Blood Res.  2021 Dec;56(4):252-258. 10.5045/br.2021.2021016.

Mutation analysis and characterisation of F9 gene in haemophilia- B population of India

• Kulkarni S
• Hegde R
• Hegde S
• Kulkarni SS
• Hanagvadi S
• Das KK
• Kolagi S
• Gai PB
• Bulagouda R

• KMID: 2524077
• DOI: http://doi.org/10.5045/br.2021.2021016

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.

Keyword

Hemophilia B; F9 gene; Stop-gain mutation; Missense mutation; India

Figure

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  Fig. 1 Graphical representation of the distribution of the mutations within the Factor 9 (F9) gene.

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  Fig. 2 Multiple sequence analysis of the Factor IX (FIX) protein. The arrow indicates the position of the p.E66D mutation.

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  Fig. 3 Segments of a 3D model of wild type (upper left panel) and mutant (p.E66D; upper right panel) Factor IX. A superimposed 3D model of wild type (red) and mutant (p.S365*; blue) Factor IX are shown in the lower panel.

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