A Strategy for the Molecular Diagnosis in Hemophilia A in Chinese Population

Hemophilia A is an x-linked recessive inherited bleeding disorder. So far, more than 1,885 disease-causing mutations of factor VIII gene have been identified. Clinic confers a great challenge for the molecular diagnosis. We aim to make a better strategy for the molecular diagnosis in Hemophilia A. First, factor VIII intron 22 inversion and intron 1 inversion mutations were detected using Inversion-PCR and double-tube multiple PCRs. And then, non-inversion mutations were analyzed by denaturing high performance liquid chromatography and/or direct sequencing. Novel mutations were further analyzed the conservation and 3D structures by a B domain deleted crystallographic model and bioinformatics. Finally, we can indirectly confirm the diagnosis by linkage analysis for the patients with the confusing diagnosis by the techniques mentioned above. Eleven patients with the factor VIII Inv 22 were found, and the remaining 16 patients were found with 11 different mutations, of which 3 was novel mutations affecting A1, B domains and splicing site. Moreover, the prenatal diagnosis was performed on 14 fetuses. Ten fetuses were successfully confirmed to be normal, 1 fetus to be a heterozygote with factor VIII c.3275–3276 ins A and 3 fetuses to be hemizygotes with factor VIII Inv 22 mutation.     

Nonsense and missense mutations in hemophilia A: estimate of the relative mutation rate at CG dinucleotides.

Hemophilia A is an X-linked disease of coagulation caused by deficiency of factor VIII. Using cloned cDNA and synthetic oligonucleotide probes, we have now screened 240 patients and found CG-to-TG transitions in an exon in nine. We have previously reported four of these patients; and here we report the remaining five, all of whom were severely affected. In one patient a TaqI site was lost in exon 23, and in the other four it was lost in exon 24. The novel exon 23 mutation is a CG-to-TG substitution at the codon for amino acid residue 2166, producing a nonsense codon in place of the normal codon for arginine. Similarly, the exon 24 mutations are also generated by CG-to-TG transitions, either on the sense strand producing nonsense mutations or on the antisense strand producing missense mutations (Arg to Gln) at position 2228. The novel missense mutations are the first such mutations observed in association with severe hemophilia A. These results provide further evidence that recurrent mutations are not uncommon in hemophilia A, and they also allow us to estimate that the extent of hypermutability of CG dinucleotides is 10-20 times greater than the average mutation rate for hemophilia A.     

Exon skipping caused by an intronic insertion of a young Alu Yb9 element leads to severe hemophilia A

Short interspersed elements, such as Alu elements, have propagated to more than one million copies in the human genome. They affect the genome in several ways, caused by retrotransposition, recombination between elements, gene conversion, and alterations in gene expression. These events, including novel insertions into active genes, have been associated with a number of human disorders. Hemophilia A is an X-linked severe bleeding disorder and is caused by mutations in the Factor VIII gene. The spectrum of mutations includes point mutations, rearrangements, insertions, and deletions. Recently, an Alu retrotransposition event in a coding exon has been reported in a family with a severe form of hemophilia A. This was the first report of an Alu insertion in the Factor VIII gene. Here, we report a second Alu insertion event that lies in an intron of the same gene that causes exon skipping and the complete disruption of gene expression.     

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.     

Screening for nonsense mutations in patients with severe hemophilia A can provide rapid,direct carrier detection

Summary Despite marked genetic heterogeneity in families with hemophilic patients, transition mutations in CG dinucleotides occur frequently. Of 71 CG dinucleotides in the factor VIII cDNA, a C-to-T transition in 12 would lead to a new Stop codon (CGA to TGA). Using restriction enzyme digestion of 11 amplified DNA fragments, seven point mutations were localized among 60 patients with severe hemophilia A. Five were detected as loss of a natural or introduced TaqI site at codons -5, 583, 1941, 2116, and 2209 and were confirmed as CGA (Arg) to TGA (Stop) nonsense mutations by DNA sequencing. A novel C-to-T nonsense mutation was detected as loss of the RsaI site at codon 1966 and confirmed by sequence in two unrelated individuals. Two partial gene deletions were detected as selective failure to amplify exon 1 and exons 15–22, respectively. In an additional (61st) patient who was subsequently found to have mild (instead of severe) hemophilia, digests suggested a mutation in codon 1696. Upon sequencing, this codon contained a novel missense mutation, a C-to-G transversion changing CGA (Arg 1696) to GGA (Gly). In four families with women available for testing, carrier status was rapidly determined by direct screening for the point mutation. In two of three with sporadic occurrences, the mother was a carrier as were two of four sisters. In the other family, the mother and a sister were homozygous for the TaqI cleavage site in their amplified exon 24 fragment, indicating a de novo C-to-T transition in codon 2209 in the patient's factor VIII gene. This final patient's sister was a noncarrier even though by linkage analysis she inherited the same factor VIII gene as her brother.These results have already been published in part in abstract form: Reiner AP, Thompson AR (1990) Circulation Research 82:304     

Mutations in argininosuccinate synthetase mRNA of Japanese patients,causing classical citrullinemia.

Citrullinemia is an autosomal recessive disease caused by a genetic deficiency of argininosuccinate synthetase. In order to characterize mutations in Japanese patients with classical citrullinemia, RNA isolated from 10 unrelated patients was reverse-transcribed, and cDNA amplified by PCR was cloned and sequenced. The 10 mutations identified included 6 missense mutations (A118T, A192V, R272C, G280R, R304W, and R363L), 2 mutations associated with an absence of an exon 7 or exon 13, 1 mutation with a deletion of the first 7 bp in exon 16 (which might be caused by abnormal splicing), and 1 mutation with an insertion of 37 bp within exons 15 and 16 in cDNA. The insertion mutation and the five missense mutations (R304W being excluded) are new mutations described in the present paper. These are in addition to 14 mutations (9 missense mutations, 4 mutations associated with an absence of an exon in mRNA, and 1 splicing mutation) that we identified previously in mainly American patients with neonatal citrullinemia. Two of these 20 mutations, a deletion of exon 13 sequence and a 7-bp deletion in exon 16, were common to Japanese and American populations from different ethnic backgrounds; however, other mutations were unique to each population. Furthermore, the presence of a frequent mutation--the exon 7 deletion mutation in mRNA, which accounts for 10 of 23 affected alleles--was demonstrated in Japanese citrullinemia. This differs from the situation in the United States, where there was far greater heterogeneity of mutations.     

Characterization and significance of nine novel mutations in exon 16 of the neurofibromatosis type 1 (NF1) gene

Nine novel mutations have been characterized as the result of screening exon 16 of the human NF1 gene in 465 unrelated neurofibromatosis type 1 patients. These lesions include three nonsense and two missense mutations, two deletions, one duplication, and one mutation in the 5′ splice site of intron 16. Although exon 16 is the largest NF1 exon, no mutations have so far been reported in this region. This apparent paucity of lesions may be due either to a reduced functional importance of exon 16 or a screening bias or both. However, consideration of the mutability of exon 16 in comparison with other exons suggests that, at least for single base pair substitutions, no such factors need be invoked. Any previous lack of exon 16 mutations in this category would be explicable in terms of a lower propensity to mutate for codons in this gene region. Received: 1 November 1996 / Revised: 5 December 1996     

Molecular analysis in Brazilian cystic fibrosis patients reveals five novel mutations

We have performed molecular genetic analyses on 160 Brazilian patients diagnosed with cystic fibrosis (CF). Screening of mutations in 320 CF chromosomes was performed through single strand conformation polymorphism (SSCP) and heteroduplex analyses assay followed by DNA sequencing of the 27 exons and exon/intron boundaries of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The frequency of CFTR variants of T-tract length of intron 8 (IVS8 Tn) was also investigated. This analysis enabled the detection of 232/320 CF mutations (72.2%) and complete genotyping of 61% of the patients. The deltaF508 mutation was found in 48.4% of the alleles. Another fifteen mutations (previously reported) were detected: G542X, R1162X, N1303K, R334W, W1282X, G58E, L206W, R553X, 621+1G-->T, V232D, 1717-1G-->A, 2347 delG, R851L, 2789+5G-->A, and W1089X. Five novel mutations were identified, V201M (exon 6a), Y275X (exon 6b), 2686 insT (exon 14a), 3171 delC (exon 17a), and 3617 delGA (exon 19). These results contribute to the molecular characterization of CF in the Brazilian population. In addition, the identification of the novel mutation Y275X allowed prenatal diagnosis in a high-risk fetus.     

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.     

First report of molecular diagnosis of Tunisian hemophiliacs A: identification of 8 novel causative mutations

ABSTRACT: INTRODUCTION: Hemophilia A is an X linked recessive hemorrhagic disorder caused by mutations in the F8 gene that lead to qualitative and/or quantitative deficiencies of coagulation factor VIII (FVIII). Molecular diagnosis of hemophilia A is challenging because of the high number of different causative mutations that are distributed throughout the large F8 gene. Molecular studies of these mutations are essential in order to reinforce our understanding of their pathogenic effect responsible for the disorder. Aim In this study we have performed molecular analysis of 28 Tunisian hemophilia A patients and analyzed the F8 mutation spectrum. METHODS: We screened the presence of intron 22 and intron 1 inversion in severe hemophilia A patients by southern blotting and polymerase chain reaction (PCR). Detection of point mutations was performed by dHPLC/sequencing of the coding F8 gene region. We predict the potential functional consequences of novel missense mutations with bioinformatics approaches and mapping of their spatial positions on the available FVIII 3D structure. RESULTS: We identified 23 different mutations in 28 Tunisian hemophilia A patients belonging to 22 unrelated families. The identified mutations included 5 intron 22 inversions, 7 insertions, 4 deletions and 7 substitutions. In total 18 point mutations were identified, of which 9 are located in exon 14, the most mutated exonic sequence in the F8 gene. Among the 23 mutations, 8 are novel and not deposited in the HAMSTeRS database nor described in recently published articles. CONCLUSION: The mutation spectrum of Tunisian hemophilia A patients is heterogeneous with the presence of some characteristic features. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1693269827490715.     

Mild hemophilia A associated with a cryptic donor splice site mutation in intron 4 of the factor VIII gene

Hemophilia A, an X-linked disease caused by deficiency of factor VIII, is characterized by variation in clinical severity and coagulation activity. This variation is though to reflect heterogeneity of mutations in the factor VIII gene. Here we describe a CG-to-CA mutation within a potential cryptic donor splice site in intron 4 of the factor VIII gene from a patient with mild disease. This mutation makes the cryptic sequence resemble more closely the consensus sequence for donor splice sites. We infer that the mutation activates the cryptic donor splice site, which in turn causes a defect in RNA processing.     

A novel mutation (4040-4045 nt. delA) in exon 14 of the factor VIII gene causing severe hemophilia A

Hemophilia A is an X-linked congenital bleeding disorder caused by Factor VIII deficiency. Different mutations including point mutations, deletions, insertions and inversions have been reported in the FVIII gene, which cause hemophilia A. In the current study, with the use of conformational sensitive gel electrophoresis (CSGE) analysis, we report a novel 1-nt deletion in the A6 sequence at codons 1328-1330 (4040-4045 nt delA) occurring in exon 14 of the FVIII gene in a seven-year-old Iranian boy with severe hemophilia A. This mutation that causes frameshift and premature stop-codon at 1331 has not previously been reported in the F8 Hemophilia A Mutation, Structure, Test and Resource Site (HAMSTeRS) database.     

Clustering of missense mutations in the C-terminal region of factor H in atypical hemolytic uremic syndrome

Hemolytic-uremic syndrome (HUS) is a microvasculature disorder leading to microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Most cases of HUS are associated with epidemics of diarrhea caused by verocytotoxin-producing bacteria, but atypical cases of HUS not associated with diarrhea (aHUS) also occur. Early studies describing the association of aHUS with deficiencies of factor H suggested a role for this complement regulator in aHUS. Molecular evidence of factor H involvement in aHUS was first provided by Warwicker et al., who demonstrated that aHUS segregated with the chromosome 1q region containing the factor H gene (HF1) and who identified a mutation in HF1 in a case of familial aHUS with normal levels of factor H. We have performed the mutational screening of the HF1 gene in a novel series of 13 Spanish patients with aHUS who present normal complement profiles and whose plasma levels of factor H are, with one exception, within the normal range. These studies have resulted in the identification of five novel HF1 mutations in four of the patients. Allele HF1 Delta exon2, a genomic deletion of exon 2, produces a null HF1 allele and results in plasma levels of factor H that are 50% of normal. T956M, W1183L, L1189R, and V1197A are missense mutations that alter amino acid residues in the C-terminal portion of factor H, within a region--SCR16-SCR20--that is involved in the binding to solid-phase C3b and to negatively charged cellular structures. This remarkable clustering of mutations in HF1 suggests that a specific dysfunction in the protection of cellular surfaces by factor H is a major pathogenic condition underlying aHUS.     

Identification of one novel mutation in the EVC2 gene in a Chinese family with Ellis–van Creveld syndrome

Ellis–van Creveld syndrome (EvC) is a rare autosomal recessive skeletal dysplasia characterized by short limbs, short ribs, postaxial polydactyly, and dysplastic nails and teeth. It is caused by biallelic mutations in the EVC or EVC2 gene. Here, we identified a novel nonsense mutation p.W828X (c.2484G>A) in exon 14 and a recurrent nonsense mutation p. R399X (c.1195C>T) in exon 10 of EVC2 gene in a Chinese boy with EvC. Identification of a novel genotype in EvC will provide clues to the phenotype–genotype relations and may assist not only in the clinical diagnosis of EvC but also in the interpretation of genetic information used for prenatal diagnosis and genetic counseling.     

Characterization of mutations in the factor VIII gene by direct sequencing of amplified genomic DNA

In order to search for mutations resulting in hemophilia A that are not detectable by restriction analysis, three regions of the factor VIII gene were chosen for direct sequence analysis. Short segments of genomic DNA of 127 unrelated patients with hemophilia A were amplified by polymerase chain reaction. A total of 136,017 nucleotides were sequenced, and four mutations leading to the disease were found: a frameshift at codon 360 due to deletion of two nucleotides (GA), a nonsense codon 1705 due to a C----T transition, and two missense codons at positions 1699 and 1708. The first missense mutation (A----T) results in a Tyr----Phe substitution at a putative von Willebrand factor binding site. The second results in an Arg----Cys substitution at a thrombin cleavage site. In addition, we identified three rare sequence variants: a silent C----T transition at codon 34 which does not result in an amino acid change, a G----C change at codon 345 (Val----Leu), and an A----G change at the third nucleotide of intron 14. Direct sequence analysis of amplified DNA is a powerful but labor-intensive method of identifying mutations in large genes such as the human factor VIII gene.     

Characterization of a splicing mutation in the factor VIII gene at the RNA level

Haemophilia A is an X-linked bleeding disorder caused by mutations in the coagulation factor VIII (FVIII) gene. The identification and characterization of naturally occurring disease-producing mutations allows the recognition of new mechanisms of pathogenesis in haemophilia A. Analysis of the illegitimately transcribed FVIII mRNA in a severely affected patient has revealed that the AG transition at position –2 of the acceptor splice site of intron 4 results in the skipping of exon 5 in 90% of the processed pre-mRNA. Another minor mRNA species arising from the skipping of exons 4 and 5 has also been observed. The skipping of exon 5 predicts the removal of the corresponding 13 amino acids from the A1 domain of FVIII. A novel missense mutation, C329S, in exon 8 of FVIII gene has been identified in another patient.     

Mutational analysis of the human dihydropyrimidine dehydrogenase gene by denaturing high-performance liquid chromatography

Mutations in the DPYD gene, which encodes dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme in the catabolism of pyrimidines, are responsible for an inborn error of metabolism associated with thymine-uraciluria and neurological symptoms. Because the antimetabolite 5-fluorouracil (5-FU) is metabolized by the same enzyme, deficient DPYD alleles may also constitute a risk factor for severe toxicity following treatment with this anticancer drug. The aim of this study was to develop a comprehensive and rapid method to detect sequence variations within the DPYD gene. Using polymerase chain reaction (PCR) amplification and denaturing high-performance liquid chromatography (DHPLC), we established a protocol that makes it possible to screen all 23 exons of the DPYD gene and their exon-intron boundaries for both known and unknown mutations under identical conditions. A novel one-step PCR mutagenesis procedure was developed to generate heterozygous mutant amplicons as positive controls to optimize DHPLC detection of any sequence variation. DHPLC analysis was shown to result in mutation-specific elution profiles and to be able to distinguish different base changes within the same exon or different heterozygous combinations of mutations within the same exon. By analyzing the DPYD gene in 16 affected individuals, a total of 47 base changes were detected, representing eight known mutations and three novel intronic base changes. Sequence analysis confirmed all base changes detected. This method will be useful in identifying patients at risk for toxicity prior to 5-FU treatment, as well as in the analysis of individual patients with thymine-uraciluria.