Hemophilia is an X-linked blood disorder that causes sustained bleeding after injuries or trauma. Symptoms mainly include bleeding in the joints and muscles. The term hemophilia was first used by Friedrich Hopff, University of Zurich, in 1828 [1, 2]. Hemophilia B (HB) is caused by defects in coagulation factor IX. It is also called “Christmas disorder”, after it was identified by Stephen Christmas in 1952 . FIX activity can be classified as severely (<1%), moderately (1–5%), and mildly (5–30%) impaired [1, 4, 5]. HB is less common than hemophilia A (HA) (1 in 25,000 males vs. 1 in 5,000 males worldwide) [6, 7]. Approximately 14,000 people with hemophilia have been registered at the Haemophilia Federation of India; however, hemophilia remains under-diagnosed and many cases are thus not registered. HA occurs in 1 out of 10,000 male births, while HB occurs in 1 out of 30,000 male births .
Recently, more than 3,000 pathogenic mutations and neutral polymorphisms have been identified in the
This study was approved by the Institutional Ethical Committee of Shri B. M. Patil Medical College, Hospital and Research Centre, BLDE (Deemed to be University), Vijayapura (Ref. No.: BLDE (DU)/IEC/340/2018-19), and the SNMC Institutional Ethics Committee of Human Subject Research, Bagalkot (Ref. No.: SNMC/IECHSR/2018-19/A-B2/1.1). Written informed consent was obtained from all HB patients before blood sample collection. A total of 150 HB patients were included in this study, which were followed up at 12 different hemophilia societies across the Karnataka state of India. A detailed clinical history was obtained from all HB patients.
Peripheral blood from HB patients was collected in EDTA vacutainers (BD, Franklin Lakes, NJ, USA). Prior to DNA isolation, the FIX concentration was measured and inhibitor assay were performed. DNA was extracted from peripheral blood using a blood and tissue DNA extraction kit (QIAGEN, Hilden, Germany). All exonic regions were amplified and the products were sequenced on an Applied Biosystems (ABI) 3500 Sanger sequencing platform using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Waltham, MA, USA). Results were analyzed using DNA sequence analysis software v5.4.
The pathogenicity of the novel non-synonymous variants was analyzed using bioinformatics tools, such as PROVEAN (http://provean.jcvi.org/seq_submit.php), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/index.shtml), PHD SNP (https://snps.biofold.org/phd-snp/phd-snp.html), SNPs & GO (https://snps.biofold.org/snps-and-go/snps-and-go.html), PANTHER (http://www.pantherdb.org/), and SNAP2 (https://www.rostlab.org/services/snap/). The conservation property of missense variants was investigated using the Clustal Omega multiple sequence alignment tool (https://www.ebi.ac.uk/Tools/msa/clustalo/). A 3D model of wild type and mutant FIX proteins was predicted using the Swiss model (https://swissmodel.expasy.org/), and the results were visualized and analyzed using the UCSF Chimera program.
Of the 150 HB patients included in this study, 102 (68%; FIX concentration, 0.6±0.2; age of onset, 2.0±1.0 y), 30 (20%; FIX concentration, 2.5±1.3; age of onset, 7.5±2.8 y), and 18 (12%; FIX concentration, 8.0±2.6; age of onset, 10.0±3.5y) suffered from severe, moderate, and mild HB, respectively. The detailed clinicopathological parameters are summarized in Table 1. In our study, we recorded 16 mutations. Of those, 1 was a synonymous mutation, 12 were missense mutations, 2 were stop-gain mutations, and 1 was a 3’ UTR variant. Notably, 13 (81.25%) mutations were previously reported, but 3 (18.75%) were novel mutations, which had not been entered into any of the human SNP databases. The majority of the mutations were found in exon 8 of the gene and largely comprised missense mutations. Exon 8 showed a high number of mutations compared to other exons of the
The novel missense mutation c.198A>T was found in the
The novel missense mutation p.E66D was shown to be harmful to FIX protein function by PROVEAN, SNAP2, PholyPhen2, SNP&GO, PHD-SNP, and PANTHER (Table 4). Multiple sequence alignment of this novel missense mutation indicated that it was present in the highly conserved residue of the FIX protein (Fig. 2). Homology modeling of the protein structure was conducted using the Swiss model server, which was then visualized and analyzed using chimera program. In p.E66D, the mutant residue is smaller than wild type residue, which might lead to the loss of interactions with the metal ion: “calcium 4 or magnesium 1; via 4-carboxyglutamate” (Fig. 3). The novel stop-gain mutation, p.S365*, results in a premature stop codon that leads to a truncated FIX variant of 365 amino acids, corresponding to a loss of approximately 21% of the wild type FIX protein (Fig. 3).
HB is a bleeding disorder that causes abnormal or poor blood clotting. Pathogenic variants of the
In the present study, we recorded a total of 16 mutations, of which 15 (93.75%) were coding sequence variants. The majority of them (81.25%) were missense variants.
We recorded the stop-gain mutation p.R75W in 2 patients with severe HB, which have been described previously by Parrado Jara
The novel missense mutation p.E66D was recorded in 4 patients with moderate HB, with a mean FIX concentration of 2.0±0.5, and in 7 patients with severe HB, with a mean FIX concentration of 0.3±0.5. It was found to be harmful to the function of the FIX protein by
Of the 150 HB patients, 90 (60%) had mutations in the
Additionally, 2 stop-gain mutations and 7 missense mutations were major pathogenic variations or disease-causing mutations recorded in our study; patients who were carriers of these mutations showed more severe conditions compared to the other 2 groups (mild and moderate). Likely pathogenic and benign mutations were also missense mutations, and all the patients carrying these mutations were mildly to moderately affected by HB, except for 2 patients. Only 3 mutations (c.127C>T, c.470G>A, and c.1070G>A) were associated with different severities. Moreover, 2 mutations were only associated with mild HB (c.304C>T and c.580A>G), 2 mutations were associated with moderate HB (c.195G>A and c.1385A>G), and 3 mutations were only associated with severe HB (c.223C>T, c.1187G>A, and c.1232G>A). One mutation each was associated with mild-moderate (c.*1110A>G) and mild-severe HB (c.197A>T). In addition, 4 mutations were associated with moderate-severe HB (c.314A>G, c.198A>T, c.676C>T, and c.1094C>A). Among the patients with severe HB, 48% (49/102) featured mutations in the
In our study, we also observed clinical differences between the mutated and wild type
Our study strongly suggests that the majority of HB cases feature pathogenic single nucleotide variations, which may be novel or previously recorded. In many cases, novel single nucleotide variants are involved. Population-based screening of mutations will help establish inhibitor risk prediction and carrier detection strategies in India.
We thank all hemophilia societies and hemophilia B patients and their families for participating in this study. We thank the Karnataka Institute for DNA Research, Dharwad, India, for providing instrumentation support for the research work. We also thank Shri B. M. Patil Medical College, Hospital and Research Centre, BLDE (Deemed to be University), Vijayapura and S. Nijaliangappa Medical College, HSK Hospital and Research Center, Bagalkot, for their continuous support throughout this research project.
No potential conflicts of interest relevant to this article were reported.