Mapping and Characterization of the X-Linked Dyskeratosis Congenita (DKC) Gene

We report the precise mapping and characterization of the genomic structure of the human homolog of the rat gene for the nucleolar protein NAP57, which has been reported to be responsible for X-linked dyskeratosis congenita (DKC). This single-copy gene, now called DKC, is transcribed from a CpG island 60 kb centromeric to the factor VIII gene in distal Xq28 and lies tail to tail with the palmitoylated erythrocyte membrane protein gene, MPP1. DKC comprises 15 exons spanning at least 16 kb and is transcribed into a widely expressed 2.6-kb message. Several functional motifs of DKC are assigned to coding sequences specified by individual exons. Analysis of normal female DNA revealed the presence of two polymorphisms in the DKC exons, while mutation analysis of a DKC patient identified a novel single amino acid missense mutation in exon 4. The latter together with exon 3 contain five of the six missense mutations reported so far in the DKC gene.     

X-linked dyskeratosis congenita is predominantly caused by missense mutations in the DKC1 gene.

Dyskeratosis congenita is a rare inherited bone marrow-failure syndrome characterized by abnormal skin pigmentation, nail dystrophy, and mucosal leukoplakia. More than 80% of patients develop bone-marrow failure, and this is the major cause of premature death. The X-linked form of the disease (MIM 305000) has been shown to be caused by mutations in the DKC1 gene. The gene encodes a 514-amino-acid protein, dyskerin, that is homologous to Saccharomyces cerevisiae Cbf5p and rat Nap57 proteins. By analogy to the homologues in other species, dyskerin is predicted to be a nucleolar protein with a role in both the biogenesis of ribosomes and, in particular, the pseudouridylation of rRNA precursors. We have determined the genomic structure of the DKC1 gene; it consists of 15 exons spanning a region of 15 kb. This has enabled us to screen for mutations in the genomic DNA, by using SSCP analysis. Mutations were detected in 21 of 37 additional families with dyskeratosis congenita that were analyzed. These mutations consisted of 11 different single-nucleotide substitutions, which resulted in 10 missense mutations and 1 putative splicing mutation within an intron. The missense change A353V was observed in 10 different families and was shown to be a recurring de novo event. Two polymorphisms were also detected, one of which resulted in the insertion of an additional lysine in the carboxy-terminal polylysine domain. It is apparent that X-linked dyskeratosis congenita is predominantly caused by missense mutations; the precise effect on the function of dyskerin remains to be determined.     

Identification of three neurofibromatosis type 2 (NF2) gene mutations in vestibular schwannomas

Vestibular schwannomas (VSs) are common benign tumors of Schwann cell origin and are frequently found in patients with neurofibromatosis type 2 (NF2). We analyzed 15 sporadic VSs for mutations in the NF2 gene. We detected mutations in three of the tumors, two of which contained loss of heterozygosity (LOH). One of the tumors contained a novel mutation, a 19-bp deletion in exon 4. The two other tumors contained an identical mutation, a complete exon 4 deletion. The exon 4 deletion represents the second most frequently reported mutation of the NF2 gene in VSs.     

Identification of one novel causative mutation in exon 4 of WFS1 gene in two Italian siblings with classical DIDMOAD syndrome phenotype

The aim of the present paper is to describe a novel missense mutation (G107R) of WFS1 gene that was unexpectedly detected, in two siblings from Southern Italy, outside exon 8; a very unusual finding which has previously been reported only twice in Italian patients with Wolfram syndrome (WS). Although in Spanish pedigrees' WFS1 mutations are frequently located in exon 4, this finding is very infrequent in other pedigrees, particularly in Italian patients. Conclusions: a) our report of two siblings with one novel WSF1 mutation (G107R) expands the molecular spectrum of WS; b) this is the 3rd report of Italian patients harbouring one mutation outside exon 8 and the 2nd with one mutation in exon 4; c) on the basis of the present observations, and literature data we can infer that mutation locations outside exon 8 do not seem to be clearly associated with peculiar phenotype expressions of WFS1 gene.     

Mutation screening of the FSH receptor gene in infertile men.

Follicle stimulating hormone (FSH) is important for controlling spermatogenesis through binding with its receptor. However, little information is available on mutations of the FSH and its receptor gene in infertile men. To study the genetic defects, which caused problems in spermatogenesis, we screened the point mutations of the FSH receptor gene in infertile men with high serum FSH concentrations. Seventy male infertile patients with high FHS levels (> 12 mIU/ml) were screened for mutations in each of the 10 exons of the FSH receptor gene, using genomic DNA PCR and a single-strand conformation polymorphism (SSCP) analysis. From this study, three shifted bands were detected by SSCP. The first shifted band was found in the PCR product of exon 4, including the exon-intron boundary sequence in only one patient. The sequence analysis revealed a nucleotide A to T substitution in intron 3 (IVS3-4A-->T). The second shifted band was detected in exon 10 with high frequency (33%). A nucleotide A to G substitution was found at the position of the 994th nucleotide, predicting a Thr to Ala substitution at the position of the 307th amino acid (Thr307Ala). The third shifted band in the 3' region of exon 10 was detected frequently in infertile patient and normal groups. It was tightly linked to the Thr307Ala variant. Thus, all of the abnormalities represent neutral polymorphisms, and not pathological mutations of the FSH receptor gene. In conclusion, we did not confirm that the genomic mutation of the FSH receptor is a major genetic cause in Korean infertile patients with high FSH levels.     

Novel mutations in the SLC12A3 gene causing Gitelman's syndrome in Swedes.

PURPOSE: Gitelman's syndrome (GS) is an inherited autosomal recessive disorder due to loss of function mutations in the SLC12A3 gene encoding the Na-Cl co-transporter (NCCT), the target of thiazide diuretics. The defective function of the NCCT, which normally is expressed in the apical membrane of the distal convolute tubule in the kidney, leads to mild hypotension, hypokalemia, hyperreninemic hyperaldosteronism, mild metabolic alkalosis, hypomagnesemia and hypocalciuria. Up to now, more than 100 mutations of the SLC12A3 gene have been described in GS patients. METHODS: We have collected 30 patients from Sweden with a clinical diagnosis of GS and undertaken a mutation screening by SSCP and successive sequencing of the 26 exons and intronic boundaries. Both mutations were identified in most (n = 28, 93%) and at least one mutation was identified in all patients. RESULTS: We found 22 different mutations evenly distributed throughout the gene, 11 of which have not been described previously. The new variants include 8 missense mutations (Glu68Lys, His69Asn, Argl45His, Vall53Met, Gly230Asp, Gly342Ala, Val677Leu and Gly867Ser), 1 insertion (c.834_835insG on exon 6) and 2 splice-site mutations (c.2667 + lT>G substitution in splicing donor site after exon 22, c.1569-1G>A substitution in the splicing acceptor site before exon 13). CONCLUSION: In Swedish patients with the clinical features of GS, disease-causing mutations in the SLC12A3 gene were identified in most patients. The spectrum of GS mutations is wide making full mutation screening of the SLC12A3 gene necessary to confirm the diagnosis.     

APC germ-line mutations in southern Spanish patients with familial adenomatous polyposis: genotype-phenotype correlations and identification of eight novel mutations

Familial adenomatous polyposis (FAP) is a disease characterized by the presence of hundreds of adenomatous polyps in the colon and rectum which, if not treated, develop into colorectal cancer. FAP is an autosomal dominantly inherited disorder caused by mutation in the APC gene. The aim of this study was to search for germ-line mutations of the APC gene in unrelated FAP families from southern Spain. By direct sequencing of all APC gene exons, we found the mutation in 13 of 15 unrelated FAP families studied. We identified eight novel mutations: 707delA (exon6), 730_731delAG (exon7), 1787C-->G and 1946_1947insG (exon14), 2496delC, 2838_2839delAT, 2977A-->T, and 3224dupA (exon15). Two patients presented de novo germ-line mutations. Genotype-phenotype correlations for extraintestinal and extracolonic manifestations were studied. Intrafamilial phenotypic variability was observed in two families with mutations in exon/intron boundary, probably due to alternative splicing.     

Identification of 13 new mutations in the vasopressin-neurophysin II gene in 17 kindreds with familial autosomal dominant neurohypophyseal diabetes insipidus.

Familial neurohypophyseal diabetes insipidus (FNDI) is an autosomal dominant disorder characterized by progressive postnatal deficiency of arginine vasopressin as a result of mutation in the gene that encodes the hormone. To determine the extent of mutations in the coding region that produce the phenotype, we studied members of 17 unrelated kindreds with the disorder. We sequenced all 3 exons of the gene by using a rapid, direct dye-terminator method and found the causative mutation in each kindred. In four kindreds, the mutations were each identical to mutations described in other affected families. In the other 13 kindreds each mutation was unique. There were two missense mutations that altered the cleavage region of the signal peptide, seven missense mutations in exon 2, which codes for the conserved portion of the protein, one nonsense mutation in exon 2, and three nonsense mutations in exon 3. These findings, together with the clinical features of FNDI, suggest that each of the mutations exerts an effect by directing the production of a pre-prohormone that cannot be folded, processed, or degraded properly and eventually destroys vasopressinergic neurons.     

Clinical and Molecular Genetic Analysis of 19 Wolfram Syndrome Kindreds Demonstrating a Wide Spectrum of Mutations in WFS1

Wolfram syndrome is an autosomal recessive neurodegenerative disorder characterized by juvenile-onset diabetes mellitus and progressive optic atrophy. mtDNA deletions have been described, and a gene (WFS1) recently has been identified, on chromosome 4p16, encoding a predicted 890 amino acid transmembrane protein. Direct DNA sequencing was done to screen the entire coding region of the WFS1 gene in 30 patients from 19 British kindreds with Wolfram syndrome. DNA was also screened for structural rearrangements (deletions and duplications) and point mutations in mtDNA. No pathogenic mtDNA mutations were found in our cohort. We identified 24 mutations in the WFS1 gene: 8 nonsense mutations, 8 missense mutations, 3 in-frame deletions, 1 in-frame insertion, and 4 frameshift mutations. Of these, 23 were novel mutations, and most occurred in exon 8. The majority of patients were compound heterozygotes for two mutations, and there was no common founder mutation. The data were also analyzed for genotype-phenotype relationships. Although some interesting cases were noted, consideration of the small sample size and frequency of each mutation indicated no clear-cut correlations between any of the observed mutations and disease severity. There were no obvious mutation hot spots or clusters. Hence, molecular screening for Wolfram syndrome in affected families and for Wolfram syndrome-carrier status in subjects with psychiatric disorders or diabetes mellitus will require complete analysis of exon 8 and upstream exons.     

粘多糖贮积症Ⅱ型患者IDS基因的2个新突变

为了研究粘多糖贮积症II型(MPS Ⅱ)患者发病的分子遗传学机制, 采用PCR扩增艾杜糖-2-硫酸酯酶(IDS)基因突变热点区(外显子2、3、5、7、8和9)、DNA测序分析和限制性内切酶图谱分析的方法, 对2个粘多糖贮积症Ⅱ型家系进行了遗传突变分析。结果表明, 2个家系患者的IDS 基因分别出现IVS 6-1g→a和c.1587~1588 ins T 2个新突变。前者属于单碱基替换, 位于内含子6的3′端剪接位点, 导致跨外显子剪接; 后者属于插入突变, 插入点位于外显子9的cDNA 1,587和1,588碱基之间, 是迄今为止报道的人类IDS基因插入突变中最接近肽链末端的突变, 导致移码突变和转录提前终止。经限制性酶切分析, 证实2个家系中的患者母亲是突变基因的携带者, 符合该病X染色体隐性遗传的规律。另外,在对随机抽取的50名正常人及另外6名不相关的粘多糖病人的测序分析中, 未检测到这2个突变, 说明不是多态性。对于筛查所得的2个新突变是否是患者的致病原因, 尚需进一步证实。     

Dyskeratosis congenita: molecular insights into telomerase function, ageing and cancer

Dyskeratosis congenita (DC) is a severe, inherited, bone marrow failure syndrome, with associated cutaneous and noncutaneous abnormalities. DC patients also show signs of premature ageing and have an increased occurrence of cancer. DC can originate through: (1) mutations in DKC1, which result in X-linked recessive DC; (2) mutations in the RNA component of telomerase (TERC), which result in autosomal dominant DC (AD-DC); and (3) mutations in other, currently uncharacterized, genes, which result in autosomal recessive DC (AR-DC). As DKC1 encodes dyskerin, a protein component of small nucleolar ribonucleoprotein (snoRNP) particles, which are important in ribosomal RNA processing, DC was initially described as a disorder of defective ribosomal biogenesis. Subsequently, dyskerin and TERC were shown to closely associate with each other in the telomerase complex, and DC has since come to be regarded as a telomerase deficiency disorder characterised by shorter telomeres. These findings demonstrate the importance of telomerase in humans and highlight how its deficiency (through DKC1 and TERC mutations) results in multiple abnormalities including premature ageing, bone marrow failure and cancer. Identification of the gene(s) involved in AR-DC will help to define the pathophysiology of DC further, as well as expand our insights into telomere function, ageing and cancer.     

Structure of human holocarboxylase synthetase gene and mutation spectrum of holocarboxylase synthetase deficiency

Holocarboxylase synthetase (HLCS) is an enzyme that catalyzes the incorporation of biotin into apo-carboxylases, and its deficiency causes biotin-responsive multiple carboxylase deficiency. The reported sequences of cDNA for human HLCS from liver, lymphocyte, and KG-1 myeloid cell lines differ at their 5' regions. To elucidate variations of the human HLCS mRNA and longer 5' cDNA ends, we performed screening of the human liver cDNA library and rapid amplification of the cDNA ends (RACE). Our results suggest the existence of three types of HLCS mRNA that start at different exons. The first type starts at exon 1, and the second type starts at exon 3, and both are found in various human tissues. The third type, corresponding to the cDNA from the KG-1 cell, starts at exon 2 of the HLCS gene. Various splicing patterns from exons 3-6 were also observed. None of the variations of cDNA found created a new initiation codon. Mutation screening from exons 6-14, therefore, was sufficient to detect amino acid changes in HLCS in patients. Our direct sequencing strategy for screening mutations in the HLCS gene revealed mutations in five Japanese patients and seven non-Japanese patients. Our analyses involving 12 Japanese and 13 non-Japanese patients and studies by others indicate that (1) there is no panethnically prevalent mutation; (2) the Arg508Trp, Gly581Ser, and Val550Met mutations are found in both Japanese and non-Japanese populations; (3) the IVS10+5G-->A mutation is predominant and probably a founder mutation in European patients; (4) the 655-656insA, Leu237Pro, and 780delG mutations are unique in Japanese patients; (5) the spectrum of the mutations in the HLCS gene may vary substantially among different ethnic groups.     

Regional distribution of mutations of the ATP7B gene in patients with Wilson disease: impact on genetic testing

Wilson disease is an autosomal recessive inherited disorder of copper metabolism. The Wilson disease gene codes for a copper transporting P-type ATPase (ATP7B). Molecular genetic analysis reveals at least 300 distinct mutations. While most reported mutations occur in single families, a few are more common. The most common mutation in patients from Central, Eastern, and Northern Europe is the point mutation H1069Q (exon 14). About 50–80% of Wilson disease (WD) patients from these countries carry at least one allele with this mutation with an allele frequency ranging between 30 and 70%. Other common mutations in Central and Eastern Europe are located on exon 8 (2299insC, G710S), exon 15 (3400delC) and exon 13 (R969Q). The allele frequency of these mutations is lower than 10%. In Mediterranean countries there is a wide range of mutations, the frequency of each of them varies considerably from country to country. In Sardinia, a unique deletion in the 5′ UTR (−441/−427 del) is very frequent. In mainland Spain the missense mutation M645R in exon 6 is particularly common. Data from non-European countries are scarce. Most data from Asia are from Far Eastern areas (China, South Korea and Japan) where the R778L missense mutation in exon 8 is found with an allele frequency of 14–49%. In summary, given the constant improvement of analytic tools genetic testing will become an integral part for the diagnosis of WD. Knowledge of the differences in the worldwide distribution of particular mutations will help to design shortcuts for genetic diagnosis of WD.     

394delTT: a Nordic cystic fibrosis mutation

In a systematic screening for mutations in the gene encoding the cystic fibrosis transmembrane regulator among Danish cystic fibrosis (CF) patients, we identified a mutation in exon 3 (394delTT); this mutation was found to be relatively common in Denmark. We therefore screened for 394delTT in Sweden and Norway, where it turned out to be the second most frequent mutation, accounting for 4% of all CF mutations. It also occurs with a high frequency in Finland, but has not been found in larger surveys of mutations in the CFTR gene. Thus, 394delTT seems to be a specific Nordic CF mutation.     

Mutation analysis of Taiwanese Wilson disease patients

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism, which is caused by mutation in copper-transporting ATPase (ATP7B). In the present study, we report a molecular diagnosis method to screen the WD chromosome in patients or in heterozygotic carriers in Taiwan. Exons 8, 11, 12, 13, 16, 17, and 18 of ATP7B are selected for the screening of mutations. The most common mutation, Arg778Leu or Arg778Gln, was first screened by PCR-RFLP then we combined single-stranded conformation polymorphism (SSCP) analysis followed by direct DNA sequencing on the DNA fragments with mobility shift on SSCP analysis. The diagnostic rate was compared with standard ATP7B whole gene sequencing analysis. Ten different mutations were identified among 29 WD patients; among them four were novel (Ala1168Pro, Thr1178Ala, Ala1193Pro, and Pro1273Gln). The false positive rates were tested against 100 normal individuals and listed as follows: exon 8: 5%; exon 11: 4%; exon 12: 6%; exon 13: 5%; exon 16: 5%; exon 17: 3%; exon 18: 4%. The Arg778Leu mutation exhibited the highest allelic frequency (43.1%). The detection rate of WD chromosomes is 65.52%, which is as sensitive as whole gene sequencing scanning. According to our results, WD chromosomes in Taiwan are predominantely located at exons 8, 11, 12, 13, 16, 17, and 18. The standard sequencing analysis on the entire gene is time consuming. We recommend screening these 7 exons first on those individuals who have a higher risk in having WD, before whole gene and promoter sequencing analysis in Taiwan.     

Mutation analysis of Korean patients with citrullinemia

Citrullinemia is an autosomal recessive disease due to the mutations in the argininosuccinate synthetase (ASS) gene. Mutation analysis was performed on three Korean patients with citrullinemia. All of the three patients had the splicing mutation previously reported as IVS6-2A>G mutation. Two had Gly324Ser mutation and the other patient had a 67-bp insertion mutation in exon 15. The IVS6-2A>G mutation was reported to be found frequently in Japanese patients with citrullinemia, but Caucasian patients showed the extreme mutational heterogeneity. Although a limited number of Korean patients were studied, the IVS6-2A>G mutation appears to be one of the most frequent mutant alleles in Korean patients with citrullinemia. The Gly324Ser mutation identified in two patients also suggests the possible high frequency of this mutation in Korean patients as well.     

APC mutation in the alternatively spliced region of exon 9 associated with late onset familial adenomatous polyposis

Germ-line mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP). Genotype-phenotype correlation studies in patients with FAP have demonstrated associations of certain variants of the disease with mutations at specific sites within the APC gene. In a large FAP family, we identified a frameshift mutation located in the alternatively spliced region of exon 9. Phenotypic studies of affected family members showed that the clinical course of FAP was delayed, with gastrointestinal symptoms and death from colorectal carcinoma occurring on average 25 and 20 years later than usual, respectively. The numbers of colorectal adenomas differed markedly among affected individuals and the location of colorectal cancer lay frequently in the proximal colon. Our findings suggest that the exon 9 mutation identified in the pedigree is associated with late onset of FAP. The atypical phenotype may be explained by the site of the mutation in the APC gene. Analysis of the APC protein product indicated that the exon 9 mutation did not result in a detectable truncated APC protein. Given the location of the mutation within an alternatively spliced exon of APC, it is conceivable that normal APC proteins are produced from the mutant allele by alternative splicing.