Identification of recurrent and novel mutations in the LDL receptor gene in Japanese familial hypercholesterolemia

We used the denaturing gradient gel electrophoresis (DGGE) method to investigate 120 Japanese patients with familial hypercholesterolemia (FH) for mutations in the promoter region and the 18 exons and their flanking intron sequence of the low density lipoprotein (LDL) receptor gene. Fourteen aberrant DGGE patterns were found, and the underlying mutations were characterized by DNA sequencing. Five novel missense mutations (C317S, F382L A410T, L547V, and E693K), two nonsense mutations (W512X and K790X), four frameshift mutation (355del7, 1246ins5, 1687ins1, and 2035ins1), one splicing mutation (1845+2 T→C), and two inframe mutations (661ins21 and 1115del9/ins6) were identified. Six of these mutations (L547V, E693K, W512X, 355del7, 1687ins1, and 20354ins1) have not been described before in FH. These newly identified mutations cosegregated in their family members with defective LDL receptor activity and hypercholesterolemia, and are thought to be causal for the FH phenotype. These results demonstrate that there is a broad spectrum of mutations in the LDL receptor gene in the Japanese population. Hum Mutat 14:87, 1999. © 1999 Wiley‐Liss, Inc.     

Screening of 383 unrelated patients affected with Menkes disease and finding of 57 gross deletions in ATP7A

Menkes disease (MD) is an X-linked multisystemic lethal disorder of copper metabolism dominated by neurodegenerative symptoms and connective tissue disturbances. MD results from mutations in the ATP7A gene, which encodes a membrane-bound copper transporting P-type ATPase located in the trans-Golgi network. In this study we describe screening of 383 unrelated patients affected with Menkes disease for gross deletions in ATP7A gene and finding of 57 patients. The present data suggests that gross deletion of ATP7A is the disease-causing mutation in 14.9% of the Menkes disease patients. Except for a few cases, gross gene deletions result in the classical form of Menkes disease with death in early childhood.     

三例Menkes病患儿的家系及基因变异分析

目的:对3例Menkes病患儿家系的 ATP7A基因进行变异分析,明确其致病原因,为临床诊断提供依据。 方法:应用二代测序(next-generation sequencing,NGS)对3个Menkes病家系的先证者进行Menkes病相关致病基因 ATP7A基因外显子检测,发现可疑致...     

Identification of four novel mutations in classical Menkes disease and successful prenatal DNA diagnosis

Menkes disease is an X-linked recessive disorder of the copper metabolism and affected males suffer a systemic copper deficiency due to malabsorption and defective distribution of dietary copper. It is caused by a defect in the Menkes (ATP7A) gene, which encodes a transmembrane copper-transporting P-type ATPase. A variety of mutations were reported; however, only a few mutations were reported in Asian patients. We identified four novel mutations and one known mutation in five Korean patients. Arg646Ter in exon 8, a novel mutation transmitted from his carrier mother, was identified in one patient. Prenatal DNA diagnosis on an unaffected fetus in this carrier mother was successfully accomplished. An additional three novel mutations, Leu706Arg in exon 9, Gly1118Asp in exon 17, and Gly1255Arg in exon 19, were identified. Splicing mutation was not identified. Menkes disease in Korean patients appears to be caused by heterogeneous mutations with different spectrums from Caucasian patients.     

Occipital horn syndrome and classical Menkes Syndrome caused by deep intronic mutations,leading to the activation of ATP7A pseudo-exon

Menkes disease is an X-linked disorder of copper metabolism caused by mutations in the ATP7A gene. Whereas most of the patients exhibit a severe classical form, about 9% of the patients exhibit a milder form of Menkes disease. The mildest form is called occipital horn syndrome (OHS). Mutations in the ATP7A gene can be identified in 95–98% of the Menkes disease patients by standard screening techniques. Investigation of RNA isolated from the fibroblasts of eleven patients with no identified mutations was performed, and revealed inclusion of new pseudo-exons into the ATP7A mRNA from three unrelated patients: two patients with OHS and one patient with classical Menkes disease. The pseudo-exons were inserted between exons 10 and 11, between exons 16 and 17 and between exons 14 and 15 in the three patients, as a result of deep intronic mutations. This is the first time the activation of pseudo-exons is demonstrated in the ATP7A gene, and it demonstrates the usefulness of RNA analysis, in terms of revealing disease-causing mutations in noncoding regions. The fact that three different mutations cause disease by the activation of pseudo-exon inclusion also indicates that in Menkes disease this is an important mechanism, which has hitherto been overlooked.     

Identification of a novel mutation in the ATP7A gene in a Korean patient with Menkes disease

Menkes disease is an infantile-onset X-linked recessive neurodegenerative disorder caused by diverse mutations in a copper-transport gene, ATP7A. Affected patients are characterized by progressive hypotonia, seizures, failure to thrive and death in early childhood. Here, we report a case of Menkes disease presented by intractable seizures and infantile spasms. A 3-month-old male infant had visited our pediatric clinic for lethargy, floppy muscle tone, poor oral intake and partial seizures. His hair was kinky, brown colored and fragile. Partial seizures became more frequent, generalized and intractable to antiseizure medications. An EEG showed frequent posteriorly dominant generalized spikes that were consistent with a generalized seizure. From a genetic analysis, a c.2743C>T (p.Gln915X) mutation was detected and diagnosed as Menkes disease. The mutation is a novel one that has not been previously reported as a cause of Menkes disease.     

Mutational analysis of ATP7B and genotype-phenotype correlation in Japanese with Wilson's disease

The gene ATP7B responsible for Wilson's disease (WD) produces a protein which is predicted to be a copper-binding P-type ATPase, homologous to the Menkes disease gene (ATP7A). Various mutations of ATP7B have been identified. This study aimed to detect disease-causing mutations, to clarify their frequency and distribution, to determine whether genotype correlates with phenotype, and to determine the rate of abnormal findings in heterozygotes for the WD gene. We analyzed 41 unrelated Japanese WD families, including 47 patients. Twenty-one mutations, including nine novel ones, were identified. 2871delC (15.9%), 1708-5T-->G (11. 0%), and Arg778Leu (13.4%) were the most common mutations. 2871delC was detected mainly in eastern Japan and 1708-5T-->G in western Japan. The homozygotes for the 1708-5T-->G, 2871delC, or Arg778Leu mutations did not show a correlation with their phenotypes. Ceruloplasmin and copper levels were abnormally low in 28.6% and 35. 0% of heterozygotes, respectively. When patients and their families are screened for WD, a high rate of abnormal laboratory data in heterozygotes must be taken into account.     

Clinical features and mutational analysis in 114 young children with Wilson disease from South China

Wilson disease (WD) is a rare autosomal recessive disorder caused by mutations in the ATP7B gene. Clinical features and mutational analysis of Chinese children with WD at early age were rarely described. Herein, we retrospectively examined 114 children with WD at the mean of 5.9 years old age at diagnosis. Eight patients developed acute liver failure at mean age of 9.7 years old, 4 of whom died. Among the 114 patients, 86.0% were presymptomatic with isolated elevation of transaminases at diagnosis, 99.1% had decreased ceruloplasmin, and 68.4% had urinary copper excretion over 100 μg/24 hr. Bi‐allele pathogenic ATP7B mutations were identified in all patients. Among the 60 mutations detected, 10 were novel, including 7 missense mutations (p.I566N, p.T704I, p.C980F, p.G1030 V, p.A1096Q, p.L1327P, and p.L1373F), 1 nonsense mutation (p.K866X), 1 small insertion (p.Y44LfsX2), and 1 small deletion (p.R1118PfsX10). The most frequent mutations were p.R778L, p.P992L, and p.I1148T, which affected 27.2, 25.4, and 20.2% of the 114 WD children, respectively. The patients carrying p.R778L presented a higher rate of acute liver failure than the patients without p.R778L (9.7% vs. 4.8%). These results will be helpful in establishing early diagnosis of WD at the gene level, offering beneficial information for genetic counseling and providing clues to genotype/phenotype correlation of ATP7B mutations.     

中国人共济失调毛细血管扩张症ATM基因突变研究

目的　探讨中国人共济失调毛细血管扩张症(ataxia- telangiectasia,AT) ATM基因的突变特点。方法　应用聚合酶链反应、逆转录-聚合酶链反应、聚丙烯酰胺凝胶电泳结合DNA序列分析方法对2例中国人临床诊断AT的患者ATM基因进行突变的筛选与检测。结果　在1例患者中发现第11外显子的1346 (G>C)的错义突变,为一种纯合突变;在另1例患者中发现第6外显子的6 10 (G>T)的无义突变和第4 7外显子的6 6 79(C>T)的错义突变,为一种复合性杂合突变。突变位点均位于ATM基因功能域。结论　在2例中国人AT患者中发现了3种新的ATM基因突变。     

Intracellular localization and loss of copper responsiveness of Mnk, the murine homologue of the Menkes protein, in cells from blotchy (Mo blo) and brindled (Mo br) mouse mutants.

Menkes disease is an X-linked copper deficiency disorder that results from mutations in the ATP7A ( MNK ) gene. A wide range of disease-causing mutations within ATP7A have been described, which lead to a diversity of phenotypes exhibited by Menkes patients. The mottled locus ( Mo, Atp7a, Mnk ) represents the murine homologue of the ATP7A gene, and the mottled mutants exhibit a diversity of phenotypes similar to that observed among Menkes patients. Therefore, these mutants are valuable models for studying Menkes disease. Two of the mottled mutants are brindled and blotchy and their phenotypes resemble classical Menkes disease and occipital horn syndrome (OHS) in humans, respectively. That is, the brindled mutant and patients with classical Menkes disease are severely copper deficient and have profound neurological problems, while OHS patients and the blotchy mouse have a much milder phenotype with predominantly connective tissue defects. In this study, in an attempt to understand the basis for the brindled and blotchy phenotypes, the copper transport characteristics and intracellular distribution of the Mnk protein were assessed in cultured cells from these mutants. The results demonstrated that the abnormal copper metabolism of brindled and blotchy cells may be related to a number of factors, which include the amount of Mnk protein, the intracellular location of the protein and the ability of Mnk to redistribute in elevated copper. The data also provide evidence for a relationship between the copper transport function and copper-dependent trafficking of Mnk.     

Mutation analysis in PKD1 of Japanese autosomal dominant polycystic kidney disease patients

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic renal disorder (incidence, 1:1,000). The mutation of PKD1 is thought to account for 85% of ADPKD. Although a considerable number of studies on PKD1 mutation have been published recently, most of them concern Caucasian ADPKD patients. In the present study, we examined PKD1 mutations in Japanese ADPKD patients. Long-range polymerase chain reaction (LR-PCR) with PKD1-specific primers followed by nested PCR was used to analyze the duplicated region of PKD1. Six novel chain-terminating mutations were detected: three nonsense mutations (Q2014X transition in exon 15, Q2969X in exon 24, and E2810X in exon 23), two deletions (2132del29 in exon10 and 7024delAC in exon 15), and one splicing mutation (IVS21-2delAG). There was also one nonconservative missense mutation (T2083I). Two other potentially pathogenic missense mutations (G2814R and L2816P) were on the downstream site of one nonsense mutation. These three mutations and a following polymorphism (8662C>T) were probably the result of gene conversion from one of the homologous genes to PKD1. Six other polymorphisms were found. Most PKD1 mutations in Japanese ADPKD patients were novel and definitely pathogenic. One pedigree did not link to either PKD1 or PKD2.     

Identification of two novel mutations in the OCRLI gene in Japanese families with Lowe syndrome

Kubota T, Sakurai A, Arakawa K, Shimazu M, Wakui K, Furihata K, Fukushima Y. Identification of two novel mutations in the OCRL1 gene in Japanese families with Lowe syndrome. Clin Genet 1998: 54: 199–202. 0 Munksgaard, 1998
The oculocerebrorenal syndrome of Lowe (OCRL) is a rare X-linked disorder with features of congenital cataracts. Fanconi syndrome of the renal tubule, and mental retardation. The OCRLI gene has been positionally cloned and shown to encode a phosphatidylinositol 4.5–biphos-phate-5–phosphatase. OCRL is thus thought to be an inborn error of inositol polyphosphate metabolism. We analyzed the gene in two Japanese OCRL patients and their families by DNA sequencing and mismatch polymerase chain reaction (PCR) followed by restriction digestion. A novel nonsense mutation (C1399T) replacing the glutamine of codon 391 (Gln 391 Stop) was identified in exon 12 in 1 patient and also in his mother. A novel missense mutation (C1743G) was identified in exon 15 in the second patient, his mother and maternal grandmother. The missense mutation predicts a substitution of serine for arginine (Ser 505 Arg) in a domain highly conserved among the inosi-tol-5–phosphatase family. Our observations expand the range of OCRLI mutations that cause Lowe syndrome. and will be useful for genetic counseling in these two Fdmilies.     

感音神经性耳聋患者大前庭导水管综合征相关SLC26A4基因IVS7-2A＞G的全序列分析

目的 对携带SLC26A4基因IVS7-2A＞G单杂合突变感音神经性耳聋患者进行 SLC26A4基因的全序列检测,以期发现除IVS7-2A＞G以外的其他突变.方法 应用直接测序法对80例携带IVS7-2A＞G单杂合突变的感音神经性耳聋患者进行SLC26A4基因进行全序列测序.结果 80例患者中47例发现另1个突变位点,其余33例未发现复合杂合突变,IVS7-2A＞G单杂合突变找到另外1个突变的比例为58.8%(47/80).发现了 3个新的突变,分别是5+2T＞A、14-2A＞G和1825del G,最为常见的5种突变为H723R(20%)、T410M(5%)、15+5G＞A(5%)、L676Q(5%)、N392Y(3.75%).第17外显子是突变发生种类最多的外显子.结论 SLC26A4基因IVS7-2A＞G单杂合突变者应该进行其他突变的筛查,SLC26A4基因复合突变可以解释部分的耳聋原因.     

Mutations of the PKD1 gene among Japanese autosomal dominant polycystic kidney disease patients, including one heterozygous mutation identified in members of the same family

More than 80 mutations of the PKD1 gene have been reported, mostly in patients from Western Europe. New techniques are being used to detect an increasing number of mutations, even in the homologous region of the PKD1 gene. Polymerase chain reaction–single-strand conformation polymorphism (PCR-SSCP) or denaturing high-performance liquid chromatography (DHPLC) analyses were performed in the present study to screen mutations from exon 23 to exon 46 in the PKD1 gene and in the entire PKD2 gene. When an abnormal pattern was found in PCR-SSCP or DHPLC, the PCR products were directly sequenced. Four mutations were identified in the PKD1 gene: a missense mutation (C47413T causing T3509M in exon 35), a splicing mutation (del 20 bp in 75 bp of intron 43), and two nonsense mutations (C48566A causing C3693X in exon 38, and C51237T causing Q4124X in exon 45). The nonsense mutation Q4124X existed in only two of three affected sib members in family K68. The pattern of the restriction enzyme digest and the haplotype analysis confirmed the presence of a heterozygous mutation in the family. Fifteen single nucleotide polymorphisms were identified in this study. Two of them (C50439A and C51659T) can be used as intragenic polymorphic markers. Received: April 21, 2001 / Accepted: June 11, 2001     

Identification of novel MLC1 mutations in Chinese patients with megalencephalic leukoencephalopathy with subcortical cysts (MLC)

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is an autosomal, recessively inherited disease caused by mutations in the MLC1 gene. Most of the previously published studies have been carried out in ethnic populations other than the Chinese. In this study, the analysis of clinical features and MLC1 mutation screening were performed in 13 Chinese patients for the first time. A total of 10 MLC1 mutations were identified in these patients, including five novel missense mutations (c.65G>A, p.R22Q; c.95C>T, p.A32V; c.218G>A, p.G73E; c.823G>A, p.A275T; c.832T>C, p.Y278H), one novel splicing mutation (c.772-1G>C in IVS9-1), one novel small deletion (c.907_930del, p.V303_L310del), one known nonsense mutation (c.593delCTCA, p.Y198X) and two known missense mutations (c.206C>T, p.S69L; c.353C>T, p.T118M). Mutation c.772-1G>C in IVS9-1, accounting for 27.3% (3/11) of the total number of genetically confirmed patients found in this study, is thus a putative hot-spot mutation in the present study group. The existence of a unique MLC1 mutation spectrum in Chinese MLC patients was shown. A systemic study to assess the mutation spectra in different populations should be undertaken.     

Identification of six novel WASP gene mutations in patients suffering from Wiskott-Aldrich syndrome

Mutation in the gene encoding the Wiskott-Aldrich Syndrome protein (WASP) has been identified as the genetic defect responsible for WAS, an X-linked primary immunodeficiency disease characterized by eczema, thrombocytopenia, and recurrent infections. In this study, the WASP gene of 7 unrelated patients with classical WAS of Dutch descent was examined by single-strand conformation polymorphism and sequence analysis. We have identified 6 novel mutations that involve nonsense mutations (196C-->A, 344C-->T), or small deletions (553delG, 768del19, IVS8+1delGTGA, 911delT), all of which result in predicted truncation of WASP protein synthesis.     

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.