequency in carcinoma of the bladder and rarely in multiple myeloma and carcinoma of the cervix. Almost all reported mutations in carcinomas corresponded to the mutations identified in thanatophoric dysplasia. We here screened a series of 297 bladder tumours and found three FGFR3 somatic mutations (G380/382R; K650/652M and K650/652T) that were not previously identified in carcinomas or thanatophoric dysplasia. Another novel finding was the occurrence of two simultaneous FGFR3 mutations in four tumours. Two of the three new mutations in bladder cancer, the G380/382R and the K650/652M mutations, were previously reported in achondroplasia and SADDAN, respectively. These syndromes entail a longer life span than thanatophoric dysplasia. The K650/652T mutation has not previously been detected in patients with skeletal disorders, but affects a codon that has been shown to be affected in some cases of thanatophoric dysplasia, SADDAN and hypochondroplasia. From a clinical perspective, the patients with FGFR3-related, non-lethal skeletal disorders might be at a higher risk for development of bladder tumours than the general population.     

Three new mutations account for the prevalence of glucose 6 phosphate deshydrogenase (G6PD) deficiency in Tunisia

A previous study on G6PD deficiency carried out on Tunisian population, led to the finding of seven different mutations with the prevalence of G6PD A- variant. This present study reports 23 new unrelated deficient subjects studied at the molecular level to determine the mutation that causes G6PD deficiency. Using PCR-SSCP of coding regions followed by direct sequencing of abnormal pattern, three new mutations were detected. Two of them are polymorphic intronic mutations. The first is IVS-V 655C→C/T, found in four female subjects with mild deficiency of class III variant. The second is IVS-VIII 43 G→A, found in three male subjects with mild deficiency of class III variant. The third mutation is in the exon region so that it changes the primary structure of the molecule. It is cited for the first time and named G6PD Tunisia. This variant affects the exon 7 of the gene at genomic position 15435 G→T. Its cDNA position is 93 G→G/T, it changes arg 246 to leu. This mutation was found in one heterozygote female with deficiency of class II who have had hemolytic anemia due to ingestion of fava beans. Finally, G6PD Med variant, reported before in three cases, was also found in five other cases (four heterozygote females and one male hemizygote). These findings first enlarge the spectre of mutations to be ten variant mutations, characterizing the Tunisian population and also contribute with hemoglobin gene research in our laboratory to trace the whole genetic map of Tunisian population.     

β地中海贫血杂合子基因突变及Gγ珠蛋白基因-158位点SNP与Hb F的关系

目的　探讨 β地中海贫血 (简称 β地贫 )杂合子基因突变类型和 Gγ珠蛋白基因启动子 - 15 8位点 (Gγ- 15 8)单核苷酸多态性与胎儿血红蛋白 (fetal hemoglobin,Hb F)水平的关系。方法　抗碱 -比色法测定 Hb F水平 ;PCR-寡核苷酸斑点杂交法检测β地贫基因型 ;限制性内切酶 Xmn 消化经 PCR扩增的Gγ基因启动子 DNA片段 ,分析Gγ- 15 8位点的单核苷酸多态性。结果　 6 3例受检的轻型β地贫中 15例 Hb F≥ 2 % (2 .0 6 %～ 10 .4 4 % )。共检出 6种β地贫基因突变 ,分别是 :CD4 1/42 (- TTCT)、CD17(A→T)、nt- 2 8(A→ G)、CD71/72 ( A)、IVS- II- 6 5 4 (C→ T)、IVS- I- 1(G→ T)。 CD4 1/42、CD17、CD71/72、IVS- II-6 5 4的杂合子在 15例 Hb F升高组和 4 8例 Hb F正常组各自所占比例相同。 6 3例个体中有 10例为Gγ-15 8(C→T)突变的杂合子 ,总检出率为 15 .9% ;其中 15例高 Hb F个体中检出 8例 (检出率 5 3.33% ) ,HbF正常的 4 8例检出 2例 (检出率 4 .17% ) ,两组检出率差异有显著性 (P<0 .0 0 1)。结论　 β地贫基因突变CD4 1/42、CD17、CD71/72、IVS- II- 6 5 4与 β地贫杂合子的 Hb F水平无关 ;而 Gγ- 15 8(C→ T)突变与广西地区 β地贫杂合子 Hb F升高密切相关。     

Frequency of mitochondrial DNA point mutations among patients with familial sensorineural hearing impairment

Several point mutations in mitochondrial DNA (mtDNA) have been shown to cause sensorineural hearing impairment (SNHI), but the frequency of these mutations among patients is not known. We identified 117 patients with possible matrilineal SNHI from population-based registers and found the 3243A > G mutation to be present in 4.3% and 1555A > G in 2.6%, while 7445T > C, 7472insC and 8344A > G were absent. Patients with 3243A > G and 1555A > G were clinically distinct. The prevalence of 1555A > G in the general adult population was estimated to be at least 4.7/100,000, but these and previous data suggest that the figure may vary between populations. Screening for mtDNA mutations is worthwhile in connection with the diagnosis of SNHI.     

Mutations in POMT1 are found in a minority of patients with Walker-Warburg syndrome

Walker-Warburg syndrome (WWS) is an autosomal recessive disorder of infancy characterized by hydrocephalus, agyria, retinal dysplasia, congenital muscular dystrophy, and over migration of neurons through a disrupted pial surface resulting in leptomeningeal heterotopia. Although previous work identified mutations in the o-mannosyl transferase, POMT1, in 6 out of 30 WWS families [Beltran-Valero de Bernabe et al., 2002], the incidence of POMT1 mutations in WWS is not known. We sequenced the entire coding region of POMT1 in 30 consecutive, unselected patients with classic WWS. Two novel heterozygous mutations were found in two patients from non-consanguineous parents, whereas 28 other patients failed to show any POMT1 mutations. One patient was found to be heterozygous for a transition, g.1233T > A, which predicts p.Y352X. A second patient was found also to be heterozygous for a transition g.1790C > G, which predicts p.S537R. As an additional determination of the frequency of the POMT1 mutations in WWS, we tested for linkage of WWS to POMT1 in six consanguineous families. All six demonstrated heterozygosity and negative LOD scores at the POMT1 locus. From these data we show that POMT1 is an uncommon cause of WWS, the incidence of coding region mutations in this population of WWS being less than 7%. We conclude that while the incidence of POMT1 mutations in WWS can be as high as 20% as reported by Beltran-Valero de Bernabe et al. [2002] and it can be as low as approximately 7%, as reported here.     

Myocilin analysis by DHPLC in French POAG patients: increased prevalence of Q368X mutation

Primary open-angle glaucoma (POAG) is a prevalent optic neuropathy with complex genetics. A small number of patients carry a mutation in the coding region of the myocilin (MYOC) gene. The nature and the frequency of these mutations, however, vary substantially, notably with the age at onset and the ethnic origin of the patients. Here, we showed that denaturing high performance liquid chromatography (DHPLC) is an appropriate method for screening carriers of MYOC mutations. We have applied the method to a group of 237 POAG patients and 108 control subjects from France. Mutations were found in 17 (7.5%) patients and in none of the controls. A single mutation, Q368X (c.1102C>T), accounted for the majority (12/17) of these mutations, corresponding to a frequency of 5% among POAG patients, the highest ever reported for this mutation. Furthermore, analysis of allelic associations at closely linked microsatellite markers indicated that most, if not all, patients inherited Q368X from a same ancestor. Five other patients carried four distinct mutations, including N480K (c.1440C>A) (2 cases), I499F (c.1495A>T), G367R (c.1099G>A) and T438I (c.1313C>T), which is reported here for the first time. Altogether, MYOC mutations in French patients were associated with a significantly increased intraocular pressure at diagnosis. In addition, the age at diagnosis of patients with a mutation other than Q368X was significantly younger than that of Q368X carriers or of patients with a normal MYOC. Based on these observations, a screening strategy of MYOC mutations in French POAG patients is briefly outlined.     

Homozygous EXOSC3 Mutation c.92G→C,p.G31A is a Founder Mutation Causing Severe Pontocerebellar Hypoplasia Type 1 Among the Czech Roma

Abstract

Pontocerebellar hypoplasia type 1 (PCH1) is characterized by cerebellar and anterior horn motor neuron degeneration and loss, signs of spinal muscular atrophy plus. Patients manifest severe perinatal weakness, hypotonia, and respiratory insufficiency, causing death frequently before the age of 1 year. Recently, causative mutations in EXOSC3 were reported in a majority of PCH1 patients, but the detailed clinical phenotype caused by EXOSC3 mutations, genotype-phenotype correlations, and prevalent mutations in specific ethnic groups is not yet known.

Three unrelated Czech Roma patients with PCH1 were investigated clinically, electrophysiologically, neuroradiologically, and neuropathologically (patients 1 and 2). The entire coding region of the EXOSC3 gene, including the adjacent intron sequences, was sequenced in all three patients. The same mutation c.92G→C, p.G31A in EXOSC3 was found in all three affected patients in homozygous state and in heterozygous state in the parents from two of the families. Haplotype analysis with four flanking microsatellite markers showed identical haplotype in 9 out of 11 haplotypes carrying the c.92G→C, p.G31A mutation. Furthermore, four heterozygotes for this mutation were found in anonymous DNA samples from 90 unrelated Roma individuals. All four of these samples shared the same haplotype. No heterozygous sample was found among 120 anonymous DNA samples from Czech non-Roma individuals with no familial relation. It may therefore be concluded that EXOSC3 c.92G→C, p.G31A mutation is a founder mutation with high prevalence among the Czech Roma causing a similar and particularly severe phenotype of PCH1. These observations from the Czech Roma may have consequences also for other Roma from other countries.

PCH1 caused by EXOSC3 founder mutation c.92G→C, p.G31A extends the list of autosomal recessive disorders rare among the general population but more frequent among Roma at least in the Czech Republic.     

G6PD deficiency assessment in Freetown,Sierra Leone,reveals further insight into the molecular heterogeneity of G6PD A-

Glucose-6-phosphate dehydrogenase (G6PD) deficiency in Africa is of high prevalence, although precise data are lacking in many individual nations. We investigated 129 unrelated subjects (71 male subjects, 58 female subjects) visiting a teaching hospital in Freetown, Sierra Leone, to collect baseline data on the distribution of G6PD deficiency among respective ethnic groups in the country. We confirmed eight G6PD-deficient male subjects by two formazan-based blood tests (11.3% of the male subjects examined), and also detected the common 376A > G mutation in 11 male subjects and eight female subjects by sequencing exons 3-5 of the G6PD gene. Selected samples were further sequenced for exons 2-13 and introns 5, 7, 8, and 11. Among the deficient male subjects, six were G6PD A- carrying the double mutations (202G > A and 376A > G), all of whom were in the Temne and Mende ethnic groups. Others included A- Betica, and a novel variant having double mutations in exon 5 (311G > A and 376A > G forming 104 Arg > His and 126 Asn > Asp, respectively), which we designate as G6PD Sierra Leone. Subsequent haplotype analysis linked this novel variant to the G6PD A- "family".     

RFLP haplotypes of beta-globin gene complex of beta-thalassemic chromosomes in Koreans

Korea is in the low-prevalence area of beta-thalassemia and the Korean population has relatively homogenous racial characteristics. Recently, we identified some causative mutations of the Korean beta-thalassemia patients. In order to elucidate the genetic background of beta-thalassemia alleles in Koreans, we determined the restriction fragment length polymorphism (RFLP)-haplotype and framework (FW) in nine beta-thalassemia chromosomes of five different causative mutations by PCR-based method and family linkage study. The result that the haplotype and the framework linked to the initiation codon ATG-->AGG mutation were -+-++-+ and FW3A, respectively, in all of three families in this study suggests a common origin of this mutation at least in Koreans. A novel beta-thalassemia mutation, codons 89/90 -TG, showed discrepancy between -++--++- and FW1, which could be explained by gene conversion. A case of codons 8/9 +G frameshift mutation had +----++ and FW1. The linkage of the two beta-thalassemia mutations, codon 17 AAG-->TAG and codons 41/42 -TTCT, with specific haplotypes and frameworks common to the Koreans and the neighboring countries suggests that those mutations are influenced by the genetic flow from the south China.     

Mutation spectrum of the PCCA and PCCB genes in Japanese patients with propionic acidemia

Propionic acidemia (PA) is an inborn error of organic acid metabolism caused by a deficiency of propionyl-CoA carboxylase. This enzyme is composed of two non-identical subunits, alpha and beta, which are encoded by the PCCA and PCCB genes, respectively. An enzyme deficiency can result from mutations in either PCCA or PCCB. To elucidate the mutation spectrum in Japanese patients, we have performed a mutation analysis of 30 patients with PA, which included nine previously reported patients. The study revealed that 15 patients were alpha-subunit deficient and 15 patients were beta-subunit deficient. Seven novel mutations were found (IVS18-6C >G, 1746G >A, C398R, G197E and IVS18+1G >A in the PCCA; A153P and IVS9+1G >T in the PCCB). Among these Japanese patients with alpha-subunit deficiencies, 923-924insT, IVS18-6C >G, and R399Q mutations were frequent and the total allelic frequency of these three mutations combined was 56% (17/30). This is in sharp contrast to the mutation spectrum found in Caucasian patients, where no prevalent mutations have been identified. Among the beta-subunit deficiencies, there were three frequent mutations; R410W, T428I, and A153P, whose allelic frequencies were 30, 26.7, and 13.3%, respectively. In conclusion, a limited number of mutations are predominant in both PCCA and PCCB genes among Japanese patients with propionic acidemia.     

The role of HFE mutations on iron metabolism in beta-thalassemia carriers

Hereditary hemochromatosis (HH) is an autosomal recessive disorder of iron metabolism characterized by increased iron absorption and progressive storage resulting in organ damage. HFE gene mutations C282Y and H63D are responsible for the majority of HH cases. A third HFE mutation, S65C, has been associated with the development of a mild form of hemochromatosis. The beta-thalassemia trait is characterized by mild, ineffective erythropoiesis that can induce excess iron absorption and ultimately lead to iron overload. The aim of this study was to evaluate the effect of genetic markers (HFE mutations C282Y, H63D, and S65C) on the iron status of beta-thalassemia carriers. A total of 101 individuals heterozygous for beta-thalassemia and 101 normal control individuals were studied. The allelic frequencies of C282Y (1.5 versus 3.5%), H63D (15.3 versus 18.3%), and S65C (1.0 versus 1.5%) did not differ significantly between beta-thalassemia carriers and normal controls. Serum iron (P=0.029) and transferrin saturation (P=0.009) were increased in beta-thalassemia carriers heterozygous for H63D mutation. The number of subjects carrying C282Y or S65C mutations was too low to conclude their effect on the iron status. These results suggest that the beta-thalassemia trait tends to be aggravated with the coinheritance of H63D mutation, even when present in heterozygosity.     

Novel mutations in the 7-dehydrocholesterol reductase gene of 13 patients with Smith–Lemli–Opitz syndrome

Smith–Lemli–Opitz syndrome (SLOS) is caused by mutations in the DHCR7 gene leading to deficient activity of 7-dehydrocholesterol reductase (DHCR7; EC 1.3.1.21), the final enzyme of the cholesterol biosynthetic pathway, resulting in low cholesterol and high concentrations of its direct precursor 7-dehydrocholesterol in plasma and tissues. We here report mutations identified in the DHCR7 gene of 13 children diagnosed with SLOS by clinical and biochemical criteria. We found a high frequency of the previously described IVS8–1 G > C splice acceptor site mutation (two homozygotes, eight compound heterozygotes). In addition, 13 missense mutations and one splice acceptor mutation were detected in eleven patients with a mild to moderate SLOS-phenotype. The mutations include three novel missense mutations (W182L, C183Y, F255L) and one novel splice acceptor site mutation (IVS8–1 G > T).
Two patients, homozygous for the IVS8–1 G > C mutation, presented with a severe clinical phenotype and died shortly after birth. Seven patients with a mild to moderate SLOS-phenotype disclosed compound heterozygosity of the IVS8–1 G > C mutation in combination with different novel and known missense mutations.     

Mutations in the optineurin gene in Japanese patients with primary open-angle glaucoma and normal tension glaucoma

The optineurin gene (OPTN) was identified as a gene that causes primary open-angle glaucoma (POAG) and normal tension glaucoma (NTG). To investigate the frequency of sequence changes in OPTN in Japanese glaucoma patients, single-strand conformation polymorphism analysis and subsequent sequence analysis were performed for genotyping OPTN in 165 unrelated Japanese patients with POAG and 148 patients with NTG, with 196 control subjects without glaucoma as reference subjects. Out of four mutations reported to be associated with risk and to cause disease in Caucasian patients, sequence alterations in 458G > A and 691_692insAG were not detected in any investigated Japanese patients with glaucoma, and alterations in 1944G > A and 603T > A, were present in similar frequencies in glaucoma patients and control subjects. The current results suggest that there may be certain racial differences between Japanese and Caucasians with respect to OPTN genotypes.     

Factors that affect hearing level in individuals with the mitochondrial 1555A>G mutation

The mitochondrial 1555A>G mutation is one of the most common mutations responsible for hearing loss in Asians. Although the association with aminoglycoside exposure is well known, there is great variation in the severity of hearing loss. We analyzed hearing levels in 221 Japanese individuals with this mutation and attempted to identify relevant covariants including (i) age, (ii) aminoglycoside exposure, (iii) heteroplasmy ratio, and (iv) other gene mutations. At every age, average hearing levels were worse than those in normal subjects, suggesting that mitochondrial function itself may affect the severity of hearing loss. Although the hearing loss in individuals with the 1555A>G mutation progressed with age, the rate did not differ from that of the normal subjects. Those who had reported aminoglycoside exposure had moderate-to-severe hearing impairment regardless of age, confirming that such exposure is the most important environmental variable. We also confirmed the presence of heteroplasmy, which is known to modify the expression of other mitochondrial diseases, but found no evidence for a significant correlation with hearing impairment. A high prevalence of GJB2 heterozygous mutations was noted, indicating that these mutations may exhibit epistatic interaction with the 1555A>G mutation.