Molecular genetics of hereditary prothrombin deficiency in Indian patients: identification of a novel Ala362→Thr (Prothrombin Vellore 1) mutation

Prothrombin deficiency is a rare (1:200 000) autosomal recessive disorder caused by diverse mutations in prothrombin gene. We have studied the molecular basis of this disorder in four unrelated Indian patients. The diagnosis was based on prolonged prothrombin (PT) and activated partial thromboplastin times and low factor II coagulant activity (FII: C) measured using a PT based assay. FII: C levels ranged between 4.7% and 17.5%. Mutations were identified in all the four patients. Five different causative mutations including four (80%) missense and an in-frame deletion (20%) were identified. One of them was a novel, Ala362→Thr aminoacid change affecting 'B' chain of α-thrombin. This mutation was present in a compound heterozygous state with a previously reported Arg-1→Gln missense change affecting pro-peptide cleavage site. Ala362→Thr occurred at a codon, evolutionarily conserved in all the 24 different prothrombins or its related serine proteases studied. Molecular modeling of this mutation was found to cause a conformational change around the region involving a catalytic triad residue His363 and a cysteine residue at codon 364. The FII: C level in this patient was 17.5%. Three other previously reported mutations were also detected in the homozygous state: Arg271→Cys in Kringle-2 region, a Glu309?Lys in 'A' chain of α-thrombin and an in-frame deletion of 3 bp (AAG) leading to Del Lys301/302 in 'A' chain of α-thrombin. This is the first report of the molecular basis of prothrombin deficiency in Indian patients and we suggest the eponym 'Prothrombin Vellore 1' for Ala362→Thr mutation

Six novel mutations including triple heterozygosity for Phe31Ser, 514delT and 516T→G factor X gene mutations are responsible for congenital factor X deficiency in patients of Nepali and Indian origin

Factor X (FX) deficiency is a rare (1 : 100000) autosomal recessive disorder caused by heterogeneous mutations in FX gene. We have studied the molecular basis this disease in six Indian and one Nepali patients. Diagnosis was confirmed by measuring the FX coagulant activity (FX: C) using a PT based assay. Six of them had a FX: C of < 1% and one patient had 24% coagulant activity. Mutations were identified in all the seven patients. These included eight (88.8%) missense and one frame-shift (11.2%) mutations of which six were novel. Three of the novel mutations, a Phe31Ser affecting 'Gla' domain and 514delT and 516T?G mutations affecting Cys132 in 'connecting region' were identified in a triple compound heterozygous state in a Nepali patient presenting with a severe phenotype. Two other novel mutations, Gly133Arg, may affect the disulphide bridge between Cys132-Cys302 in the connecting region while Gly223Arg may perturb the catalytic triad (His236, Asp282 and Ser379). The other novel mutation, Ser354Arg, involves the replacement of a small-buried residue by a large basic aminoacid and is likely to have steric or electrostatic effects in the pocket involving Lys351-Arg347-Lys414 that contributes to the core epitope of FXa for binding to FVa. Three previously reported mutations, Thr318Met; Gly323Ser; Gly366Ser were also identified. This is the first report of the molecular basis of FX deficiency in patients from the Indian subcontinent

Mutation spectrum of 122 hemophilia A families from Taiwanese population by LD-PCR, DHPLC, multiplex PCR and evaluating the clinical application of HRM

<p>Abstract</p> <p>Background</p> <p>Hemophilia A represents the most common and severe inherited hemorrhagic disorder. It is caused by mutations in the F8 gene, which leads to a deficiency or dysfunctional factor VIII protein, an essential cofactor in the factor X activation complex.</p> <p>Methods</p> <p>We used long-distance polymerase chain reaction and denaturing high performance liquid chromatography for mutation scanning of the F8 gene. We designed the competitive multiplex PCR to identify the carrier with exonal deletions. In order to facilitate throughput and minimize the cost of mutation scanning, we also evaluated a new mutation scanning technique, high resolution melting analysis (HRM), as an alternative screening method.</p> <p>Results</p> <p>We presented the results of detailed screening of 122 Taiwanese families with hemophilia A and reported twenty-nine novel mutations. There was one family identified with whole exons deletion, and the carriers were successfully recognized by multiplex PCR. By HRM, the different melting curve patterns were easily identified in 25 out of 28 cases (89%) and 15 out of 15 (100%) carriers. The sensitivity was 93 % (40/43). The overall mutation detection rate of hemophilia A was 100% in this study.</p> <p>Conclusion</p> <p>We proposed a diagnostic strategy for hemophilia A genetic diagnosis. We consider HRM as a powerful screening tool that would provide us with a more cost-effective protocol for hemophilia A mutation identification.</p

Identification of factor IX gene defects using a multiplex PCR and CSGE strategy_a first report

This article does not have an abstract

Mutations in the MCFD2 gene are predominant among patients with hereditary combined FV and FVIII deficiency (F5F8D) in India

Combined FV and FVIII deficiency (F5F8D) is a rare (1:1.000.000) autosomal recessive disorder caused by a defect in the LMAN1 or MCFD2 genes, encoding for a FV and FVIII cargo receptor complex. We report the phenotype and genotype analyses in nine unrelated Indian patients with low FV and FVIII coagulant activity [FV:C, range: 5.6-22.4% and FVIII:C, range: 8.3-27.1%]. Four homozygous mutations, including two frame shift, one missense and one splice site, were identified in all the nine patients. Three of them, a 72-bp deletion in LMAN1 (c.813_822 + 62del72, p.K272fs), a 35-bp deletion in MCFD2 (c.210_244del35) and a missence mutation in MCFD2 (p.D122V), identified in four patients, were novel mutations. A previously reported c.149 + 5G &gt; A transition in MCFD2 was identified in the remaining five patients. Haplotype analysis of MCFD2 gene in patients with p.E71fs and c.149 + 5G &gt; A defects suggested an independent origin of both these mutations. The identification of two common mutations (p.E71fs, c.149 + 5G &gt; A) in MCFD2 gene in seven of nine patients, particularly the c.149 + 5G &gt; A (55,6% of patients), suggests that this gene could be the first to be analysed during the genetic diagnosis of F5F8D in this population. This is the first report describing the molecular analysis of a consistent number of F5F8D patients of South Indian origin, a population with a high frequency of such recessive bleeding disorders

Mutations in the MCFD2 gene are predominant among patients with hereditary combined FV and FVIII deficiency (F5F8D) in India

Combined FV and FVIII deficiency (F5F8D) is a rare (1:1.000.000) autosomal recessive disorder caused by a defect in the LMAN1 or MCFD2 genes, encoding for a FV and FVIII cargo receptor complex. We report the phenotype and genotype analyses in nine unrelated Indian patients with low FV and FVIII coagulant activity [FV:C, range: 5.6-22.4% and FVIII:C, range: 8.3-27.1%]. Four homozygous mutations, including two frame shift, one missense and one splice site, were identified in all the nine patients. Three of them, a 72-bp deletion in LMAN1 (c.813_822 + 62del72, p.K272fs), a 35-bp deletion in MCFD2 (c.210_244del35) and a missence mutation in MCFD2 (p.D122V), identified in four patients, were novel mutations. A previously reported c.149 + 5G > A transition in MCFD2 was identified in the remaining five patients. Haplotype analysis of MCFD2 gene in patients with p.E71fs and c.149 + 5G > A defects suggested an independent origin of both these mutations. The identification of two common mutations (p.E71fs, c.149 + 5G > A) in MCFD2 gene in seven of nine patients, particularly the c.149 + 5G > A (55,6% of patients), suggests that this gene could be the first to be analysed during the genetic diagnosis of F5F8D in this population. This is the first report describing the molecular analysis of a consistent number of F5F8D patients of South Indian origin, a population with a high frequency of such recessive bleeding disorder

A new multiplex PCR and conformation-sensitive gel electrophoresis strategy for mutation detection in the platelet glycoprotein &#945;IIb and &#946;3 genes

This article does not have an abstract

Novel missense mutations in two patients with factor XI deficiency (Val271Leu and Tyr351Ser) and one patient with combined factor XI and factor IX deficiency (Phe349Val)

This article does not have an abstract

Identification of factor VIII gene mutations in 101 patients with haemophilia A: mutation analysis by inversion screening and multiplex PCR and CSGE and molecular modelling of 10 novel missense substitutions

Haemophilia A (HA) is an X-linked bleeding disorder caused by diverse mutations in the human coagulation factor VIII (FVIII) gene. We have analysed DNA from 109 unrelated Indian patients with HA for their FVIII gene defects. Among these patients 89 (82%) had severe (FVIII:C &gt;1%) HA, 11 (10%) had moderate (FVIII:C 1-5%) HA and nine (8%) had mild (FVIII:C 5-30%) HA. These patients were first screened for the common intron 22 and intron 1 inversions. Inversion negative samples were screened for point mutations by a multiplex PCR and conformation sensitive gel electrophoresis strategy. Mutations were identified in 101 of the 109 patients. These included two (2%) intron 1 and 51 (51%) intron 22 inversions, four (4%) gross deletions and 44 (43%) point mutations. Twenty-nine novel causative mutations, including 11 missense, seven frameshift, five nonsense mutations, three splice site defects and three gross deletions were detected. Ten of the novel missense mutations were studied by molecular modelling. Two different (Thr2253Pro and Pro1392fs) mutations were seen in four unrelated families and FVIII gene haplotyping suggested a common founder effect. Seven of these 109 patients had inhibitors. Among them, four had intron 22 inversions, one had a novel gross deletion (delexon 2-9) and one a nonsense mutation (Trp1535Stop). In one of these patients, no mutation could be identified in the FVIII gene. A Thr2253Pro novel mutation and an intron 22 inversion were identified in two female haemophiliacs. The data from this study suggests that the spectrum of gene defects in Indian patients with HA is as heterogeneous as reported in other populations