Four novel mutations in the thiazide-sensitive Na-Cl co-transporter gene in Japanese patients with Gitelman's syndrome.

BACKGROUND: Gitelman's syndrome (GS) is an autosomal recessive disorder resulting from inactivating mutations in the thiazide-sensitive Na-Cl co-transporter (NCCT) gene. To date, almost 90 mutations have been identified. It is possible that there is a population-specific distribution of mutations. In this study, we analysed mutations in the NCCT gene of seven Japanese patients with GS. METHODS: Peripheral blood mononuclear cells were isolated from patients with GS, their family members and healthy control subjects. A mutation analysis of the NCCT gene was performed completely by direct automated sequencing of polymerase chain reaction-amplified DNA products. In patients with a deletion or splice site mutation, we undertook cDNA sequence analysis. RESULTS: We identified nine mutations. Five of them [c.185C>T (Thr60Met), c.1712C>T (Ala569Val), c.1930C>T (Arg642Cys), c.2552T>A (Leu849His) and c.1932delC] have been reported in Japanese patients, but not in GS patients from other ethnic groups. The remaining four mutations [c.7A>T (Met1Leu), c.1181_1186+20del26, c.1811_1812delAT and IVS16+1G>A] were novel. In cDNA derived from a patient with c.1181_1186+20del26, a deletion of exon 9 and a frameshift at the start of exon 10 were observed. In cDNA derived from patients with IVS16+1G>A, an additional 96 bp insertion between exons 16 and 17 was observed. Six out of seven patients were compound heterozygotes, and the remaining one carried a single heterozygous mutation. CONCLUSIONS: We found four novel mutations in the NCCT gene in seven Japanese patients with GS. Moreover, our study suggests that the distribution of mutations in the NCCT gene in Japanese GS patients potentially differs from that in other populations.     

Identification of a novel R642C mutation in Na/Cl cotransporter with Gitelman's syndrome.

Gitelman's syndrome, a variant of Bartter's syndrome, is an inherited disorder characterized by hypokalemic metabolic alkalosis, hypomagnesemia, and hypocalciuria, and these abnormalities have recently been linked to the thiazide-sensitive Na/Cl cotransporter (TSC) gene. We evaluated three unrelated patients affected with this syndrome whose diagnosis was made based on clinical and biochemical features. The data of clearance studies in these patients were compatible with Gitelman's syndrome. We then investigated possible mutations of the TSC gene. In one patient whose parents are consanguineous, we identified a novel missense mutation in the TSC gene, which causes alteration of arginine to cysteine at codon 642 (R642C mutation) located in the cytoplasmic tail of the product. This mutation results in the loss of an MspI site in exon 15 of the TSC gene. MspI digestion analysis of genomic DNA fragments from the family was consistent with the autosomal recessive inheritance of the disorder, and presence of this mutation correlated with the clinical manifestations. Such mutation was not detected in 47 normal healthy subjects. In the second patient, we found another missense mutation in one allele of the TSC gene, which results in alteration of arginine to glutamine at codon 955. In the third patient, no mutation causing amino acid substitution was found in the TSC gene. These results indicate that the R642C mutation in TSC is critically important for impairment of this cotransporter function and also suggest the necessity of further investigations in the genetic background of Gitelman's syndrome.     

JAK3 in clear cell renal cell carcinoma: Mutational screening and clinical implications

ObjectivesJanus Kinase 3 (JAK3) mediates cytokine signaling and T-cell activation. We hypothesized that JAK3 mutations may contribute to the development and progression of clear cell renal cell carcinoma (ccRCC). To test this hypothesis, we performed mutational screening and functional studies.Patients and methodsThis hospital-based case-control study included 50 patients with ccRCC and 100 age- and gender-matched controls. Both genomic and tumor DNA were extracted. All 23 JAK3 exons were amplified by PCR and analyzed by denaturing high-performance liquid chromatography and automatic sequencing. Effects of JAK3 mutations on interleukin-2-stimulated peripheral T-cells were analyzed by confocal laser-scanning microscopy and immunoprecipitation.ResultsFour different JAK3 germline missense mutations (p.Gln13Lys; p.Arg925Ser; p.Ala677Thr, p.Val722Ile) were found in a total of 7 ccRCC patients (14%), but in none of the controls (P = 0.0006). All germline mutations were similarly detected in the tumors. An additional somatic missense mutation (p.Tyr238Cys) was found in a patient who had a germline mutation. Four of the mutations have not been previously described (p.Gln13Lys; p.Arg925Ser; p.Ala677Thr, p.Tyr238Cys). Patients with JAK3 mutations more frequently presented with metastases (3 out of 4 [75%] vs. 4 out of 46 [9%]; P = 0.004) and had poorer survival (P = 0.049). In p.Arg925Ser and p.Ala677Thr/p.Val722Ile, functional analyses showed abnormal JAK3 and STAT5 tyrosine phosphorylation and a reduction of JAK3/STAT5 interaction.ConclusionsJAK3 mutations are found in a subset of ccRCC patients and may be associated with ccRCC development and a greater risk of metastases. JAK3 function is compromised in p.Arg925Ser and p.Ala677Thr/p.Val722Ile. Future studies with a larger number of patients need to confirm these findings.     

Gitelman综合征SLC12A3基因突变研究

目的分析和确定Gitelman综合征相关基因SLC12A3突变位点,以提高对该病的认识和理解。方法通过直接测序的方法寻找和确定12例Gitelman综合征患者相关基因SLC12A3的突变位点。选取50例健康正常人作为对照,评估发现的突变位点。结果共确定SLC12A3基因8个突变位点,其中5个为新突变位点,包括2个错义突变：Cys430Gly和Leu571Pro;2个缺失突变：1384delG和346～353delACTGATGG;1个非移码插入突变： 997insCys。3个已报道过的突变,其中包括2个错义突变：Thr60Met和Asp486Asn;1个缺失突变：2883-2884delAG。12例患者中8例携带Thr60Met纯合或杂合突变,大部分患者为复合杂合突变。结论基因突变分析对诊断Gitelman综合征有重要价值。Thr60Met可能是中国Gitelman综合征患者较常见的突变。Gitelman综合征特异的表型和基因型之间的联系目前较难确定。     

A novel splice site mutation of the thiazide-sensitive NaCl cotransporter gene in a Japanese patient with Gitelman syndrome

Gitelman syndrome (GS, MIM 263800) is an inherited disorder characterized by metabolic alkalosis with hypokalemia, hypomagnesemia, and hypocalciuria. The genetic abnormalities causing GS are known to lie in the thiazide-sensitive NaCl cotransporter (TSC), which is expressed in the distal tubule of the kidney. The TSC gene, located at chromosome 16, consists of 26 exons and encodes the protein containing 12 putative transmembrane domains with long intracellular amino and carboxy termini. Most of the abnormalities identified in GS were missense mutations, distributed throughout the TSC gene without a hot spot. A 42-year-old Japanese man was introduced for close examination of hypokalemia. In renal clearance studies using furosemide or thiazide, chloride clearance was increased after furosemide but not after thiazide administration. Furthermore, the distal fractional chloride reabsorption was dramatically decreased by furosemide but not thiazide administration, suggesting a defect in the distal tubule. We then analyzed the TSC gene to confirm the diagnosis of GS, and identified a novel G to T mutation at the acceptor splice site preceding exon 14, resulting in disruption of a conventional 3'AG consensus splice site. Abnormal splicing by this mutation is predicted to cause the formation of truncated TSC with a partial deletion of the transmembrane domain, which will loose the function of transporter. In conclusion, we have identified a unique novel splice site mutation of the TSC gene in GS. The predicted structure of this mutant TSC can conceivably cause an impairment of the transporter activity and thereby be responsible for the development of GS in our patient.     

Characterization of glucokinase mutations associated with maturity-onset diabetes of the young type 2 (MODY-2): different glucokinase defects lead to a common phenotype.

Glucokinase (GK) is expressed in the pancreatic beta-cells and liver, and plays a key role in the regulation of glucose homeostasis. The enzymatic activity and thermal stability of wild-type (WT) GK and several mutant forms associated with maturity-onset diabetes of the young type 2 (MODY-2) were determined by a steady-state kinetic analysis of the purified expressed proteins. The eight MODY-2 mutations studied were Ala53Ser, Val367Met, Gly80Ala, Thr168Pro, Arg36Trp, Thr209Met, Cys213Arg, and Val226Met. These missense mutations were shown to have variable effects on GK kinetic activity. The Gly80Ala and Thr168Pro mutations resulted in a large decrease in Vmax and a complete loss of the cooperative behavior associated with glucose binding. In addition, the Gly80Ala mutation resulted in a sixfold increase in the half-saturating substrate concentration (S0.5) for ATP, and Thr168Pro resulted in eight- and sixfold increases in the S0.5 values for ATP and glucose, respectively. The Thr209Met and Val226Met mutations exhibited three- and fivefold increases, respectively, in the S0.5 for ATP, whereas the Cys213Arg mutation resulted in a fivefold increase in the S0.5 for glucose. These mutations also led to a small yet significant reduction in Vmax. Of all the mutations studied, only the Cys213Arg mutation had reduced enzymatic activity and decreased thermal stability. Two mutants, Ala53Ser and Val367Met, showed kinetic and thermal stability properties similar to those of WT. These mutants had increased sensitivities to the known negative effectors of GK activity, palmitoyl-CoA, and GK regulatory protein. Taken together, these results illustrate that the MODY-2 phenotype may be linked not only to kinetic alterations but also to the regulation of GK activity.     

Denaturing high performance liquid chromatography screening of ryanodine receptor type 1 gene in patients with malignant hyperthermia in Taiwan and identification of a novel mutation (Y522C)

We performed the present study to identify the mutation in patients in Taiwan with malignant hyperthermia (MH). We also test the hypothesis that a denaturing high-performance liquid chromatography (DHPLC) protocol can be used for mutation detection in these patients. We identified five Taiwanese patients with typical clinical presentations of MH after general anesthesia. We also enrolled 50 healthy volunteers. Polymerase chain reaction was used to amplify the ryanodine receptor (RYR1) gene mutation hot spots and DHPLC techniques were used to screen for mutations. Upon detection of a heterozygous elution pattern in DHPLC analysis, DNA sequencing reaction was performed to identify the nucleotide variations. We identified a RYR1 mutation in all 5 patients with MH. There were 4 different mutations in the 5 patients: Tyr522Cys, Arg552Trp, Val2168Met, and Thr2206Arg. Among the 5 patients, 2 unrelated patients had the same Thr2206Arg mutation. Three of the mutations had been reported before, but the Tyr522Cys mutation was novel. None of the MH-related mutations were found in the control group. In conclusion, we identified RYR1 mutations in 5 Taiwanese patients with MH using a DHPLC-based approach. A DHPLC-based genetic test may be developed as a noninvasive and convenient test for MH.     

Comparison of the Segregation of the RYR1 C1840T Mutation with Segregation of the Caffeine/Halothane Contracture Test Results for Malignant Hyperthermia Susceptibility in a Large Manitoba Mennonite Family

Background: Malignant hyperthermia (MH) is an important cause of anesthesia-induced death. Malignant hyperthermia susceptibility is diagnosed using the in vitro caffeine/halothane contracture test (CHCT) in fresh muscle biopsy specimens. The CHCT test is highly invasive, expensive, and lacks 100% specificity. Genetic and biochemical evidence provide strong support for the view that the substitution of cysteine for arginine 614 (Arg614Cys) in the human ryanodine receptor gene is one of several mutations that are likely to cause human MH. DNA testing was compared with CHCT as a means of predicting MH susceptibility in a large MH family in which the Arg614Cys mutation was detected.

Methods: A comparison of CHCT and DNA-based diagnosis was conducted in a large Manitoba Mennonite MH kindred identified by an index patient who died at age 45 yr of an MH crisis after general anesthesia. The presence of the Arg614Cys mutation was detected through a combination of polymerase chain reaction and restriction endonuclease digestion. Blood samples for DNA analysis were obtained from 68 family members, including 19 who had undergone muscle biopsies and 1 who had a documented crisis but did not undergo biopsy. Family members were classified as MH-susceptible or MH-normal on the basis of the CHCT.

Results: Twenty-two persons were found to be heterozygous for the Arg614Cys mutation. Five of these persons had prior positive CHCT results and one had an MH crisis but did not undergo biopsy. On DNA testing, 44 persons were found to be homozygous for the normal allele. Of these, ten had been classified as MH-normal and five as MH-susceptible on the basis of the CHCT. On reevaluation of the data obtained in our earlier CHCT diagnoses, we found that the condition of the muscle was poor, with no twitch, for three of five individuals homozygous for the normal allele but originally classified as MH-susceptible and for one who was homozygous for the normal allele and originally classified as MH-normal. Caffeine/halothane contracture test results for these four persons were considered invalid. The twitch response was good for the two remaining persons who were homozygous for the normal allele but classified as MH-susceptible, because contracture was observed with appropriately low levels of both caffeine and halothane.     

Molecular characterization of 6 unrelated Italian patients with 5alpha-reductase type 2 deficiency

Steroid 5alpha-reductase (5alphaR) deficiency (OMIM number #264600) is a rare 46,XY disorder of sex differentiation caused by mutations in the 5alphaR type 2 gene (SRD5A2) resulting in dihydrotestosterone deficiency during fetal development. We report on the analysis of the SRD5A2 gene in 6 unrelated 46,XY Italian patients with external genitalia morphology ranging from predominantly female to nearly completely male. Three subjects were seen and assessed at birth, 1 patient was referred to us before puberty, and 2 at postpubertal age. Six different causative mutations (5 missense and 1 nonsense) and a rare polymorphism were identified. Four patients presented homozygous single-base substitutions. These SRD5A2 mutations were located in exon 2 (variant Cys133Gly), exon 4 (Gly196Ser and Ala207Asp) and exon 5 (Tyr235Phe). A fifth subject was a compound heterozygote who carried a nonsense mutation in exon 1 (Trp53X) and a second SRD5A2 alteration in exon 5 (Tyr235Phe). The final patient presented a mutation in only 1 allele (Gly34Trp) together with the Ala49Thr variant. The molecular characterization of these patients made it possible to identify novel mutations and to confirm, before gender assignment or any surgical approach, the suspected 5alphaR deficiency in 2 newborns, 1 of whom had inconclusive hormonal data. 5alphaR deficiency in subjects without parental consanguinity and the presence of compound heterozygotic patients suggest that SRD5A2 mutations carrier frequency may be higher than previously thought.     

Clinical implications of mutation analysis in primary hyperoxaluria type 1

BACKGROUND: Primary hyperoxaluria type 1 (PH1) is an inborn error of glyoxylate metabolism with an extensive clinical and genetic heterogeneity. Although over 50 disease-causing mutations have been identified, the relationship between genotype and clinical outcome remains unclear. The aim of this study was to determine this association in order to find clues for improvement of patient care. METHODS: AGXT mutation analysis and assessment of biochemical characteristics and clinical outcome were performed on patients from a Dutch PH1 cohort. RESULTS: Thirty-three of a cohort of 57 PH1 patients, identified in The Netherlands over a period of 30 years, were analyzed. Ten different mutations were found. The most common mutations were the Gly170Arg, Phe152Ile, and the 33insC mutations, with an allele frequency of 43%, 19%, and 15%, respectively. Homozygous Gly170Arg and Phe152Ile mutations were associated with pyridoxine responsiveness and a preserved renal function over time when treatment was timely initiated. All patients homozygous for the 33insC mutation had end-stage renal disease (ESRD) before the first year of age. In two unrelated patients, a new Val336Asp mutation was found coupled with the Gly170Arg mutation on the minor allele. We also found 3 patients homozygous for a novel Gly82Arg mutation with adverse outcome in 2 of them. CONCLUSION: Early detection of Gly170Arg and Phe152Ile mutations in PH1 has important clinical implications because of their association with pyridoxine responsiveness and clinical outcome. The association of a homozygous 33insC mutation with severe infantile ESRD, resulting in early deaths in 2 out of 3 cases, warrants a choice for prenatal diagnostics in affected families.     

Polymorphism of cytochrome P450 2B6 and prostate cancer risk: A significant association in a Japanese population

Objectives:   To explore whether Lys262Arg polymorphism of the Cytochrome P450 2B6 (CYP2B6) gene could act as a genetic marker for prostate cancer risk among Japanese men.
Methods:   A total of 350 patients with sporadic prostate cancer and 328 controls were examined. A single nucleotide polymorphism with non-synonymous amino acid change located at Lys262Arg of the CYP2B6 gene was genotyped using a TaqMan assay.
Results:   The frequency of the Arg/Arg genotype among prostate cancer patients was significantly higher than that among the controls ( P  = 0.027). The frequency of the G allele of the Lys262Arg polymorphism was also significantly higher in prostate cancer patients than in the controls (30.4% vs 24.8%, P  = 0.025). Patients with the Lys/Arg plus Arg/Arg genotypes carried a low Gleason score more frequently than those with the Lys/Lys genotype ( P  = 0.042). The frequency of the G allele of the Lys262Arg polymorphism was significantly higher in the low Gleason score group than that in the high Gleason score group (34.3% vs 26.8%, P  = 0.038).
Conclusions:   Lys262Arg polymorphism of the CYP2B6 gene may be a genetic marker for evaluating the risk of sporadic prostate cancer in native Japanese men.     

Mutation screening in the ryanodine receptor 1 gene (RYR1) in patients susceptible to malignant hyperthermia who show definite IVCT results: identification of three novel mutations

BACKGROUND: The ryanodine receptor of the skeletal muscle (RYR1) seems to be of outstanding importance in the pathogenesis of malignant hyperthermia (MH). It has been shown that point mutations in the RYR1 gene are strongly associated with the MH phenotype. A correctly determined phenotype is the basic prerequisite for adequate genetic MH screening. In this study we examined only those MH susceptible patients for the presence of potential RYR1 mutations who showed strong pathological muscle responses in the in vitro contracture test (IVCT). METHODS: A total of 56 MHS index patients who complied with the following IVCT criteria were included in the molecular genetic investigation: Contracture forces > or =4 mN at a caffeine concentration of 2.0 mmol/l and > or =8 mN at a halothane concentration of 0.44 mmol/l. DNA sequences of exons 2, 6, 9, 11, 12, 14, 15, 17, 39, 40, 45, 46, 102 of the RYR1 gene were analysed by the direct sequencing technique. Furthermore, if an MH mutation was identified in an index patient, all relatives were screened for their family specific RYR1 defect. RESULTS: In 39 index patients an RYR1 mutation was detected: Arg163Cys (n = 2), Asp166Asn (n = 1), Gly341Arg (n = 2), Arg401His (n = 2), Arg614Cys (n = 12), Asp2129Glu (n = 1),Vol2168Met (n = 1), Thr2206Met (n = 9), Ala2428Thr (n = 1), Gly2434Arg (n = 2), Arg2435His (n = 1), Arg2452Trp (n = 1), Arg2454His (n = 4). Three new RYR1 mutations were identified. We found a potential MH mutation in a further 130 relatives of the 39 index patients. Thirty-seven individuals were classified as MHS exclusively by molecular genetic techniques and did not have to undergo the IVCT. CONCLUSIONS: The ascertained high rate of successful MH mutation screening (69.64%) is obviously associated with the more clearly defined MHS diagnosis in the IVCT. According to the EMHG guidelines for the molecular genetic detection of MH susceptibility, a positive MH disposition could be determined in numerous persons by a less invasive technique.     

Mutations in Known Monogenic High Bone Mass Loci Only Explain a Small Proportion of High Bone Mass Cases

High bone mass (HBM) can be an incidental clinical finding; however, monogenic HBM disorders (eg, LRP5 or SOST mutations) are rare. We aimed to determine to what extent HBM is explained by mutations in known HBM genes. A total of 258 unrelated HBM cases were identified from a review of 335,115 DXA scans from 13 UK centers. Cases were assessed clinically and underwent sequencing of known anabolic HBM loci: LRP5 (exons 2, 3, 4), LRP4 (exons 25, 26), SOST (exons 1, 2, and the van Buchem's disease [VBD] 52‐kb intronic deletion 3′). Family members were assessed for HBM segregation with identified variants. Three‐dimensional protein models were constructed for identified variants. Two novel missense LRP5 HBM mutations ([c.518C>T; p.Thr173Met], [c.796C>T; p.Arg266Cys]) were identified, plus three previously reported missense LRP5 mutations ([c.593A>G; p.Asn198Ser], [c.724G>A; p.Ala242Thr], [c.266A>G; p.Gln89Arg]), associated with HBM in 11 adults from seven families. Individuals with LRP5 HBM (～prevalence 5/100,000) displayed a variable phenotype of skeletal dysplasia with increased trabecular BMD and cortical thickness on HRpQCT, and gynoid fat mass accumulation on DXA, compared with both non‐LRP5 HBM and controls. One mostly asymptomatic woman carried a novel heterozygous nonsense SOST mutation (c.530C>A; p.Ser177X) predicted to prematurely truncate sclerostin. Protein modeling suggests the severity of the LRP5‐HBM phenotype corresponds to the degree of protein disruption and the consequent effect on SOST‐LRP5 binding. We predict p.Asn198Ser and p.Ala242Thr directly disrupt SOST binding; both correspond to severe HBM phenotypes (BMD Z‐scores +3.1 to +12.2, inability to float). Less disruptive structural alterations predicted from p.Arg266Cys, p.Thr173Met, and p.Gln89Arg were associated with less severe phenotypes (Z‐scores +2.4 to +6.2, ability to float). In conclusion, although mutations in known HBM loci may be asymptomatic, they only account for a very small proportion (～3%) of HBM individuals, suggesting the great majority are explained by either unknown monogenic causes or polygenic inheritance. © 2015 The Authors Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).     

四例脂蛋白肾病患者载脂蛋白E基因突变筛查

目的 通过对4例脂蛋白肾病患者及家系成员载脂蛋白E(apoE)基因的分析， 研究脂蛋白肾病的发病机制。 方法 调查患者家系情况，对其中2例患者的家系成员进行尿常规筛查及血肌酐、血脂和血清脂蛋白的检测。PCR法扩增4例患者apoE基因的外显子， DNA测序， 发现突变后，寻找限制性内切酶酶切位点。使用聚合酶链反应-限制性酶切片段长度多态性(PCR-RFLP)的方法筛查正常对照及其家系成员。 结果 4例脂蛋白肾病患者中有2例携带杂合的apoE基因缺失突变 (142-144-0)，2例患者携带杂合的apoE点突变 (Arg25Cys)，2种突变均可见于尿液检查正常的亲属，并均表现为apoE升高。 结论 4例脂蛋白肾病的患者中发现两种apoE基因的突变，apoE Arg25Cys和apoE(142-144-0)。结合既往的研究结果，apoE(142-144-0)同时见于5例(本研究2例，前期研究3例)患者，为中国脂蛋白肾病患者常见的致病性基因突变。患者家系成员中的携带者可以不表现出肾脏病。     

A novel mutation in the chloride channel gene,CLCNKB, as a cause of Gitelman and Bartter syndromes

BACKGROUND: Gitelman syndrome (GS) and Bartter syndrome (BS) are hereditary hypokalemic tubulopathies with distinct phenotypic features. GS has been considered a genetically homogeneous disorder caused by mutation in the gene encoding the NaCl cotransporter (TSC) of the distal convoluted tubule. In contrast, BS is caused by mutations in the genes encoding either the Na-K-2Cl cotransporter (NKCC2), the K+ channel (ROMK) or the Cl- channel (ClC-Kb) of the thick ascending limb. The purpose of this study was to examine the clinical, biochemical and genetic characteristics of a very large inbred Bedouin kindred in Northern Israel with hereditary hypokalemic tubulopathy. METHODS: Twelve family members affected with hypokalemic tubulopathy, as well as 26 close relatives were clinically and biochemically evaluated. All study participants underwent genetic linkage analysis. Mutation analysis was performed in affected individuals. RESULTS: Evaluation of affected family members (age range 3 to 36 years) revealed phenotypic features of both GS and classic Bartter syndrome (CBS). Features typical of GS included late age of presentation (>15 years) in 7 patients (58%), normal growth in 9 (75%), hypomagnesemia (SMg <0.7mmol/L) in 5 (42%), hypermagnesiuria (FEMg>5%) in 6 (50%) and hypocalciuria (urinary calcium/creatinine mmol/mmol <0.15) in 5 (42%). Features typical of CBS included early age of presentation (<1 year) in 3 (25%), polyuria/dehydration in 4 (33%), growth retardation in 3 (25%), hypercalciuria (urinary calcium/creatinine mmol/mmoverline>0.55) in 4 (33%) and nephrolithiasis in 1 (8%). Linkage analysis in affected patients excluded the TSC gene, SLC12A3, as the mutated gene, but demonstrated linkage to the Cl- channel gene, CLCNKB, on chromosome 1p36. Mutation analysis by direct sequencing revealed a novel homozygous missense mutation, arginine 438 to histidine (R438H), in exon 13 of CLCNKB in all patients. A restriction fragment length polymorphism (RFLP) analysis has been developed to aid in genotyping of family members. CONCLUSIONS: Our findings demonstrate intrafamilial heterogeneity, namely the presence of GS and CBS phenotypes, in a kindred with the CLCNKB R438H mutation. We conclude that GS can be caused by a mutation in a gene other than SLC12A3. The exact role of the CLCNKB R438H mutation in the pathogenesis of the electrolyte and mineral abnormalities in GS and CBS remains to be established.     

Identification of five novel SLC3A1 (rBAT) gene mutations in Japanese cystinuria

Identification of five novel SLC3A1 (rBAT) gene mutations in Japanese cystinuria. BACKGROUND: Cystinuria is an inheritable amino aciduria and has been classified into three subtypes: I, II, and III. One of the genes responsible for cystinuria has recently been identified as SLC3A1 or rBAT, but only type I cystinuria seems to be caused by genetic alterations in rBAT. To our knowledge, thus far 38 mutations in rBAT gene have been described. In this study, we investigated rBAT mutations in Japanese patients and compared the results with the previously reported mutations in other races. METHODS: We investigated 36 Japanese cystinuria patients by mutational analysis of rBAT gene. To identify newly mutated alleles, genomic DNA was analyzed by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). When an abnormal migration was observed on SSCP, a nucleotide sequence determination was performed. RESULTS: Five novel mutations were identified in five patients, three with missense mutations (L346P, I445T, C673R), one with a 1 bp deletion (1820delT), and one with a 2 bp insertion (1898insTA), and we detected three previously reported polymorphisms. Three of the mutations were homozygous, in whom parents had intermarried, and two were heterozygous for each mutations. Analysis of rBAT in family of the 1898insTA patient revealed that the patient had inherited the mutated allele from his parents. CONCLUSION: Five novel mutations in the rBAT gene have been identified in Japanese patients with cystinuria. A racial difference was not apparent in the position and frequency of the mutations.     

A novel mutation of the thiazide-sensitive sodium chloride cotransporter gene in a Japanese family with Gitelman syndrome

Gitelman syndrome is an inherited renal disorder characterized by impaired NaCl reabsorption in the distal convoluted tubule leading to hypokalemia, hypomagnesemia and normocalcemic hypocalciuria. It has been shown that this syndrome results from mutations in the gene encoding the thiazide-sensitive sodium chloride cotransporter (TSC). We performed the mutational analysis in the TSC gene of a 30-year-old Japanese woman with Gitelman syndrome and found two mutations at adjacent spots in both alleles. One was a frame shift mutation which generated stop codon at position 671, the other was a single nucleotide mutation, which resulted in an aminoacid substitution at position 672, Met to Ile. Her 52-year-old mother and two daughters had neither hypokalemia nor hypomagnesemia. However, her mother and her 8-year-old daughter had the Met672Ile mutation as heterozygotes. Her 4-year-old daughter had the same frame shift mutation as her mother, a heterozygotic mutation. These results suggest that Gitelman syndrome requires 2 compound heterozygotic mutations and the coexistence of the large deletion in the C-terminal domain with Met672Ile substitution of the TSC could impair the transporter activity underling the hypokalemia and hypomagnesemia in this patient.     

Mutational analysis of ETV6 in prostate carcinoma

BACKGROUND: In an effort to better understand the molecular events responsible for progression of prostate carcinoma to metastatic disease, we have recently identified a homozygous deletion at 12p12-13 involving ETV6 (tel). Although mutational analysis of ETV6 has not been examined previously in prostate carcinoma, it is an attractive candidate prostate cancer tumor suppressor gene since as it previously has been implicated in malignancy. Therefore, we decided to analyze 43 prostate cell lines, xenografts, and metastatic foci for inactivating mutations. METHODS: DNA was isolated from 7 cell lines, 18 xenografts, and 18 metastatic deposits. Single-strand conformational polymorphism (SSCP) analysis of ETV6, was performed by polymerase chain reaction (PCR) amplification of each exon by using intron specific primers. PCR products were then resolved by gel electrophoresis, and aberrantly migrating PCR products were then sequenced. RESULTS: Two previously described polymorphisms and four novel sequence changes were identified. Polymorphisms at nucleotide 258 (G --> A, Thr --> Thr) and 602 (T --> C, Leu --> Pro) were identified in eight and one specimen(s), respectively. Analysis of noncancerous DNA confirmed the presence of the polymorphisms in the germ-line. Four possible mutations were identified at nucleotides 24 (T --> G, Cys --> Trp), 380 (G --> A, Arg --> Glu), 776 (G --> T, Arg --> Leu), and 876 (C --> T, Leu --> Leu). Three were in xenografts or cell lines. Because normal DNA was not available, these could represent rare polymorphisms. The sole mutation in a clinical specimen, at nucleotide 876, did not result in an amino acid change. CONCLUSION: Our data suggest that mutational inactivation ETV6 may occur in prostate carcinoma. The functional significance of these potential inactivating mutations remains to be determined.