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Phenotypic and genotypic characterization of Factor VII deficiency patients from Western India
Clinica Chimica Acta 409 (2009) 106–111
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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Phenotypic and genotypic characterization of Factor VII deficiency patients from Western India Leenam Mota a, Shrimati Shetty a, Susan Idicula-Thomas b, Kanjaksha Ghosh a,⁎ a b
National Institute of Immunohaematology, 13th floor of KEM Hospital Campus, Parel, Mumbai, 400012, India Biomedical Informatics Center of Indian Council of Medical Research, National Institute for Research in Reproductive Health, Parel, Mumbai, 400012, India
a r t i c l e
i n f o
Article history: Received 13 July 2009 Received in revised form 4 September 2009 Accepted 4 September 2009 Available online 13 September 2009 Keywords: Factor VII deficiency Mutations India
a b s t r a c t Background: Congenital factor VII (FVII) deficiency is a rare coagulation deficiency caused due to defects in the FVII gene. Methods: We analyzed 14 unrelated Indian patients with congenital FVII deficiency for mutations in FVII gene by conformation sensitive gel electrophoresis (CSGE) followed by DNA sequencing. Results: A total of 11 different missense mutations were identified, of which 5 were novel (Ala191Pro, Asp338Glu, Ile138Thr, Leu263Arg and Trp284Arg) and 6 had been previously reported (Cys22Arg, Arg152Gln, Cys310Phe, Thr324Met, Gly117Arg and His348Arg). Six of the 11 mutations were located in the catalytic serine protease domain, 3 in the activation domain and 1 each in the Gla and the second epidermal growth factor domain respectively. Multiple sequence alignment using ClustalW2 analysis showed that all the mutations were found in residues that are highly conserved across species. Implications of mutations on the structural stability and function of human factor VIII (hFVII) using Swiss-Pdb Viewer and the intra-molecular interactions of the mutant residues using PIC showed that there is a structural instability in all the mutants either by steric hindrance or instability in the protein molecule folding. Conclusion: A wide spectrum of mutations was detected in the FVII gene; the presence of 6 out of 11 mutations in the serine protease domain suggests the crucial role of catalytic domain in FVII functional activity. © 2009 Elsevier B.V. All rights reserved.
1. Introduction FVII is a vitamin K-dependant serine protease synthesized in the liver and occupies a very important position in the coagulation cascade. Normal hemostasis is initiated by the formation of a complex between tissue factor (TF) and FVII in the circulating blood [1]. FVII deficiency is a rare, autosomally inherited, recessive disorder which affects approximately 1 in 500,000 of the population [2]. The clinical features are quite variable in patients with FVII deficiency. Reports from all over the world have highlighted the lack of correlation between the measured factor level and the clinical manifestation [3]. Common clinical manifestations include epistaxis, gum bleeding and prolonged bleeding from cuts and injuries. Menorrhagia and chronic iron deficiency are frequently seen in homozygous women with FVII deficiency, the prevalence being as high as 60% and a similar prevalence has been reported for central nervous system (CNS) bleeding [4]. The FVII gene is located on chromosome 13 (13q34), consists of 9 exons and 8 introns and is approximately 12 kb in size [5,6] encoding a mature protein of 406 amino acids. The FVII protein consists of an
⁎ Corresponding author. Tel.: +91 22 24138518/19; fax: +91 22 24138521. E-mail address: [email protected] (K. Ghosh). 0009-8981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2009.09.007
amino-terminal (light chain) Gla domain, carboxy-terminal (heavy chain) catalytic domain and 2 epidermal growth factor domains. To date about 250 cases of congenital FVII deficiency have been reported [7] ; approximately two thirds of the mutations affect the protease domain, indicating that loss of protease function is the commonest cause of the clinical phenotype [8]. Very few reports are available on the mutation profile of Indian patients with congenital FVII deficiency [9,10], Although a rare disorder, yet due to the high incidence of consanguinity in many parts of India, a substantial number of patients are expected with congenital FVII deficiency, who require appropriate management and genetic diagnosis. Characterization of mutations in these patients assumes great significance in the background of rarity of the condition, the clinical diversity and the importance of the FVII in the coagulation cascade.
2. Materials and methods 2.1. Patients Fourteen patients with FVII coagulant activities ranging from <1% to 13%, 8 males (age 2 months–62 y) and 6 females (age 1–45 y) were referred for molecular genetic analysis. The cases were selected only after ruling out the acquired causes i.e. liver dysfunction, malignancy
L. Mota et al. / Clinica Chimica Acta 409 (2009) 106–111
and vitamin K deficiency and other acquired causes of FVII deficiency. The study was approved by the Institutional Ethics Committee. 2.2. Blood collection After obtaining informed consent, the blood samples were collected in 3.18% tri Sodium Citrate (1: 9 anticoagulant to blood). The clinical details were obtained in a well designed clinical proforma. 2.3. Screening coagulation tests Prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT) were performed as described earlier [10] followed by one-stage factor assay using specific deficient plasma. Mixing studies were performed in all the cases to rule out the presence of inhibitors against FVII. 2.4. FVII:C and FVII:Ag assays FVII:C was measured by one-stage assay using commercial deficient plasma (Diagnostic Stago, Asnieres, France) using a semi automated coagulometer (ST Art , Diagnostic Stago). FVII:Ag was assayed by ELISA using commercial kits (Asserachrom FVII:Ag, Diagnostic Stago). 2.5. DNA analysis Genomic DNA was extracted from peripheral blood leukocytes according to standard protocols. Following DNA extraction, the coding region, intron/exon boundaries and the untranslated regions of the FVII gene were amplified by polymerase chain reaction (PCR) and screened for mutations by CSGE as described earlier [9]. Direct sequencing of amplified DNA was performed to confirm the nature of mutation.
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3. Haplotype analysis Haplotypes of FVII were constructed using the following polymorphisms: promoter − 323 decanucleotide insertion/no insertion (A2/A1); Intron 1a 73 (G/A); exon 8 Arg353Gln (M1/M2) and exon 5 His115His (H1/H2). 3.1. Conservation of residues The homologs of human Factor VII (hFVII) were identified using PSI-BLAST algorithm [11]. Proteins, annotated as factor VII and shared full-length similarity with hFVII were selected. The homologs were then subjected to multiple sequence alignment using ClustalW2 [12]. Sequence logo was also constructed for easy visualization of the conservation of residues (Fig. 1) [13]. 3.2. Structural analyses The structure of mature hFVII has been elucidated experimentally using X-ray crystallographic methods with a resolution of 2.0 Å (PDB ID:1dan) [14]. The structures of the mutants were modeled and energy minimized using Swiss-Pdb Viewer [15]. The intra-molecular interactions of the mutant residues were studied using PIC [16] and were compared with the native interactions as present in the wildtype. The surface accessibility of the residues present in hFVII was determined using GETAREA software [17]. 4. Results 4.1. Clinical manifestations Table 1 shows the details of the FVII deficiency cases. Among the 14 cases diagnosed with FVII deficiency, all patients showed mild to
Fig. 1. Sequence logo depicting the conservation of residues in the selected homologs of human Factor VII. Ala191, Leu263, Trp284 and Asp338 are fully conserved amongst the homologs. Ala191, Leu263 and Trp284 residues are also flanked with fully conserved residues suggesting that they might be part of an important motif. Ala191 is in the immediate neighbourhood of His193 which is a constituent of the catalytic triad of serine protease.
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severe bleeding tendencies except 3 patients who were clinically asymptomatic. Of these 3 patients, 2 male patients (ID 8, 10) had major surgeries without any excessive bleeding and the third female patient (ID 12) had 2 uneventful normal vaginal deliveries. Two patients had a family history of bleeding; both patients had a sibling who had died of intra-cranial bleeding. Seven out of 14 (50%) cases had history of first degree consanguinity among parents. 4.2. Phenotypic analysis The phenotypic and the clinical data of the patients are shown in Table 1. Four out of 14 patients were type 1 deficiency with concomitant reduction in both FVII:C and FVII:Ag levels. Remaining 10 patients with discrepant FVII:C and FVII:Ag levels were classified as type 2 FVII deficiency. The FVII:C levels ranged between <1% and 25% while the levels of FVII:Ag ranged from 1. 3% to 130%. 4.3. Mutations detected in FVII deficiency cases Among the 14 FVII deficiency patients analyzed, 11 different mutations were detected out of which five were novel. All were missense mutations, of which nine were homozygotes, 2 heterozygotes and three compound heterozygotes. Five novel mutations were detected i.e. Asp338Glu (ID 6), Ile138Thr (ID 10), Trp284Arg (ID 14), Ala191Pro (ID 1) and Leu263Arg (ID 10, 4, 5) of which the former 2 were found to be present in homozygous state where as the last three in heterozygous states. Cys310Phe mutation was detected in three patients (ID 7,11,12) while Arg152Gln (ID 2, 3) and Thr324Met (ID 8,9) were detected in 2 patients each. Remaining mutations were seen either in homozygous or heterozygous states in different patients. 4.4. Polymorphisms detected in FVII deficiency cases T-122C (rs561241), G73A (rs6039) were 2 polymorphisms found in the promoter region of FVII gene in 2 patients (ID 11, 12). His115His
(C7880T) was observed in homozygous state in 2 patients (ID 8, 14). Arg353His (G10976A), a dimorphism in the catalytic region of the FVII region, was seen in homozygous state in 4 patients (ID 10, 4, 5, 14). 4.5. Haplotype analysis Three haplotypes were seen in 14 patients studied of which the M1M1,A1A1,GG,H1H1 haplotype was most common (n = 10), 2 patients had the M2M2,A1A1,GG,H2H2 haplotype while the remaining 2 patients had M2M2,A1A1,AA,H1H1 haplotype. 4.6. Effect of the mutations on sequence conservation The multiple sequence alignment and sequence logo created using the selected homologs revealed that the residues Ala191, Leu263, Trp284 and Asp338 are fully conserved across these organisms. Out of the 14 sequences, 13 of them have Ile and only 1 of them had Val at position 138. The sequence logo clearly indicates that Ala191, Leu263 and Trp284 all form part of a conserved motif since these residues along with their flanking neighbours are highly conserved in the homologs (Fig. 1). 5. Implications of the mutations on the structural stability and function of hFVII In case of Ile138Thr mutant, it is to be noted that while the wild types have either Ile or Val which are non-polar hydrophobic amino acids, the mutant has Thr, which is a polar and hydrophilic amino acid at the corresponding position. Structural analysis revealed that Ile138 forms a part of a buried hydrophobic core and participates in hydrophobic interactions with Pro165, Trp166 and Trp356 (Fig. 2A). Substitution of Ile138 with Thr138 disrupts these native hydrophobic interactions (Fig. 2B). hFVII is a serine protease and His193 is part of the catalytic triad. Ala191 is involved in non-covalent interactions with His193. Any structural perturbations of the neighbours of the catalytic triad can severely affect the serine protease activity of the protein and hence as
Table 1 Clinical, laboratory and molecular data of FVII deficiency patients. No. Age /Sex
Consangunity Clinical manifestations
FVII: FVII: C Ag
Mutation
Exon Domain
1
16 y/M
No
<1
46
2
2 months/M
Yes
Gum bleed, ecchymosis, hematuria, GI bleeds, haemarthrosis, died of IC bleed Subdural haematoma, sibling died if IC bleed
<1
2
Cys22Arg Ala191Proa Arg152Gln
2 6 6
3
9 months/M
Yes
Umbilical cord bleed, multiple haematomas
75
Arg152Gln
4
3 y/M
No
5
1 y/F
6
5.4
8 a
<1
1.3 Leu263Arg hetero
No
Multiple heamatomas, ankle and knee haemarthrosis, IC bleed Malena, GI bleeds
<1
2
9 y/M
Yes
Epistaxis, sibling died of IC bleed
<1
95
Gly117Arg 6 Leu263Arg heterozygousa 8 Asp338Glua 6
7
19 y/F
No
Menorrhagia
<1
36
Cys310Phe
8
8
62 y/M
No
Asymptomatic, 2 surgeries uneventful
13
36
Thr324Met
8
9
45 y/F
Yes
Easy bruisability
4
70
Thr324Met
8
10
54 y/M
No
Asymptomatic, 3 surgeries uneventful
4
11
25 y/F
Yes
Ecchymosis, menorrhagia
3.3
5.5 Ile138Thr* 6 Leu263Arg heterozygousa 8 23 Cys310Phe 8
12
25 y/F
Yes
Gum bleeds, uneventful vaginal delivery
<1
13
2 y/F
Yes
Gum bleeds, epistaxis, malena
<1
14
6 y/M
No
Epistaxis
2.8
130
8
Cys310Phe
8
5.5 His348Arg 65
8 a
Trp284Arg heterozygous
8
Arg353Gln Arg/Gln (M1/M2), − 323 decanucleotide insertion/no insertion (A2/A1), Intron 1a 73 G to A (G/A), His115His (C7880T) C/T H1/H2. a Novel mutations detected.
Haplotype
Gla Activation M1M1,A1A1, GG,H1H1 Activation M1M1,A1A1, GG,H1H1 Catalytic M1M1,A1A1, GG,H1H1 Catalytic M1M1,A1A1, GG,H1H1 Activation, M1M1,A1A1, Catalytic GG,H1H1 Activation M1M1,A1A1, GG,H1H1 Catalytic M1M1,A1A1, GG,H1H1 Catalytic M2M2,A1A1, GG,H2H2 Catalytic M2M2,A1A1, AA,H1H1 Activation, M1M1,A1A1, Catalytic GG,H1H1 Catalytic M2M2,A1A1, AA,H1H1 Catalytic M1M1,A1A1, GG,H1H1 Catalytic M1M1,A1A1, GG,H1H1 Catalytic M2M2,A1A1, GG,H2H2
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Fig. 2. (A) Ile138 forms a part of a buried hydrophobic core and interacts with Pro165, Trp166 and Trp356. (B) These native hydrophobic interactions are lost in I138T mutant. The hydrophobic and polar residues have been colored grey and pink respectively. Helices, strands and loops are colored as cyan, yellow and green respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
expected along with His193, the flanking residues are also highly conserved in the homologs (Fig. 1). The replacement of Ala191 by Pro191 might disturb the interactions and geometry of catalytic triad and thus lead to loss of protease activity. Structural studies on the Leu263Arg mutant revealed that this mutation would create a steric strain in the protein. Leu263 is present in a loop structure and is surrounded by bulky residues (Fig. 3A). Substitution of Leu with Arg, which has a bulky side chain would bring about steric clashes with the neighbouring residues and thus drastically decrease the stability of the structure (Fig. 3B). Asp338 interacts with Ala369 through a side chain–main chain hydrogen bond (Fig. 4A) whereas in the Asp338Glu mutant, the difference in the lengths of side chains of Asp and Glu results in a shift of the hydrogen bond registry. Glu338 instead forms side chain–main chain hydrogen bond with Gly375 (Fig. 4B). In the wild-type, side chain–main chain hydrogen bonds are also seen between Trp284and His211 (Fig. 5A). In case of the Trp284Arg mutant, substitution of the hydrophobic Trp with a hydrophilic positively charged Arg, creates a non-native ionic interaction between Arg284 and Glu210 (Fig. 5B). 6. Discussion There is generally a broad consensus that in patients with hemophilia bleeding tendency correlates with severity of deficiency. However, this is not true for FVII deficiency. One of the reasons cited
for this is that FVII deficiency when measured as <1% is very often associated with substantial circulating factor VII antigen [18]. Ten out of 14 cases were cross reacting material (CRM) positive; thus prediction of the clinical course of the disease is often difficult in these patients. Except in 3 cases all other patients had severe bleeding manifestations. IC bleeding has been found to a common clinical manifestation in FVII deficient cases observed in as high as 60% of the cases [19]. In the present series, 4 out of 14 patients (29%) had suffered hemorrhage in the brain, out of which 3 could not survive the bleed. Seven out of 14 patients in our study (50%) had a history of consanguinity; slightly lower than that reported in a study from Iran, where 78.6% of the cases history of parental consanguinity [7]. Many marriages in India are endogamous making distant consanguinity a possibility. In Patient (ID 1) a compound heterozygote mutation Cys22Arg and Ala191Pro was detected. Of these 2 mutations the Cys22Arg mutation in exon 2 coding for the Gla domain has previously been reported [20]. Cys17 and Cys22 are conserved residues in vitamin K-dependant coagulation factors forming a disulfide bond for holding together 2 Gla residues in proper position. It also enables capturing the Ca+ 2 which is very important for the proper function. Substitution at this site abolishes this specific function. The expression studies by these authors also reveal absence of secretion of FVII from BHK cells. The other heterozygous mutation Ala191Pro is a novel mutation not reported earlier. Ala191 is a conserved (11 out of 13 related serine proteases) amino acid buried in the hydrophobic core. The structural
Fig. 3. (A) Leu263 in the loop structure along with its structural neighbours. Leu263 is flanked by bulky amino acids. (B) The substitution of Leu by Arg leads to steric hindrance. The residues have been colored as Met (orange), aliphatic residues—Val, Leu, Ile (grey), Arg (red), Gln (pink). Helices, strands and loops are colored as cyan, yellow and green respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. The shift in the hydrogen bond registry in Asp338Glu mutant. (A) Asp338 participates in side chain–main chain hydrogen bonds with Ala369. (B) In the mutant, the difference in the lengths of the side chains of Asp and Glu causes Glu376 to form side chain–main chain hydrogen bond with Gly375. The implication of this non-native hydrogen bond registry needs to be studied further. Helices, strands and loops are colored as cyan, yellow and green respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
analysis reveals that the replacement of Ala191 by Pro191 might disturb the interactions and geometry of catalytic triad and thus leads to the loss of protease activity. Arg152Gln is a recurrent mutation and is found in 2 patients (ID 2 and ID 3). It occurs at a site (Arg 152-Ile 153) that is normally cleaved to generate activated FVII (FVIIa). Analysis of purified FVII from patient plasma showed that FXa and cofactors could not activate the material [21]. A common mutation Cys310Phe was detected in three patients (ID 7, 11 & 12). Cys-310 to Phe, suppresses a disulphide bond conserved in the catalytic domain of all serine proteases also Cys310Phe displays diminished binding to TF and does not activate FX [22].Thr324Met is previously reported in a heterozygous state [23] while in the present study, in both the patients (ID 8, 9) this mutation was in homozygous state with moderately deficient levels of FVII:C and were clinically asymptomatic. Gly117Arg (Patient ID 5) was previously reported in a patient of Indian origin. Extensive sequence comparison in a wide evolutionary context suggested that the Gly117 residue is critical for structure of FVII i.e. the mutation primarily affected the folding / secretion or stability of the protein [24]. His348Arg mutation was previously reported from India as a heterozygous mutation [9]. All the residues in which the novel mutations were detected i.e. Ile138, Ala191, Leu263, Trp284 and Asp338 were highly conserved across the species. Residues that are conserved during evolution generally play a crucial role in the
stability and function of the protein. Hence, it can be safely assumed that any substitution of these fully/highly conserved residues can lead to structural and/or functional loss of the protein. A novel Ile138Thr mutation in homozygous state along with the Leu263Arg heterozygous mutation was detected in patient 10. Surprisingly, this patient was asymptomatic with 3 uneventful surgeries. Structural analyses revealed that Ile138 forms a part of a buried hydrophobic core and participates in hydrophobic interactions with Pro165, Trp166 and Trp356 (Fig. 2A). Substitution of Ile138 with Thr138 disrupts these native hydrophobic interactions (Fig. 2B). The buried hydrophobic residues of a protein are known to play a vital role in the early stages of its folding and stability. Loss of these hydrophobic interactions and replacement of a hydrophobic residue by a polar residue in the buried hydrophobic core may severely destabilize the protein. Despite the predicted severe impact on the functional activity of FVII, the patient has been found to be asymptomatic. Whether there is modulating effect of thrombophilia markers to alleviate the clinical severity as it has been observed in hemophilia [25] in this patient needs to be analyzed. Leu263Arg mutation was seen in heterozygous state in three patients. Of the three patients, 2 were clinically severe with symptoms like intra-cranial and gastrointestinal bleeds along with haemarthrosis (ID 4, 5). Asp338Gln mutation was seen in boy born of consanguineous marriage (ID 6). He had symptoms such as epistaxis and had a strong
Fig. 5. The creation of a non-native ionic bond in W284R mutant. (A) Trp284 interacts with His211 through side chain–main chain hydrogen bond. (B) In the mutant, this hydrogen bond is lost and a non-native ionic interaction occurs between Arg284 with Glu210. The implication of this non-native ionic interaction needs further investigation. Helices, strands and loops are colored as cyan, yellow and green respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
L. Mota et al. / Clinica Chimica Acta 409 (2009) 106–111
family history where an elder sibling had died of IC bleed. The structural analysis shows changes in hydrogen bonding thus causing the severe phenotype. Six different polymorphisms were detected, of which 3 have been previously reported. The Arg353Gln heterozygous polymorphism is known to cause ~20–25% reduction in the FVII:C where as individuals homozygous for this polymorphism have an approximately 50% reduction in circulating plasma FVII levels [26]. There was no evidence for modulation of clinical features by this functional polymorphism in FVII deficient cases [27]; however modulation of clinical severity has been documented for hemophilia [28]. It is possible that they have a similar effect in FVII deficient cases too, however these can be confirmed only by studying a large series of patients. The novel FVII missense mutations reported here occur in amino acid residues that are conserved between the homologous FVII molecules of different organisms. Additional evidence to support the pathological involvement of these lesions comes from molecular modeling. In general, the CRM status of the probands could be related to the location of the mutations within the FVII molecule; thus, CRM+ phenotypes were usually associated with mutations in surface accessible residues and CRM− phenotypes with mutations located deep within the FVII molecule. Another important observation is the relative scarcity of nonsense mutations i.e. all the 11 mutations are missense mutations. A probable explanation for this would be presumably the inherent structure of the F VII gene or the early lethality of homozygous F VII deficiency due to nonsense mutations. The same has been observed in the mutational spectrum of F X gene mutations. In conclusion, the molecular basis of FVII deficiency in a large series of Indian patients has been unraveled and its effect on the function of FVII molecule has been elucidated. The data can now be utilized in genetic diagnosis of the affected families. Acknowledgements We thank the Lady Tata Memorial Trust for the scholarship offered to Ms. Mota to carry out this work. References [1] Osterud B, Rapaport S. Activation of factor IX by the reaction product of tissue factor and factor VII: additional pathway for initiating blood coagulation. Proc Natl Acad Sci U S A 1977;74:5260–4. [2] Peyvandi F, Duga S, Akhavan S, et al. Rare coagulation deficiencies. Hemophilia 2002;8:308–21. [3] Triplett DA, Brandt JT, Batard MA, Dixon JL, Fair DS. Hereditary factor VII deficiency: heterogeneity defined by combined functional and immunochemical analysis. Blood 1985;66:1284–7. [4] Mariani G, Dolce A, Marchetti G, Bernardi F. Clinical picture and management of congenital factor VII deficiency. Hemophilia 2004;10:180–3.
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[5] de Grouchy J, Dautzenberg MD, Turleau C, et al. Regional mapping of clotting factors VII and X to 13q34: expression of factor VII through chromosome 8. Hum Genet 1984;66:230–3. [6] O'hara PJ, Grant FJ, Halderman A, et al. Neocleotide sequence of the gene coding for human Factor VII a vitamin K dependant protein participating in blood coagulation. Proc Natl Acad Sci U S A 1987;84:5158–62. [7] FVII mutation database: europium.csc.mrc.ac.uk. [8] Jayandharan GR, Viswabandya A, Nair SC, et al. Molecular basis of hereditary factor VII deficiency in India: five novel mutations including a double missense mutation (Ala191Glu; Trp364Cys) in 11 unrelated patients. Haematologica 2007;92:1002–3. [9] Ahmed RP, Biswas A, Kannan M, et al. First report of a FVII-deficient Indian patient carrying double heterozygous mutations in the FVII gene. Thromb Res 2005;115: 535–6. [10] Dacie JV, Lewis SM. Practical hematology. 6th edn. Edinburgh: Churchill Livingstone; 1984. p. 230–2. [11] Altschul SF, Madden TL, Schäffer AA, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997;25: 3389–402. [12] Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22: 4673–80. [13] Crooks GE, Hon G, Chandonia JM, Brenner SE. WebLogo: a sequence logo generator. Genome Res 2004;14:1188–90. [14] Banner DW, D'Arcy A, Chène C, et al. The crystal structure of the complex of blood coagulation factor VIIa with soluble tissue factor. Nature 1996;380:41–6. [15] Guex N, Peitsch MC. SWISS-MODEL and the Swiss-Pdb Viewer: an environment for comparative protein modeling. Electrophoresis 1997;18:2714–23. [16] Tina KG, Bhadra R, Srinivasan N. PIC: Protein Interactions Calculator. Nucleic Acids Res 2007;35:W473–6. [17] Fraczkiewicz R, Braun W. Exact and efficient analytical calculation of the accessible surface areas and their gradients for macromolecules. J Comp Chem 1998;19:319–33. [18] Rapaport SI, Rao LV. The tissue factor pathway: how it has become a “prima ballerina”. Thromb Hemost 1995;74:7–17. [19] Peyvandi F, Mannucci PM, Asti D, et al. Clinical manifestations in 28 Italian and Iranian patients with severe factor VII deficiency. Hemophilia 1997;3:242–6. [20] Fromovich-Amit Y, Zivelin A, Rosenberg N, et al. Characterization of mutations causing factor VII deficiency in 61 unrelated Israeli patients. J Thromb Hemost 2004;2:1774–81. [21] Chaing S, Clarke B, Sridhara S, et al. Severe factor VII deficiency caused by mutations abolishing the cleavage site for activation and altering binding to tissue factor. Blood 1994;83:3524–35. [22] Giansily-Blaizot M, Verdier R, Biron-Adréani C, et al. Analysis of biological phenotypes from 42 patients with inherited factor VII deficiency: can biological tests predict the bleeding risk? Haematologica 2004;89:704–9. [23] Gomez K, Laffan MA, Kemball-Cook G, et al. Two novel mutations in severe factor VII deficiency. Br J Haematol 2004;126:105–10. [24] Sauer RT, Milla ME, Waldburger CD, et al. Sequence determinants of folding and stability for the P22 Arc repressor dimer. FASEB J 1996;10:42–8. [25] Shetty S, Vora S, Kulkarni B, et al. Contribution of natural anticoagulant and fibrinolytic factors in modulating the clinical severity of hemophilia patients. Br J Haematol 2007;138:541–4. [26] Arbini AA, Bodkin D, Lopaciuk S, Bauer KA. Molecular analysis of Polish patients with factor VII deficiency. Blood 1994;84:2214–20. [27] Mariani G, Herrmann FH, Dolce A, et al. Clinical phenotypes and factor VII genotype in congenital factor VII deficiency. Thromb Hemost 2005;93:481–7. [28] G.R. Jayandharan, S.C. Nair, P.M. Poonnoose, et al. Polymorphism in factor VII gene modifies phenotype of severe hemophilia. Hemophilia (in press).
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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Phenotypic and genotypic characterization of Factor VII deficiency patients from Western India Leenam Mota a, Shrimati Shetty a, Susan Idicula-Thomas b, Kanjaksha Ghosh a,⁎ a b
National Institute of Immunohaematology, 13th floor of KEM Hospital Campus, Parel, Mumbai, 400012, India Biomedical Informatics Center of Indian Council of Medical Research, National Institute for Research in Reproductive Health, Parel, Mumbai, 400012, India
a r t i c l e
i n f o
Article history: Received 13 July 2009 Received in revised form 4 September 2009 Accepted 4 September 2009 Available online 13 September 2009 Keywords: Factor VII deficiency Mutations India
a b s t r a c t Background: Congenital factor VII (FVII) deficiency is a rare coagulation deficiency caused due to defects in the FVII gene. Methods: We analyzed 14 unrelated Indian patients with congenital FVII deficiency for mutations in FVII gene by conformation sensitive gel electrophoresis (CSGE) followed by DNA sequencing. Results: A total of 11 different missense mutations were identified, of which 5 were novel (Ala191Pro, Asp338Glu, Ile138Thr, Leu263Arg and Trp284Arg) and 6 had been previously reported (Cys22Arg, Arg152Gln, Cys310Phe, Thr324Met, Gly117Arg and His348Arg). Six of the 11 mutations were located in the catalytic serine protease domain, 3 in the activation domain and 1 each in the Gla and the second epidermal growth factor domain respectively. Multiple sequence alignment using ClustalW2 analysis showed that all the mutations were found in residues that are highly conserved across species. Implications of mutations on the structural stability and function of human factor VIII (hFVII) using Swiss-Pdb Viewer and the intra-molecular interactions of the mutant residues using PIC showed that there is a structural instability in all the mutants either by steric hindrance or instability in the protein molecule folding. Conclusion: A wide spectrum of mutations was detected in the FVII gene; the presence of 6 out of 11 mutations in the serine protease domain suggests the crucial role of catalytic domain in FVII functional activity. © 2009 Elsevier B.V. All rights reserved.
1. Introduction FVII is a vitamin K-dependant serine protease synthesized in the liver and occupies a very important position in the coagulation cascade. Normal hemostasis is initiated by the formation of a complex between tissue factor (TF) and FVII in the circulating blood [1]. FVII deficiency is a rare, autosomally inherited, recessive disorder which affects approximately 1 in 500,000 of the population [2]. The clinical features are quite variable in patients with FVII deficiency. Reports from all over the world have highlighted the lack of correlation between the measured factor level and the clinical manifestation [3]. Common clinical manifestations include epistaxis, gum bleeding and prolonged bleeding from cuts and injuries. Menorrhagia and chronic iron deficiency are frequently seen in homozygous women with FVII deficiency, the prevalence being as high as 60% and a similar prevalence has been reported for central nervous system (CNS) bleeding [4]. The FVII gene is located on chromosome 13 (13q34), consists of 9 exons and 8 introns and is approximately 12 kb in size [5,6] encoding a mature protein of 406 amino acids. The FVII protein consists of an
⁎ Corresponding author. Tel.: +91 22 24138518/19; fax: +91 22 24138521. E-mail address: [email protected] (K. Ghosh). 0009-8981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2009.09.007
amino-terminal (light chain) Gla domain, carboxy-terminal (heavy chain) catalytic domain and 2 epidermal growth factor domains. To date about 250 cases of congenital FVII deficiency have been reported [7] ; approximately two thirds of the mutations affect the protease domain, indicating that loss of protease function is the commonest cause of the clinical phenotype [8]. Very few reports are available on the mutation profile of Indian patients with congenital FVII deficiency [9,10], Although a rare disorder, yet due to the high incidence of consanguinity in many parts of India, a substantial number of patients are expected with congenital FVII deficiency, who require appropriate management and genetic diagnosis. Characterization of mutations in these patients assumes great significance in the background of rarity of the condition, the clinical diversity and the importance of the FVII in the coagulation cascade.
2. Materials and methods 2.1. Patients Fourteen patients with FVII coagulant activities ranging from <1% to 13%, 8 males (age 2 months–62 y) and 6 females (age 1–45 y) were referred for molecular genetic analysis. The cases were selected only after ruling out the acquired causes i.e. liver dysfunction, malignancy
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and vitamin K deficiency and other acquired causes of FVII deficiency. The study was approved by the Institutional Ethics Committee. 2.2. Blood collection After obtaining informed consent, the blood samples were collected in 3.18% tri Sodium Citrate (1: 9 anticoagulant to blood). The clinical details were obtained in a well designed clinical proforma. 2.3. Screening coagulation tests Prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT) were performed as described earlier [10] followed by one-stage factor assay using specific deficient plasma. Mixing studies were performed in all the cases to rule out the presence of inhibitors against FVII. 2.4. FVII:C and FVII:Ag assays FVII:C was measured by one-stage assay using commercial deficient plasma (Diagnostic Stago, Asnieres, France) using a semi automated coagulometer (ST Art , Diagnostic Stago). FVII:Ag was assayed by ELISA using commercial kits (Asserachrom FVII:Ag, Diagnostic Stago). 2.5. DNA analysis Genomic DNA was extracted from peripheral blood leukocytes according to standard protocols. Following DNA extraction, the coding region, intron/exon boundaries and the untranslated regions of the FVII gene were amplified by polymerase chain reaction (PCR) and screened for mutations by CSGE as described earlier [9]. Direct sequencing of amplified DNA was performed to confirm the nature of mutation.
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3. Haplotype analysis Haplotypes of FVII were constructed using the following polymorphisms: promoter − 323 decanucleotide insertion/no insertion (A2/A1); Intron 1a 73 (G/A); exon 8 Arg353Gln (M1/M2) and exon 5 His115His (H1/H2). 3.1. Conservation of residues The homologs of human Factor VII (hFVII) were identified using PSI-BLAST algorithm [11]. Proteins, annotated as factor VII and shared full-length similarity with hFVII were selected. The homologs were then subjected to multiple sequence alignment using ClustalW2 [12]. Sequence logo was also constructed for easy visualization of the conservation of residues (Fig. 1) [13]. 3.2. Structural analyses The structure of mature hFVII has been elucidated experimentally using X-ray crystallographic methods with a resolution of 2.0 Å (PDB ID:1dan) [14]. The structures of the mutants were modeled and energy minimized using Swiss-Pdb Viewer [15]. The intra-molecular interactions of the mutant residues were studied using PIC [16] and were compared with the native interactions as present in the wildtype. The surface accessibility of the residues present in hFVII was determined using GETAREA software [17]. 4. Results 4.1. Clinical manifestations Table 1 shows the details of the FVII deficiency cases. Among the 14 cases diagnosed with FVII deficiency, all patients showed mild to
Fig. 1. Sequence logo depicting the conservation of residues in the selected homologs of human Factor VII. Ala191, Leu263, Trp284 and Asp338 are fully conserved amongst the homologs. Ala191, Leu263 and Trp284 residues are also flanked with fully conserved residues suggesting that they might be part of an important motif. Ala191 is in the immediate neighbourhood of His193 which is a constituent of the catalytic triad of serine protease.
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severe bleeding tendencies except 3 patients who were clinically asymptomatic. Of these 3 patients, 2 male patients (ID 8, 10) had major surgeries without any excessive bleeding and the third female patient (ID 12) had 2 uneventful normal vaginal deliveries. Two patients had a family history of bleeding; both patients had a sibling who had died of intra-cranial bleeding. Seven out of 14 (50%) cases had history of first degree consanguinity among parents. 4.2. Phenotypic analysis The phenotypic and the clinical data of the patients are shown in Table 1. Four out of 14 patients were type 1 deficiency with concomitant reduction in both FVII:C and FVII:Ag levels. Remaining 10 patients with discrepant FVII:C and FVII:Ag levels were classified as type 2 FVII deficiency. The FVII:C levels ranged between <1% and 25% while the levels of FVII:Ag ranged from 1. 3% to 130%. 4.3. Mutations detected in FVII deficiency cases Among the 14 FVII deficiency patients analyzed, 11 different mutations were detected out of which five were novel. All were missense mutations, of which nine were homozygotes, 2 heterozygotes and three compound heterozygotes. Five novel mutations were detected i.e. Asp338Glu (ID 6), Ile138Thr (ID 10), Trp284Arg (ID 14), Ala191Pro (ID 1) and Leu263Arg (ID 10, 4, 5) of which the former 2 were found to be present in homozygous state where as the last three in heterozygous states. Cys310Phe mutation was detected in three patients (ID 7,11,12) while Arg152Gln (ID 2, 3) and Thr324Met (ID 8,9) were detected in 2 patients each. Remaining mutations were seen either in homozygous or heterozygous states in different patients. 4.4. Polymorphisms detected in FVII deficiency cases T-122C (rs561241), G73A (rs6039) were 2 polymorphisms found in the promoter region of FVII gene in 2 patients (ID 11, 12). His115His
(C7880T) was observed in homozygous state in 2 patients (ID 8, 14). Arg353His (G10976A), a dimorphism in the catalytic region of the FVII region, was seen in homozygous state in 4 patients (ID 10, 4, 5, 14). 4.5. Haplotype analysis Three haplotypes were seen in 14 patients studied of which the M1M1,A1A1,GG,H1H1 haplotype was most common (n = 10), 2 patients had the M2M2,A1A1,GG,H2H2 haplotype while the remaining 2 patients had M2M2,A1A1,AA,H1H1 haplotype. 4.6. Effect of the mutations on sequence conservation The multiple sequence alignment and sequence logo created using the selected homologs revealed that the residues Ala191, Leu263, Trp284 and Asp338 are fully conserved across these organisms. Out of the 14 sequences, 13 of them have Ile and only 1 of them had Val at position 138. The sequence logo clearly indicates that Ala191, Leu263 and Trp284 all form part of a conserved motif since these residues along with their flanking neighbours are highly conserved in the homologs (Fig. 1). 5. Implications of the mutations on the structural stability and function of hFVII In case of Ile138Thr mutant, it is to be noted that while the wild types have either Ile or Val which are non-polar hydrophobic amino acids, the mutant has Thr, which is a polar and hydrophilic amino acid at the corresponding position. Structural analysis revealed that Ile138 forms a part of a buried hydrophobic core and participates in hydrophobic interactions with Pro165, Trp166 and Trp356 (Fig. 2A). Substitution of Ile138 with Thr138 disrupts these native hydrophobic interactions (Fig. 2B). hFVII is a serine protease and His193 is part of the catalytic triad. Ala191 is involved in non-covalent interactions with His193. Any structural perturbations of the neighbours of the catalytic triad can severely affect the serine protease activity of the protein and hence as
Table 1 Clinical, laboratory and molecular data of FVII deficiency patients. No. Age /Sex
Consangunity Clinical manifestations
FVII: FVII: C Ag
Mutation
Exon Domain
1
16 y/M
No
<1
46
2
2 months/M
Yes
Gum bleed, ecchymosis, hematuria, GI bleeds, haemarthrosis, died of IC bleed Subdural haematoma, sibling died if IC bleed
<1
2
Cys22Arg Ala191Proa Arg152Gln
2 6 6
3
9 months/M
Yes
Umbilical cord bleed, multiple haematomas
75
Arg152Gln
4
3 y/M
No
5
1 y/F
6
5.4
8 a
<1
1.3 Leu263Arg hetero
No
Multiple heamatomas, ankle and knee haemarthrosis, IC bleed Malena, GI bleeds
<1
2
9 y/M
Yes
Epistaxis, sibling died of IC bleed
<1
95
Gly117Arg 6 Leu263Arg heterozygousa 8 Asp338Glua 6
7
19 y/F
No
Menorrhagia
<1
36
Cys310Phe
8
8
62 y/M
No
Asymptomatic, 2 surgeries uneventful
13
36
Thr324Met
8
9
45 y/F
Yes
Easy bruisability
4
70
Thr324Met
8
10
54 y/M
No
Asymptomatic, 3 surgeries uneventful
4
11
25 y/F
Yes
Ecchymosis, menorrhagia
3.3
5.5 Ile138Thr* 6 Leu263Arg heterozygousa 8 23 Cys310Phe 8
12
25 y/F
Yes
Gum bleeds, uneventful vaginal delivery
<1
13
2 y/F
Yes
Gum bleeds, epistaxis, malena
<1
14
6 y/M
No
Epistaxis
2.8
130
8
Cys310Phe
8
5.5 His348Arg 65
8 a
Trp284Arg heterozygous
8
Arg353Gln Arg/Gln (M1/M2), − 323 decanucleotide insertion/no insertion (A2/A1), Intron 1a 73 G to A (G/A), His115His (C7880T) C/T H1/H2. a Novel mutations detected.
Haplotype
Gla Activation M1M1,A1A1, GG,H1H1 Activation M1M1,A1A1, GG,H1H1 Catalytic M1M1,A1A1, GG,H1H1 Catalytic M1M1,A1A1, GG,H1H1 Activation, M1M1,A1A1, Catalytic GG,H1H1 Activation M1M1,A1A1, GG,H1H1 Catalytic M1M1,A1A1, GG,H1H1 Catalytic M2M2,A1A1, GG,H2H2 Catalytic M2M2,A1A1, AA,H1H1 Activation, M1M1,A1A1, Catalytic GG,H1H1 Catalytic M2M2,A1A1, AA,H1H1 Catalytic M1M1,A1A1, GG,H1H1 Catalytic M1M1,A1A1, GG,H1H1 Catalytic M2M2,A1A1, GG,H2H2
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Fig. 2. (A) Ile138 forms a part of a buried hydrophobic core and interacts with Pro165, Trp166 and Trp356. (B) These native hydrophobic interactions are lost in I138T mutant. The hydrophobic and polar residues have been colored grey and pink respectively. Helices, strands and loops are colored as cyan, yellow and green respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
expected along with His193, the flanking residues are also highly conserved in the homologs (Fig. 1). The replacement of Ala191 by Pro191 might disturb the interactions and geometry of catalytic triad and thus lead to loss of protease activity. Structural studies on the Leu263Arg mutant revealed that this mutation would create a steric strain in the protein. Leu263 is present in a loop structure and is surrounded by bulky residues (Fig. 3A). Substitution of Leu with Arg, which has a bulky side chain would bring about steric clashes with the neighbouring residues and thus drastically decrease the stability of the structure (Fig. 3B). Asp338 interacts with Ala369 through a side chain–main chain hydrogen bond (Fig. 4A) whereas in the Asp338Glu mutant, the difference in the lengths of side chains of Asp and Glu results in a shift of the hydrogen bond registry. Glu338 instead forms side chain–main chain hydrogen bond with Gly375 (Fig. 4B). In the wild-type, side chain–main chain hydrogen bonds are also seen between Trp284and His211 (Fig. 5A). In case of the Trp284Arg mutant, substitution of the hydrophobic Trp with a hydrophilic positively charged Arg, creates a non-native ionic interaction between Arg284 and Glu210 (Fig. 5B). 6. Discussion There is generally a broad consensus that in patients with hemophilia bleeding tendency correlates with severity of deficiency. However, this is not true for FVII deficiency. One of the reasons cited
for this is that FVII deficiency when measured as <1% is very often associated with substantial circulating factor VII antigen [18]. Ten out of 14 cases were cross reacting material (CRM) positive; thus prediction of the clinical course of the disease is often difficult in these patients. Except in 3 cases all other patients had severe bleeding manifestations. IC bleeding has been found to a common clinical manifestation in FVII deficient cases observed in as high as 60% of the cases [19]. In the present series, 4 out of 14 patients (29%) had suffered hemorrhage in the brain, out of which 3 could not survive the bleed. Seven out of 14 patients in our study (50%) had a history of consanguinity; slightly lower than that reported in a study from Iran, where 78.6% of the cases history of parental consanguinity [7]. Many marriages in India are endogamous making distant consanguinity a possibility. In Patient (ID 1) a compound heterozygote mutation Cys22Arg and Ala191Pro was detected. Of these 2 mutations the Cys22Arg mutation in exon 2 coding for the Gla domain has previously been reported [20]. Cys17 and Cys22 are conserved residues in vitamin K-dependant coagulation factors forming a disulfide bond for holding together 2 Gla residues in proper position. It also enables capturing the Ca+ 2 which is very important for the proper function. Substitution at this site abolishes this specific function. The expression studies by these authors also reveal absence of secretion of FVII from BHK cells. The other heterozygous mutation Ala191Pro is a novel mutation not reported earlier. Ala191 is a conserved (11 out of 13 related serine proteases) amino acid buried in the hydrophobic core. The structural
Fig. 3. (A) Leu263 in the loop structure along with its structural neighbours. Leu263 is flanked by bulky amino acids. (B) The substitution of Leu by Arg leads to steric hindrance. The residues have been colored as Met (orange), aliphatic residues—Val, Leu, Ile (grey), Arg (red), Gln (pink). Helices, strands and loops are colored as cyan, yellow and green respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. The shift in the hydrogen bond registry in Asp338Glu mutant. (A) Asp338 participates in side chain–main chain hydrogen bonds with Ala369. (B) In the mutant, the difference in the lengths of the side chains of Asp and Glu causes Glu376 to form side chain–main chain hydrogen bond with Gly375. The implication of this non-native hydrogen bond registry needs to be studied further. Helices, strands and loops are colored as cyan, yellow and green respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
analysis reveals that the replacement of Ala191 by Pro191 might disturb the interactions and geometry of catalytic triad and thus leads to the loss of protease activity. Arg152Gln is a recurrent mutation and is found in 2 patients (ID 2 and ID 3). It occurs at a site (Arg 152-Ile 153) that is normally cleaved to generate activated FVII (FVIIa). Analysis of purified FVII from patient plasma showed that FXa and cofactors could not activate the material [21]. A common mutation Cys310Phe was detected in three patients (ID 7, 11 & 12). Cys-310 to Phe, suppresses a disulphide bond conserved in the catalytic domain of all serine proteases also Cys310Phe displays diminished binding to TF and does not activate FX [22].Thr324Met is previously reported in a heterozygous state [23] while in the present study, in both the patients (ID 8, 9) this mutation was in homozygous state with moderately deficient levels of FVII:C and were clinically asymptomatic. Gly117Arg (Patient ID 5) was previously reported in a patient of Indian origin. Extensive sequence comparison in a wide evolutionary context suggested that the Gly117 residue is critical for structure of FVII i.e. the mutation primarily affected the folding / secretion or stability of the protein [24]. His348Arg mutation was previously reported from India as a heterozygous mutation [9]. All the residues in which the novel mutations were detected i.e. Ile138, Ala191, Leu263, Trp284 and Asp338 were highly conserved across the species. Residues that are conserved during evolution generally play a crucial role in the
stability and function of the protein. Hence, it can be safely assumed that any substitution of these fully/highly conserved residues can lead to structural and/or functional loss of the protein. A novel Ile138Thr mutation in homozygous state along with the Leu263Arg heterozygous mutation was detected in patient 10. Surprisingly, this patient was asymptomatic with 3 uneventful surgeries. Structural analyses revealed that Ile138 forms a part of a buried hydrophobic core and participates in hydrophobic interactions with Pro165, Trp166 and Trp356 (Fig. 2A). Substitution of Ile138 with Thr138 disrupts these native hydrophobic interactions (Fig. 2B). The buried hydrophobic residues of a protein are known to play a vital role in the early stages of its folding and stability. Loss of these hydrophobic interactions and replacement of a hydrophobic residue by a polar residue in the buried hydrophobic core may severely destabilize the protein. Despite the predicted severe impact on the functional activity of FVII, the patient has been found to be asymptomatic. Whether there is modulating effect of thrombophilia markers to alleviate the clinical severity as it has been observed in hemophilia [25] in this patient needs to be analyzed. Leu263Arg mutation was seen in heterozygous state in three patients. Of the three patients, 2 were clinically severe with symptoms like intra-cranial and gastrointestinal bleeds along with haemarthrosis (ID 4, 5). Asp338Gln mutation was seen in boy born of consanguineous marriage (ID 6). He had symptoms such as epistaxis and had a strong
Fig. 5. The creation of a non-native ionic bond in W284R mutant. (A) Trp284 interacts with His211 through side chain–main chain hydrogen bond. (B) In the mutant, this hydrogen bond is lost and a non-native ionic interaction occurs between Arg284 with Glu210. The implication of this non-native ionic interaction needs further investigation. Helices, strands and loops are colored as cyan, yellow and green respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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family history where an elder sibling had died of IC bleed. The structural analysis shows changes in hydrogen bonding thus causing the severe phenotype. Six different polymorphisms were detected, of which 3 have been previously reported. The Arg353Gln heterozygous polymorphism is known to cause ~20–25% reduction in the FVII:C where as individuals homozygous for this polymorphism have an approximately 50% reduction in circulating plasma FVII levels [26]. There was no evidence for modulation of clinical features by this functional polymorphism in FVII deficient cases [27]; however modulation of clinical severity has been documented for hemophilia [28]. It is possible that they have a similar effect in FVII deficient cases too, however these can be confirmed only by studying a large series of patients. The novel FVII missense mutations reported here occur in amino acid residues that are conserved between the homologous FVII molecules of different organisms. Additional evidence to support the pathological involvement of these lesions comes from molecular modeling. In general, the CRM status of the probands could be related to the location of the mutations within the FVII molecule; thus, CRM+ phenotypes were usually associated with mutations in surface accessible residues and CRM− phenotypes with mutations located deep within the FVII molecule. Another important observation is the relative scarcity of nonsense mutations i.e. all the 11 mutations are missense mutations. A probable explanation for this would be presumably the inherent structure of the F VII gene or the early lethality of homozygous F VII deficiency due to nonsense mutations. The same has been observed in the mutational spectrum of F X gene mutations. In conclusion, the molecular basis of FVII deficiency in a large series of Indian patients has been unraveled and its effect on the function of FVII molecule has been elucidated. The data can now be utilized in genetic diagnosis of the affected families. Acknowledgements We thank the Lady Tata Memorial Trust for the scholarship offered to Ms. Mota to carry out this work. References [1] Osterud B, Rapaport S. Activation of factor IX by the reaction product of tissue factor and factor VII: additional pathway for initiating blood coagulation. Proc Natl Acad Sci U S A 1977;74:5260–4. [2] Peyvandi F, Duga S, Akhavan S, et al. Rare coagulation deficiencies. Hemophilia 2002;8:308–21. [3] Triplett DA, Brandt JT, Batard MA, Dixon JL, Fair DS. Hereditary factor VII deficiency: heterogeneity defined by combined functional and immunochemical analysis. Blood 1985;66:1284–7. [4] Mariani G, Dolce A, Marchetti G, Bernardi F. Clinical picture and management of congenital factor VII deficiency. Hemophilia 2004;10:180–3.
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