Shoma Baidya

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

Summary.  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 <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.

Determination of the breakpoint and molecular diagnosis of a common α‐thalassaemia‐1 deletion in the Indian population

Summary.  The previously described South African type α‐thalassaemia‐1 mutation was identified in Indian HbH patients using a polymerase chain reaction (PCR) strategy. A multiplex PCR assay was devised to detect heterozygotes and homozygotes. This α‐thalassaemia‐1 mutation was found to be the commonest determinant causing HbH disease in this population. In one family this mutation was found in combination with a novel splice donor mutation α2 IVS I‐1 (G→A). Characterization of the breakpoint junction sequence revealed, in addition to a 23 kb deletion, that there was an addition of ∼160 bp bridging the breakpoints. Similar to other deletions in the α‐globin gene cluster, there is an Alu repeat‐mediated mechanism for the origin of the deletion.

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.

Summary.  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.

Correlating clinical manifestations with factor levels in rare bleeding disorders: a report from Southern India.

Summary.  Data on the clinical manifestations of patients with clotting factor defects other than Haemophilia A, B and von Willebrand disease are limited because of their rarity. Due to their autosomal recessive nature of inheritance, these diseases are more common in areas where there is higher prevalence of consanguinity. There is no previous large series reported from southern India where consanguinity is common. Our aim was to analyze clinical manifestations of patients with rare bleeding disorders and correlate their bleeding symptoms with corresponding factor level. Data were collected in a standardized format from our centre over three decades on 281 patients who were diagnosed with rare bleeding disorders (fibrinogen, prothrombin, factor V (FV), FVII, FX, FXI, FXIII and combined FV or FVIII deficiency). Patients with liver dysfunction or those on medications which can affect factor level were excluded. All patients with <50% factor levels were included in this analysis. Patients were analysed for their salient clinical manifestations and it was correlated with their factor levels. The data shows that FXIII deficiency is the commonest and FXI deficiency is the rarest in Southern India. There was no significant difference in bleeding symptoms among those who were < or >1% factor coagulant activities among all disorders, except for few symptoms in FVII and FX deficiency. An international collaborative study is essential to find out the best way of classifying severity in patients with rare bleeding disorders.

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

Summary.  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.

Clinical manifestations of combined factor V and VIII deficiency: A series of 37 cases from a single center in India†

We describe here the clinical manifestations of 37 patients with combined coagulation factor (F) V and FVIII deficiency, which is the commonest multiple coagulation factor deficiency state. Only a few cases are reported in the literature from India. Prolonged bleed post injury/ surgery (62%) is the commonest manifestation in our series, and epistaxis (19%) is rare in our patients in comparison to other series described in the literature. Although the frequency of bleeding manifestations differs among various reports, there is no evidence for increased bleeding manifestation due to combined deficiency of two coagulation factors as against a single coagulation factor. To the best of our knowledge, this is the largest series of this disorder described so far.

Successful surgical haemostasis in patients with von Willebrand disease with Koate®DVI

Summary.  This report describes our experience with Koate®DVI, a factor VIII (FVIII) concentrate containing von Willebrand factor (VWF) for surgery in patients with von Willebrand’s disease (VWD). Twenty‐one patients underwent 26 procedures, 10 of which were major and 16 were minor. The median age was 27 years (3–55) and the mean weight was 52 kg (16–88). Among the ten patients (type 2–5; type 3–5) who underwent major procedures, the pre‐operative dose was 35 IU kg−1 of FVIII followed by 10–20 IU kg−1 once daily depending on FVIII:C levels. The mean total dose of FVIII used per procedures was 106 IU kg−1 (30–190) over a mean duration of 7 days (3–11). In this group, pre‐infusion FVIII:C, VWF:Ag and VWF: ristocetin cofactor (RCoF) level that were 19.5% (1–64), 20 U dL−1 (0–96) and 12% (0–66) increased to 72% (54–198), 131 U dL−1 (68–206) and 68% (27–108) postinfusion, respectively. Sixteen minor procedures were performed in 11 patients (type 1–3, type 2–6, type 3–2). The preparative dose of FVIII was 10–20 IU kg−1. The average duration of factor support was 2 days (1–3) for a mean total dose of 23 IU kg−1 (9–60). The pre‐infusion levels of FVIII:C, VWF:Ag and VWF:ristocetin cofactor (RCo) which were 31% (22–64), 25.5 U dL−1 (0–63) and 21% (0–76), respectively, increased to 76% (27–111), 73 U dL−1 (30–137) and 45% (2–106) postinfusion. Whereas surgical haemostasis was achieved in all patients, minor postoperative bleeding occurred after one procedure in each group. Both were controlled with additional doses of factor replacement. We conclude that Koate®DVI in modest doses provide adequate haemostasis for surgery in patients with VWD.

A new multiplex PCR and conformation-sensitive gel electrophoresis strategy for mutation detection in the platelet glycoprotein αIIb and β3 genes

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