Open Access

Three novel F8 mutations in sporadic haemophilia A cases

  • Rashid Hussain1Email author,
  • Noman Bin Abid2,
  • Sajjad Hussain3,
  • Zeeshan Shaukat3,
  • Mudassir Altaf3,
  • Sara Altaf3 and
  • Gulzar Niazi3
SpringerPlus20121:10

DOI: 10.1186/2193-1801-1-10

Received: 17 April 2012

Accepted: 2 July 2012

Published: 30 July 2012

Hemophilia A (HA) is an X-linked hereditary disorder characterized by bleeding of variable severity through mild, moderate to severe owing to large range of mutations in the Factor VIII (F8) gene (Bowen [2002]). All kind of F8 mutations, except repeats, have been reported for HA, in total up to 2370 (Human Genome Mutation Database [2005]). A preliminary study was conducted in our lab for identification of mutations in F8 gene in Pakistani HA patients. Correlation of F8 mutations with clinical manifestation of HA patients was the main objective of the study. Blood samples were collected from 62 HA patients from all over the Pakistan and clinical history of all HA patients was recorded (only patients frequently visiting medical centers for the replacement of Factor VIII were selected for the study). Genomic DNA was extracted from whole blood by standard organic procedure. Specific primers (Figure 1) were designed using “Primer3” (http://biotools.umassmed.edu/bioapps/primer3_www.cgi) to amplify the coding region of F8 gene; amplified products were sequenced by ABI 310 and ABI 3100 sequencer (Applied Biosystems, Carlsbad, CA, USA). The sequencing results were visualized using “Chromas 2.33” software (Applied Biosystems) and mutations were detected using “BLAST” software available on the NCBI website (http://balst.ncbi.nlm.nih.gov/Blast.cgi). Three novel mutations (1 deletion; 2 point mutations) were detected in four sporadic HA patients, all from different ethnic backgrounds (Table 1). The deletion of T in exon 7 within the A1 domain represents a frame-shift change disrupting the protein structure and function, which result in severe manifestation of the disease. A missense point mutation in the A3 domain occurs in codon 1907 at nucleotide number 5720, replacing Serine with Isoleucine, and confers a moderate type of severity. It should be noted that Serine is a polar and acidic amino acid while Isoleucine is a nonpolar and basic amino acid. A nonsense point-mutation was found in two unrelated patients in the C3 domain (exon 26) and was correlated with moderate clinical findings. Beside these mutations, 27 common SNPs were also detected in F8 gene for the studied patients (Table 2). The allelic data and accession numbers of these SNPs were collected from Ensembl Genome Browser (Ensembl [2000]). The results of the study will form the basis not only for an enlarged study but also for diagnosis and genetic counseling of classical hemophilia in Pakistan.
Figure 1

Primers used in the study.

Table 1

Novel mutations in F8 gene

Age/Sex

Severity

Exon

Nucleotide change

Amino acid change

Codon/Codon no.

Nucleotide genome ref./cDNA ref.

Affected Domain

4 yr /male

Severe

7

Deletion of T

Frame-shift

CTC → C-C/ 318

159197688/953

A1

35 yr / male

Moderate

17

G → T

Ser → Ile

AGC → ATC/ 1907

154132724/5720

A3

15 & 19 yr /male

Moderate

26

C → A

Tyr → Termination

TAC → TAA/ 2324

154065994/6972

C2

yr (years).

Table 2

Common SNPs in F8 gene (exonic region)

Sr. #

Patient

Exon

SNP ambiguity

SNP

Codon

Codon#

Comments

Accession number

1

All 62 Samples

2

W: A/T

A/A

GA T

75

European = T/T

rs1800288

2

All 62 Samples

7

K: G/T

G/G

TGG

274

European = C/C; Spanish Caucasians = C(0.995)/A(0.005); African American, Chinese, Southeast Asia, Mexican Indian = C/A

rs34371500

3

All 62 Samples

8

R: G/A

G/G

CG C

391

Ancestral: G

rs137852364

4

All 62 Samples

8

Y: T/C

T/T

T CA

392

European = C/C

rs28933669

5

All 62 Samples

8

Y: C/T

C/C

TC A

392

?

rs28933668

6

All 62 Samples

8

K: T/G

T/T

AT T

405

European = A/A

rs28933670

7

All 62 Samples

8

R: A/G

A/A

GA G

409

?

rs28933671

8

All 62 Samples

9

K: G/T

T/T

TTG

431

Ancestral: G

rs28933672

9

All 62 Samples

9

R: A/G

A/A

AA A

444

Ancestral: G

rs28937272

10

All 62 Samples

9

W: T/A

T/T

T AC

450

Ancestral: A

rs111033616

11

All 62 Samples

10

R: G/A

G/G

CG T

503

Ancestral: A

rs35383156

12

All 62 Samples

12

Y: T/C

T/T

CTT

622

Ancestral: T

rs1800290

13

All 62 samples

15

R: G/A

G/G

CAG

1764

Ancestral: A

rs5986891

14

All 62 samples

16

R: G/A

G/G

ATG

1842

European = G/G

rs28943674

15

All 62 samples

16

Y: C/T

C/C

C CC

1844

European = C/C

rs28933675

16

All 62 samples

16

M: A/C

A/A

A CT

1845

?

rs28933676

17

All 62 samples

16

Y: C/T

C/C

GC C

1853

European = C/C

rs28933677

18

All 62 samples

17

D: G/A/T

G/G

GAT

1865

Not Available

CI076951

19

All 62 samples

17

R: A/G

A/A

CA C

1867

Ancestral: G

rs28933679

20

All 62 samples

17

S: C/G

C/C

CC C

1873

European = G/G

rs28933680

21

All 62 samples

17

R: G/A

G/G

G AG

1904

European = C/C

rs28933681

22

All 62 samples

17

S: G/C

G/G

TG C

1922

European = G/G

rs4384155

23

All 62 samples

17

S: C/G

C/C

TGC

1922

European = C/C

rs4520342

24

All 62 samples

18

R: A/G

A/A

AAT

1940

?

CM083806

25

All 62 samples

18

D: G/A/T

G/G

CG A

1960

?

rs28937294

26

All 62 samples

18

R: G/A

G/G

GGC

1967

?

rs111033615

27

All 62 samples

24

Y: C/T

C/C

TAC

2214

Ancestral: C

rs1800296

Declarations

Authors’ Affiliations

(1)
National Institute for Genomics and Advance Biotechnology (NIGAB)/National Agricultural Research Centre (NARC)
(2)
Lahore University of Management Sciences, DHA Phase III Hospital Street 29
(3)
National Center of Excellence in Molecular Biology

References

  1. Bowen DJ: Hemophilia A and B: molecular insights. J clin path; mol path 2002, 55: 1-18. 10.1136/mp.55.1.1View ArticleGoogle Scholar
  2. Human Genome Mutation Database Institute of Medical Genetics, Cardiff; 2005.http://www.hgmd.cf.ac.uk Accessed 26 May 2012.
  3. Ensembl: European Molecular Biology Laboratory and Welcome Trust Sanger Institute. 2000. http://www.ensembl.org.Accessed10JuneGoogle Scholar

Copyright

© Hussain et al.; licensee Springer. 2012

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.