Clinica Chimica Acta 409 (2009) 106–111

Contents lists available at ScienceDirect

Clinica Chimica Acta

j ourna l homepage: www.e lsev ie r.com/ locate /c l inch im

Phenotypic and genotypic characterization of Factor VII deficiency patients fromWestern India

Leenam Mota a, Shrimati Shetty a, Susan Idicula-Thomas b, Kanjaksha Ghosh a,⁎a National Institute of Immunohaematology, 13th floor of KEM Hospital Campus, Parel, Mumbai, 400012, Indiab Biomedical Informatics Center of Indian Council of Medical Research, National Institute for Research in Reproductive Health, Parel, Mumbai, 400012, India

⁎ Corresponding author. Tel.: +91 22 24138518/19;E-mail address: kanjakshaghosh@hotmail.com (K. G

0009-8981/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.cca.2009.09.007

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 13 July 2009Received in revised form 4 September 2009Accepted 4 September 2009Available online 13 September 2009

Keywords:Factor VII deficiencyMutationsIndia

Background: Congenital factor VII (FVII) deficiency is a rare coagulation deficiency caused due to defects inthe FVII gene.Methods:We analyzed 14 unrelated Indian patients with congenital FVII deficiency for mutations in FVII geneby conformation sensitive gel electrophoresis (CSGE) followed by DNA sequencing.Results: A total of 11differentmissensemutationswere identified, ofwhich 5werenovel (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 proteasedomain, 3 in the activation domain and 1 each in the Gla and the second epidermal growth factor domainrespectively.Multiple sequence alignment using ClustalW2 analysis showed that all themutationswere found in

residues that are highly conserved across species. Implications of mutations on the structural stability andfunction of human factor VIII (hFVII) using Swiss-Pdb Viewer and the intra-molecular interactions of themutantresidues using PIC showed that there is a structural instability in all the mutants either by steric hindrance orinstability in the protein molecule folding.Conclusion:Awide spectrum ofmutationswas detected in the FVII gene; the presence of 6 out of 11mutations inthe 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 theliver and occupies a very important position in the coagulationcascade. Normal hemostasis is initiated by the formation of a complexbetween tissue factor (TF) and FVII in the circulating blood [1].

FVII deficiency is a rare, autosomally inherited, recessive disorderwhich affects approximately 1 in 500,000 of the population [2]. Theclinical features are quite variable in patients with FVII deficiency.Reports from all over the world have highlighted the lack ofcorrelation between the measured factor level and the clinicalmanifestation [3]. Common clinical manifestations include epistaxis,gum bleeding and prolonged bleeding from cuts and injuries.Menorrhagia and chronic iron deficiency are frequently seen inhomozygous women with FVII deficiency, the prevalence being ashigh as 60% and a similar prevalence has been reported for centralnervous system (CNS) bleeding [4].

The FVII gene is located on chromosome 13 (13q34), consists of 9exons and 8 introns and is approximately 12 kb in size [5,6] encodinga mature protein of 406 amino acids. The FVII protein consists of an

fax: +91 22 24138521.hosh).

ll rights reserved.

amino-terminal (light chain) Gla domain, carboxy-terminal (heavychain) catalytic domain and 2 epidermal growth factor domains. Todate about 250 cases of congenital FVII deficiency have been reported[7] ; approximately two thirds of the mutations affect the proteasedomain, indicating that loss of protease function is the commonestcause of the clinical phenotype [8].

Very few reports are available on the mutation profile of Indianpatients with congenital FVII deficiency [9,10], Although a rare disorder,yet due to the high incidence of consanguinity in many parts of India, asubstantial number of patients are expected with congenital FVIIdeficiency, who require appropriate management and genetic diagno-sis. Characterization of mutations in these patients assumes greatsignificance in the background of rarity of the condition, the clinicaldiversity 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) werereferred for molecular genetic analysis. The cases were selected onlyafter ruling out the acquired causes i.e. liver dysfunction, malignancy

107L. 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 collectedin 3.18% tri Sodium Citrate (1: 9 anticoagulant to blood). The clinicaldetails 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 deficientplasma. Mixing studies were performed in all the cases to rule outthe presence of inhibitors against FVII.

2.4. FVII:C and FVII:Ag assays

FVII:C wasmeasured by one-stage assay using commercial deficientplasma (Diagnostic Stago, Asnieres, France) using a semi automatedcoagulometer (ST Art , Diagnostic Stago). FVII:Ag was assayed by ELISAusing commercial kits (Asserachrom FVII:Ag, Diagnostic Stago).

2.5. DNA analysis

Genomic DNA was extracted from peripheral blood leukocytesaccording to standard protocols. Following DNA extraction, the codingregion, intron/exon boundaries and the untranslated regions of the FVIIgene were amplified by polymerase chain reaction (PCR) and screenedfor mutations by CSGE as described earlier [9]. Direct sequencing ofamplified DNA was performed to confirm the nature of mutation.

Fig. 1. Sequence logo depicting the conservation of residues in the selected homologs ofhomologs. Ala191, Leu263 and Trp284 residues are also flanked with fully conserved residuesneighbourhood of His193 which is a constituent of the catalytic triad of serine protease.

3. Haplotype analysis

Haplotypes of FVII were constructed using the following poly-morphisms: promoter −323 decanucleotide insertion/no insertion(A2/A1); Intron 1a 73 (G/A); exon 8 Arg353Gln (M1/M2) and exon 5His115His (H1/H2).

3.1. Conservation of residues

The homologs of human Factor VII (hFVII) were identified usingPSI-BLAST algorithm [11]. Proteins, annotated as factor VII and sharedfull-length similarity with hFVII were selected. The homologs werethen subjected to multiple sequence alignment using ClustalW2 [12].Sequence logo was also constructed for easy visualization of theconservation of residues (Fig. 1) [13].

3.2. Structural analyses

The structure of mature hFVII has been elucidated experimentallyusing X-ray crystallographic methods with a resolution of 2.0 Å (PDBID:1dan) [14]. The structures of the mutants were modeled andenergy minimized using Swiss-Pdb Viewer [15]. The intra-molecularinteractions of the mutant residues were studied using PIC [16] andwere compared with the native interactions as present in the wild-type. The surface accessibility of the residues present in hFVII wasdetermined using GETAREA software [17].

4. Results

4.1. Clinical manifestations

Table 1 shows the details of the FVII deficiency cases. Among the14 cases diagnosed with FVII deficiency, all patients showed mild to

human Factor VII. Ala191, Leu263, Trp284 and Asp338 are fully conserved amongst thesuggesting that they might be part of an important motif. Ala191 is in the immediate

108 L. Mota et al. / Clinica Chimica Acta 409 (2009) 106–111

severe bleeding tendencies except 3 patients who were clinicallyasymptomatic. Of these 3 patients, 2 male patients (ID 8, 10) hadmajor surgeries without any excessive bleeding and the third femalepatient (ID 12) had 2 uneventful normal vaginal deliveries. Twopatients had a family history of bleeding; both patients had a siblingwho had died of intra-cranial bleeding. Seven out of 14 (50%) caseshad history of first degree consanguinity among parents.

4.2. Phenotypic analysis

The phenotypic and the clinical data of the patients are shown inTable 1. Four out of 14 patients were type 1 deficiency withconcomitant reduction in both FVII:C and FVII:Ag levels. Remaining10 patients with discrepant FVII:C and FVII:Ag levels were classified astype 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 differentmutations were detected out of which five were novel. All weremissense mutations, of which nine were homozygotes, 2 hetero-zygotes and three compound heterozygotes. Five novel mutationswere detected i.e. Asp338Glu (ID 6), Ile138Thr (ID 10), Trp284Arg(ID 14), Ala191Pro (ID 1) and Leu263Arg (ID 10, 4, 5) of which theformer 2 were found to be present in homozygous state where as thelast 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 2patients each. Remaining mutations were seen either in homozygousor heterozygous states in different patients.

4.4. Polymorphisms detected in FVII deficiency cases

T-122C (rs561241), G73A (rs6039) were 2 polymorphisms foundin the promoter region of FVII gene in 2 patients (ID 11, 12). His115His

Table 1Clinical, laboratory and molecular data of FVII deficiency patients.

No. Age /Sex Consangunity Clinical manifestations

1 16 y/M No Gum bleed, ecchymosis, hematuria, GI bleeds,haemarthrosis, died of IC bleed

2 2 months/M Yes Subdural haematoma, sibling died if IC bleed

3 9 months/M Yes Umbilical cord bleed, multiple haematomas

4 3 y/M No Multiple heamatomas, ankle and knee haemarthrosisIC bleed

5 1 y/F No Malena, GI bleeds

6 9 y/M Yes Epistaxis, sibling died of IC bleed

7 19 y/F No Menorrhagia

8 62 y/M No Asymptomatic, 2 surgeries uneventful

9 45 y/F Yes Easy bruisability

10 54 y/M No Asymptomatic, 3 surgeries uneventful

11 25 y/F Yes Ecchymosis, menorrhagia

12 25 y/F Yes Gum bleeds, uneventful vaginal delivery

13 2 y/F Yes Gum bleeds, epistaxis, malena

14 6 y/M No Epistaxis

Arg353Gln Arg/Gln (M1/M2), −323 decanucleotide insertion/no insertion (A2/A1), Introna Novel mutations detected.

(C7880T) was observed in homozygous state in 2 patients (ID 8, 14).Arg353His (G10976A), a dimorphism in the catalytic region of the FVIIregion, 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 theM1M1,A1A1,GG,H1H1 haplotype was most common (n=10), 2patients had the M2M2,A1A1,GG,H2H2 haplotype while the remain-ing 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 usingthe selected homologs revealed that the residues Ala191, Leu263, Trp284

and Asp338 are fully conserved across these organisms. Out of the 14sequences, 13 of them have Ile and only 1 of them had Val at position138. The sequence logo clearly indicates that Ala191, Leu263 and Trp284

all form part of a conservedmotif since these residues alongwith theirflanking neighbours are highly conserved in the homologs (Fig. 1).

5. Implications of the mutations on the structural stability andfunction of hFVII

In case of Ile138Thrmutant, it is to be noted thatwhile thewild typeshave either Ile or Val which are non-polar hydrophobic amino acids, themutant has Thr, which is a polar and hydrophilic amino acid at thecorresponding position. Structural analysis revealed that Ile138 forms apart of a buried hydrophobic core and participates in hydrophobicinteractionswith Pro165, Trp166 andTrp356 (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. Anystructural perturbations of the neighbours of the catalytic triad canseverely affect the serine protease activity of the protein and hence as

FVII:C

FVII:Ag

Mutation Exon Domain Haplotype

<1 46 Cys22ArgAla191Proa

26

Gla Activation M1M1,A1A1,GG,H1H1

<1 2 Arg152Gln 6 Activation M1M1,A1A1,GG,H1H1

5.4 75 Arg152Gln 8 Catalytic M1M1,A1A1,GG,H1H1

, <1 1.3 Leu263Arg heteroa 8 Catalytic M1M1,A1A1,GG,H1H1

<1 2 Gly117ArgLeu263Arg heterozygousa

68

Activation,Catalytic

M1M1,A1A1,GG,H1H1

<1 95 Asp338Glua 6 Activation M1M1,A1A1,GG,H1H1

<1 36 Cys310Phe 8 Catalytic M1M1,A1A1,GG,H1H1

13 36 Thr324Met 8 Catalytic M2M2,A1A1,GG,H2H2

4 70 Thr324Met 8 Catalytic M2M2,A1A1,AA,H1H1

4 5.5 Ile138Thr*Leu263Arg heterozygousa

68

Activation,Catalytic

M1M1,A1A1,GG,H1H1

3.3 23 Cys310Phe 8 Catalytic M2M2,A1A1,AA,H1H1

<1 130 Cys310Phe 8 Catalytic M1M1,A1A1,GG,H1H1

<1 5.5 His348Arg 8 Catalytic M1M1,A1A1,GG,H1H1

2.8 65 Trp284Arg heterozygousa 8 Catalytic M2M2,A1A1,GG,H2H2

1a 73 G to A (G/A), His115His (C7880T) C/T H1/H2.

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. Thehydrophobic 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 thereferences to colour in this figure legend, the reader is referred to the web version of this article.)

109L. Mota et al. / Clinica Chimica Acta 409 (2009) 106–111

expected along with His193, the flanking residues are also highlyconserved in the homologs (Fig. 1). The replacement of Ala191 byPro191 might disturb the interactions and geometry of catalytic triadand thus lead to loss of protease activity.

Structural studies on the Leu263Arg mutant revealed that thismutation would create a steric strain in the protein. Leu263 is presentin a loop structure and is surrounded by bulky residues (Fig. 3A).Substitution of Leuwith Arg, which has a bulky side chain would bringabout steric clashes with the neighbouring residues and thusdrastically 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 thelengths of side chains of Asp and Glu results in a shift of the hydrogenbond registry. Glu338 instead forms side chain–main chain hydrogenbond with Gly375 (Fig. 4B).

In the wild-type, side chain–main chain hydrogen bonds are alsoseen between Trp284and His211 (Fig. 5A). In case of the Trp284Argmutant, substitution of the hydrophobic Trp with a hydrophilicpositively charged Arg, creates a non-native ionic interaction betweenArg284 and Glu210 (Fig. 5B).

6. Discussion

There is generally a broad consensus that in patients withhemophilia bleeding tendency correlates with severity of deficiency.However, this is not true for FVII deficiency. One of the reasons cited

Fig. 3. (A) Leu263 in the loop structure along with its structural neighbours. Leu263 is flankedresidues have been colored as Met (orange), aliphatic residues—Val, Leu, Ile (grey), Argrespectively. (For interpretation of the references to colour in this figure legend, the reader

for this is that FVII deficiency when measured as <1% is very oftenassociated with substantial circulating factor VII antigen [18]. Ten outof 14 cases were cross reacting material (CRM) positive; thusprediction of the clinical course of the disease is often difficult inthese patients. Except in 3 cases all other patients had severe bleedingmanifestations. IC bleeding has been found to a common clinicalmanifestation in FVII deficient cases observed in as high as 60% of thecases [19]. In the present series, 4 out of 14 patients (29%) hadsuffered hemorrhage in the brain, out of which 3 could not survive thebleed. Seven out of 14 patients in our study (50%) had a history ofconsanguinity; slightly lower than that reported in a study from Iran,where 78.6% of the cases history of parental consanguinity [7]. Manymarriages in India are endogamous making distant consanguinity apossibility.

In Patient (ID 1) a compound heterozygotemutation Cys22Arg andAla191Pro was detected. Of these 2 mutations the Cys22Arg mutationin exon 2 coding for the Gla domain has previously been reported [20].Cys17 and Cys22 are conserved residues in vitamin K-dependantcoagulation factors forming a disulfide bond for holding together 2 Glaresidues in proper position. It also enables capturing the Ca+2 whichis very important for the proper function. Substitution at this siteabolishes this specific function. The expression studies by theseauthors also reveal absence of secretion of FVII from BHK cells. Theother heterozygous mutation Ala191Pro is a novel mutation notreported earlier. Ala191 is a conserved (11 out of 13 related serineproteases) amino acid buried in the hydrophobic core. The structural

by bulky amino acids. (B) The substitution of Leu by Arg leads to steric hindrance. The(red), Gln (pink). Helices, strands and loops are colored as cyan, yellow and greenis referred to the web version of this article.)

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 differencein 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 registryneeds 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, thereader is referred to the web version of this article.)

110 L. Mota et al. / Clinica Chimica Acta 409 (2009) 106–111

analysis reveals that the replacement of Ala191 by Pro191 mightdisturb the interactions and geometry of catalytic triad and thus leadsto the loss of protease activity. Arg152Gln is a recurrent mutation andis found in 2 patients (ID 2 and ID 3). It occurs at a site (Arg 152-Ile153) that is normally cleaved to generate activated FVII (FVIIa).Analysis of purified FVII from patient plasma showed that FXa andcofactors could not activate the material [21].

A commonmutation Cys310Phe was detected in three patients (ID7, 11 & 12). Cys-310 to Phe, suppresses a disulphide bond conserved inthe catalytic domain of all serine proteases also Cys310Phe displaysdiminished binding to TF and does not activate FX [22].Thr324Met ispreviously reported in a heterozygous state [23] while in the presentstudy, in both the patients (ID 8, 9) this mutation was in homozygousstate with moderately deficient levels of FVII:C and were clinicallyasymptomatic.

Gly117Arg (Patient ID 5) was previously reported in a patient ofIndian origin. Extensive sequence comparison in a wide evolutionarycontext suggested that the Gly117 residue is critical for structure ofFVII i.e. the mutation primarily affected the folding / secretion orstability of the protein [24]. His348Arg mutation was previouslyreported from India as a heterozygous mutation [9]. All the residues inwhich the novel mutations were detected i.e. Ile138, Ala191, Leu263,Trp284 and Asp338 were highly conserved across the species. Residuesthat are conserved during evolution generally play a crucial role in the

Fig. 5. The creation of a non-native ionic bond in W284R mutant. (A) Trp284 interacts with Hbond is lost and a non-native ionic interaction occurs between Arg284 with Glu210. The impliloops are colored as cyan, yellow and green respectively. (For interpretation of the references

stability and function of the protein. Hence, it can be safely assumedthat any substitution of these fully/highly conserved residues can leadto structural and/or functional loss of the protein.

A novel Ile138Thr mutation in homozygous state along with theLeu263Arg heterozygous mutation was detected in patient 10.Surprisingly, this patient was asymptomatic with 3 uneventfulsurgeries. Structural analyses revealed that Ile138 forms a part of aburied hydrophobic core and participates in hydrophobic interactionswith Pro165, Trp166 and Trp356 (Fig. 2A). Substitution of Ile138 withThr138 disrupts these native hydrophobic interactions (Fig. 2B). Theburied hydrophobic residues of a protein are known to play a vital rolein the early stages of its folding and stability.

Loss of these hydrophobic interactions and replacement of ahydrophobic residue by a polar residue in the buried hydrophobiccore may severely destabilize the protein. Despite the predictedsevere impact on the functional activity of FVII, the patient has beenfound to be asymptomatic. Whether there is modulating effect ofthrombophilia markers to alleviate the clinical severity as it has beenobserved in hemophilia [25] in this patient needs to be analyzed.

Leu263Arg mutation was seen in heterozygous state in threepatients. Of the three patients, 2 were clinically severe with symptomslike intra-cranial and gastrointestinal bleeds along with haemarthrosis(ID 4, 5). Asp338Gln mutation was seen in boy born of consanguineousmarriage (ID 6). He had symptoms such as epistaxis and had a strong

is211 through side chain–main chain hydrogen bond. (B) In the mutant, this hydrogencation of this non-native ionic interaction needs further investigation. Helices, strands andto colour in this figure legend, the reader is referred to the web version of this article.)

111L. Mota et al. / Clinica Chimica Acta 409 (2009) 106–111

family historywhere an elder sibling had died of IC bleed. The structuralanalysis shows changes in hydrogen bonding thus causing the severephenotype.

Six different polymorphisms were detected, of which 3 have beenpreviously reported. The Arg353Gln heterozygous polymorphism isknown to cause ~20–25% reduction in the FVII:C where as individualshomozygous for this polymorphism have an approximately 50%reduction in circulating plasma FVII levels [26]. Therewas no evidencefor modulation of clinical features by this functional polymorphism inFVII deficient cases [27]; however modulation of clinical severity hasbeen documented for hemophilia [28]. It is possible that they have asimilar effect in FVII deficient cases too, however these can beconfirmed only by studying a large series of patients.

The novel FVII missense mutations reported here occur in aminoacid residues that are conserved between the homologous FVIImolecules of different organisms. Additional evidence to support thepathological involvement of these lesions comes from molecularmodeling. In general, the CRM status of the probands could be relatedto the location of themutations within the FVII molecule; thus, CRM+phenotypes were usually associated with mutations in surfaceaccessible residues and CRM− phenotypes with mutations locateddeep within the FVII molecule. Another important observation is therelative scarcity of nonsense mutations i.e. all the 11 mutations aremissense mutations. A probable explanation for this would bepresumably the inherent structure of the F VII gene or the earlylethality of homozygous F VII deficiency due to nonsense mutations.The same has been observed in the mutational spectrum of F X genemutations.

In conclusion, the molecular basis of FVII deficiency in a largeseries of Indian patients has been unraveled and its effect on thefunction of FVII molecule has been elucidated. The data can now beutilized in genetic diagnosis of the affected families.

Acknowledgements

We thank the Lady Tata Memorial Trust for the scholarship offeredto Ms. Mota to carry out this work.

References

[1] Osterud B, Rapaport S. Activation of factor IX by the reaction product of tissuefactor and factor VII: additional pathway for initiating blood coagulation. Proc NatlAcad Sci U S A 1977;74:5260–4.

[2] Peyvandi F, Duga S, Akhavan S, et al. Rare coagulation deficiencies. Hemophilia2002;8:308–21.

[3] Triplett DA, Brandt JT, Batard MA, Dixon JL, Fair DS. Hereditary factor VIIdeficiency: heterogeneity defined by combined functional and immunochemicalanalysis. Blood 1985;66:1284–7.

[4] Mariani G, Dolce A, Marchetti G, Bernardi F. Clinical picture and management ofcongenital factor VII deficiency. Hemophilia 2004;10:180–3.

[5] de Grouchy J, Dautzenberg MD, Turleau C, et al. Regional mapping of clottingfactors VII and X to 13q34: expression of factor VII through chromosome 8. HumGenet 1984;66:230–3.

[6] O'hara PJ, Grant FJ, Halderman A, et al. Neocleotide sequence of the gene coding forhuman 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] JayandharanGR, ViswabandyaA,Nair SC, et al.Molecular basis of hereditary factorVII

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 patientcarrying double heterozygous mutations in the FVII gene. Thromb Res 2005;115:535–6.

[10] Dacie JV, Lewis SM. Practical hematology. 6th edn. Edinburgh: ChurchillLivingstone; 1984. p. 230–2.

[11] Altschul SF, Madden TL, Schäffer AA, et al. Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs. Nucleic Acids Res 1997;25:3389–402.

[12] Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity ofprogressive multiple sequence alignment through sequence weighting, positionspecific 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 logogenerator. Genome Res 2004;14:1188–90.

[14] Banner DW, D'Arcy A, Chène C, et al. The crystal structure of the complex of bloodcoagulation factor VIIa with soluble tissue factor. Nature 1996;380:41–6.

[15] Guex N, Peitsch MC. SWISS-MODEL and the Swiss-Pdb Viewer: an environmentfor comparative protein modeling. Electrophoresis 1997;18:2714–23.

[16] Tina KG, Bhadra R, Srinivasan N. PIC: Protein Interactions Calculator. Nucleic AcidsRes 2007;35:W473–6.

[17] Fraczkiewicz R, Braun W. Exact and efficient analytical calculation of the accessiblesurface areas and their gradients formacromolecules. J CompChem1998;19:319–33.

[18] Rapaport SI, Rao LV. The tissue factor pathway: how it has become a “primaballerina”. Thromb Hemost 1995;74:7–17.

[19] Peyvandi F, Mannucci PM, Asti D, et al. Clinical manifestations in 28 Italian andIranian patients with severe factor VII deficiency. Hemophilia 1997;3:242–6.

[20] Fromovich-Amit Y, Zivelin A, Rosenberg N, et al. Characterization of mutationscausing factor VII deficiency in 61 unrelated Israeli patients. J Thromb Hemost2004;2:1774–81.

[21] Chaing S, Clarke B, Sridhara S, et al. Severe factor VII deficiency caused bymutations abolishing the cleavage site for activation and altering binding to tissuefactor. Blood 1994;83:3524–35.

[22] Giansily-Blaizot M, Verdier R, Biron-Adréani C, et al. Analysis of biologicalphenotypes from 42 patients with inherited factor VII deficiency: can biologicaltests predict the bleeding risk? Haematologica 2004;89:704–9.

[23] Gomez K, Laffan MA, Kemball-Cook G, et al. Two novel mutations in severe factorVII deficiency. Br J Haematol 2004;126:105–10.

[24] Sauer RT, Milla ME, Waldburger CD, et al. Sequence determinants of folding andstability 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 andfibrinolytic factors in modulating the clinical severity of hemophilia patients. Br JHaematol 2007;138:541–4.

[26] Arbini AA, Bodkin D, Lopaciuk S, Bauer KA. Molecular analysis of Polish patientswith factor VII deficiency. Blood 1994;84:2214–20.

[27] Mariani G, Herrmann FH, Dolce A, et al. Clinical phenotypes and factor VIIgenotype 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 genemodifies phenotype of severe hemophilia. Hemophilia (in press).