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Blood Res 2021; 56(4):
Published online December 31, 2021
https://doi.org/10.5045/br.2021.2021016
© The Korean Society of Hematology
Mutation analysis and characterisation of F9 gene in haemophilia- B population of India
Correspondence to : Rudragouda Bulagouda, M.D.
Human Genetics Laboratory, Department of Anatomy, Shri B.M Patil Medical College, Hospital and Research Centre, BLDE (Deemed to be University), Vijayapura 586101, India
E-mail: drravisb2012@gmail.com
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Hemophilia B (HB) is an X-linked bleeding disorder resulting from coagulation factor IX defects. Over 3,000 pathogenic, HB-associated mutations in the F9 gene have been identified. We aimed to investigate the role of F9 variants in 150 HB patients using sequencing technology.
Methods
F9 gene sequences were amplified from peripheral blood-derived DNA and sequenced on an Applied Biosystems (ABI) 3500 Sanger sequencing platform. Functional and structural predictions of mutant FIX were analyzed.
Results
Among 150 HB patients, 102 (68%), 30 (20%), and 18 (12%) suffered from severe, moderate, and mild HB, respectively. Genetic analysis identified 16 mutations, including 3 novel mutations. Nine mutations (7 missense and 2 stop-gain) were found to be pathogenic. Only 3 mutations (c.127C＞T, c.470G＞A, and c.1070G＞A) were associated with different severities. While 2 mutations were associated with mild HB cases (c.304C＞T and c.580A＞G), 2 (c.195G＞A and c.1385A＞G) and 3 mutations (c.223C＞T, c.1187G＞A, and c.1232G＞A) resulted in moderate and severe disease, respectively. Additionally, 1 mutation each was associated with mild-moderate (c.*1110A＞G) and mild-severe HB disease (c.197A＞T), 4 mutations were associated with moderate-severe HB cases (c.314A＞G, c.198A＞T, c.676C＞T, and c.1094C＞A). FIX concentrations were lower in the mutated group (5.5±2.5% vs. 8.0±2.5%). Novel p.E66D and p.S365 mutations were predicted to be pathogenic based on changes in FIX structure and function.
Conclusion
Novel single nucleotide polymorphisms (SNPs) largely contributed to the pathogenesis of HB. Our study strongly suggests that population-based genetic screening will be particularly helpful to identify risk prediction and carrier detection tools for Indian HB patients.
Keywords Hemophilia B, F9 gene, Stop-gain mutation, Missense mutation, India
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Original Article
Blood Res 2021; 56(4): 252-258
Published online December 31, 2021 https://doi.org/10.5045/br.2021.2021016
Copyright © The Korean Society of Hematology.
Mutation analysis and characterisation of F9 gene in haemophilia- B population of India
Sujayendra Kulkarni1,2, Rajat Hegde4, Smita Hegde4, Suyamindra S. Kulkarni4, Suresh Hanagvadi5, Kusal K. Das6, Sanjeev Kolagi3, Pramod B. Gai4, Rudragouda Bulagouda1
1Human Genetics Laboratory, Department of Anatomy, Shri B.M Patil Medical College, Hospital and Research Centre, BLDE (Deemed to be University), Vijayapura, 2Division of Human Genetics (Central Research Lab), 3Department of Anatomy, S. Nijaliangappa Medical College, HSK Hospital and Research Center, Bagalkot, 4Karnataka Institute for DNA Research (KIDNAR), Dharwad, 5Department of Pathology, J. J. M. Medical College, Davangere, Karnataka, 6Laboratory of Vascular Physiology and Medicine, Department of Physiology, Shri B. M. Patil Medical College, Hospital and Research Centre, BLDE (Deemed to be University), Vijayapura, India
Correspondence to:Rudragouda Bulagouda, M.D.
Human Genetics Laboratory, Department of Anatomy, Shri B.M Patil Medical College, Hospital and Research Centre, BLDE (Deemed to be University), Vijayapura 586101, India
E-mail: drravisb2012@gmail.com
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background
Hemophilia B (HB) is an X-linked bleeding disorder resulting from coagulation factor IX defects. Over 3,000 pathogenic, HB-associated mutations in the F9 gene have been identified. We aimed to investigate the role of F9 variants in 150 HB patients using sequencing technology.
Methods
F9 gene sequences were amplified from peripheral blood-derived DNA and sequenced on an Applied Biosystems (ABI) 3500 Sanger sequencing platform. Functional and structural predictions of mutant FIX were analyzed.
Results
Among 150 HB patients, 102 (68%), 30 (20%), and 18 (12%) suffered from severe, moderate, and mild HB, respectively. Genetic analysis identified 16 mutations, including 3 novel mutations. Nine mutations (7 missense and 2 stop-gain) were found to be pathogenic. Only 3 mutations (c.127C＞T, c.470G＞A, and c.1070G＞A) were associated with different severities. While 2 mutations were associated with mild HB cases (c.304C＞T and c.580A＞G), 2 (c.195G＞A and c.1385A＞G) and 3 mutations (c.223C＞T, c.1187G＞A, and c.1232G＞A) resulted in moderate and severe disease, respectively. Additionally, 1 mutation each was associated with mild-moderate (c.*1110A＞G) and mild-severe HB disease (c.197A＞T), 4 mutations were associated with moderate-severe HB cases (c.314A＞G, c.198A＞T, c.676C＞T, and c.1094C＞A). FIX concentrations were lower in the mutated group (5.5±2.5% vs. 8.0±2.5%). Novel p.E66D and p.S365 mutations were predicted to be pathogenic based on changes in FIX structure and function.
Conclusion
Novel single nucleotide polymorphisms (SNPs) largely contributed to the pathogenesis of HB. Our study strongly suggests that population-based genetic screening will be particularly helpful to identify risk prediction and carrier detection tools for Indian HB patients.
Keywords: Hemophilia B, F9 gene, Stop-gain mutation, Missense mutation, India
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Table 1 . Clinicopathological characteristics of the study population..
Clinicopathological parameters Disease severity Severe Moderate Mild (Factor IX <1%), N=102 (68%) (Factor IX 1–5%), N=30 (20%) (Factor IX >5%), N=18 (12%) Mean factor IX levels (%) 0.6±0.2 2.5±1.3 8.0±2.6 Family history of bleeding (N) 80 10 7 Mean age of onset (yr) 2.0±1.0 7.5±2.8 10.0±3.5 Mutation frequency (within group) 48% (49/102) 86.7% (26/30) 83.3% (15/18) Inhibitor-positive (N) 23 11 4 Inhibitor-negative (N) 79 19 14 Hemoglobin concentration Normal Normal Normal Platelet count Normal Normal Normal Prothrombin time Normal Normal Normal Activated partial thromboplastin time High High High Bleeding sites Joints 55 (54%) 19 (63.3%) 12 (66.7%) Gum 17 (16.7%) 6 (20%) 4 (22.2%) Muscle 13 (12.7%) 1 (3.3%) 2 (11.1%) Epistaxis 7 (6.9%) 4 (13.3%) 0 Petechiae 10 (9.8%) 0 0
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Table 2 . List of mutations recorded in the
F9 genes of our study cohort..Mutation type Nucleotide change cDNA position Amino acid change Exon Status Frequency, N (%) Missense g.11313C>T c.127C>T p.R43W 2 Reported (rs1603264205) 9 (6.0%) Missense g.11381G>A c.195G>A p.M65I 2 Reported (rs763568424) 1 (0.66%) Missense g.11383A>T c.197A>T p.E66V 2 Reported (CM940423) 3 (2.0%) Missense g.11384A>T c.198A>T p.E66D 2 Not reported 12 (8.0%) Stop-gain g.11409 C>T c.223C>T p.R75a) 2 Reported (rs137852227) 2 (1.33%) Missense g.15369 T>C c.304C>T p.C102R 4 Reported (CM960574) 2 (1.33%) Missense g.22664 T>C c.314A>G p.G143R 5 Reported (CM940499) 4 (2.66%) Missense g.22706 G>A c.470G>A p.C157Y 5 Reported (rs1367198680) 17 (11.33%) Missense g.25386 A>G c.580A>G p.T194A 6 Reported (rs6048) 3 (2.0%) Missense g.25482C>T c.676C>T p.R226W 6 Reported (rs137852240) 6 (4.0%) Missense g.36020 G>A c.1070G>A p.G357E 8 Reported (rs137852275) 8 (5.33%) Stop-gain g.36044C>A c.1094C>A p.S365a) 8 Not reported 15 (10.0%) Missense g.36137G>A c.1187G>A p.C396Y 8 Reported (rs137852273) 1 (0.66%) Missense g.36182 G>A c.1232G>A p.S411N 8 Reported (rs137852276) 3 (2.0%) Synonymous g.36335 A>G c.1385A>G p.Ter462= 8 Reported (rs561793582) 1 (0.66%) 3’ UTR g.37446A>G c.*1110A>G ……… 8 Not reported 3 (2.0%) a)NG_007994.1, NM_000133.4, and NP_000124.1 reference sequences wereused for genomic DNA variant nomenclature, coding region variant nomenclature, and protein variant nomenclature, respectively. All nomenclatures were made according to Human Genome Variation Society (HGVS) guidelines..
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Table 3 . Genotypic and phenotypic associations of the mutations recorded in the study population..
Mutation type cDNA position Patients Factor IX (IX) concentration level (%) Age of onset Inhibitor-positive (N) Inhibitor-negative (N) Clinical significance m M S Missense c.127C>T 03 04 02 6.5, 8.0, 10 (m)
2.0, 2.3. 1.8, 2.0 (M)
0.8, 0.5 (S)10, 12, 8 (m)
7, 6, 9, 9 (M)
1, 1.5 (S)2 (m)
2 (M)
-1 (m)
2 (M)
2 (S)Pathogenic Missense c.195G>A 00 01 00 3.0 (M) 7.5 (M) 1 (M) - Likely pathogenic Missense c.197A>T 02 00 01 6.0, 9.0 (m)
0.3 (S)13, 11 (m)
1 (S)- 2 (m)
1 (S)Likely benign Missense c.198A>T 00 04 07 1.5, 2.0. 2.5, 2.0 (M)
0.8, 0.5, 0.1, 0.6, 0.6, <0.01, 0.01 (S)6, 7, 7, 8 (M)
1, 2, 1, 1.5, 2, 2, 1.6 (S)4 (M)
4 (S)-
3 (S)Pathogenic Stop-gain c.223C>T 00 00 02 <0.01, <0.01 (S) 1, 1 (S) 1 (S) 1 (S) Pathogenic Missense c.304C>T 02 00 00 12.0, 10.0 (m) 13, 10 (m) 2 (m) - Likely pathogenic Missense c.314A>G 00 01 03 2.0 (M)
0.2, 0.1, 0.01 (S)8 (M)
1, 2, 1 (S)- 1 (M)
3 (S)Pathogenic Missense c.470G>A 03 05 09 5.0, 7.5, 10.3 (m)
2.0, 3.5, 2.5, 1.8, 4.0 (M)
0.8, 0.5, 0.01, 0.2, <0.01, <0.01, 0.4, 0.2, <0.01 (S)6.5, 10, 11 (m)
5, 7, 7, 9, 7 (M)
1, 2, 2, 3, 2, 2, 2, 1, 2 (S)3 (m)
1 (M)
2 (S)-
4 (M)
7 (S)Pathogenic Missense c.580A>G 03 00 00 10.7, 9.0, 9.0 (m) 10, 8, 8 (m) - 3 (m) Likely benign Missense c.676C>T 00 01 05 4.0 (M)
0.5, 0.1, <0.01, 0.01, <0.01 (S)5.5 (M)
3, 1, 2, 2, 1 (S)1 (M)
1 (S)-
3 (S)Pathogenic Missense c.1070G>A 01 03 04 6.5 (m)
2.0, 3.8, 2.5 (M)
0.01, 0.1, <0.01, <0.01 (S)12 (m)
6, 7, 7 (M)
2, 2, 2, 1 (S)-
2 (M)
2 (S)1 (m)
1 (M)
1 (S)Pathogenic Stop-gain c.1094C>A 00 03 12 2.6, 3.5, 1.8 (M)
0.01, 0.01, 0.1, 0.2, <0.01, <0.01, 0.1, 0.01, <0.01, 0.01, <0.01, <0.01 (S)7, 7, 6 (M)
1, 2, 2, 2, 2, 3, 1.5, 3, 2, 2, 2.5, 1 (S)-
10 (S)3 (M)
2 (S)Pathogenic Missense c.1187G>A 00 00 01 0.6 (S) 2 (S) - 1 (S) Benign Missense c.1232G>A 00 00 03 0.6, 0.3, 0.1 (S) 2, 1, 1.5 (S) 1 (S) 2 (S) Pathogenic Synonymous c.1385A>G 00 01 00 4.0 (M) 10 (M) 1 (M) - Benign 3’ UTR c.*1110A>G 01 03 00 12.0 (m)
5.0, 4.0, 3.8 (M)13 (m)
7, 7.5, 9 (M)1 (m)
1 (M)-
2 (M)- Abbreviations: m, mild; M, moderate; S, severe..
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Table 4 . Pathogenicity predictions of the p.E66D mutation..
Mutation PROVEANa) SNAP2b) PolyPhen2c) PHD-SNPd) SNP&GOe) PANTHER E66D Deleterious
Score: -2.540Effect
Score: 37Probably damaging
Score: 0.999Disease
Score: 4Disease
Probability: 0.705Probably damaging a)PROVEAN: “Deleterious” if the prediction score was ≤2.5, “Neutral” if the prediction score was ≥2.5. b)NAP2: “Neutral” if the score ranged from 0 to -100. “Effect” if the score was between 0 and 100. c)PolyPhen2: “Probably damaging” is the most disease-causing ability, with a score near 1. “Possibly damaging” signifies less disease-causing ability with a score of 0.5–0.8. “Benign”, which does not alter protein function, with a score closer to 0. d)PHD-SNP: if the probability is >0.5, mutation is predicted as “Disease” and if less than <0.5, mutation is predicted to be “Neutral”. e)SNP & GO: Probability of >0.5 is predicted to be a disease-causing nsSNP..
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