Mutation Analysis of F8 Gene and Phenotypic Characterization of Hemophilia A

Introduction

Hemophilia is a congenital bleeding disorder caused due to deficiency of certain clotting proteins in the blood. It is characterized by an X-linked recessive mode of inheritance and caused by mutations in the gene encoding for Factor VIII and IX clotting proteins.¹ The most common deficiency is factor VIII, known as “Classical hemophilia” or Hemophilia A [HA] with the prevalence of 1 in 5000 male births, followed by Factor IX deficiency known as “Christmas disease” or Hemophilia B [HB] with 1 in 30,000 prevalence.^2,3 As per World Bleeding Disorder Registry (WBDR)–2021 World Federation of Hemophilia (WFH), 233,577 people with hemophilia (PWH) are registered all over the world and India has 25,384 PWH enrolled cases.^4-6 But with a population of 1.32 billion and prevalence of 1/10000 male births in India, the expected number of patients with hemophilia (PwH) should have been 1,32,000, indicating low awareness, lack of diagnostic facilities, and limited "registry data".⁵

People with hemophilia (PwH) can be classified as mild, moderate or severe disease based on their residual activity of clotting factor (>5%-40%, 1%-5% or <1%, respectively), which determines the clinical manifestations.⁶ Most severe patients experience spontaneous, frequent bleeding episodes, predominantly in the joints and muscles, whereas moderate hemophilic patients present with traumatic bleeds. Mild patients bleed only during major challenges like surgeries or road traffic accidents.^7-9 In general, most severe patients develop progressive musculoskeletal complications in the form of chronic synovitis, arthropathy, joint contractures and pseudotumor formation. Some patients may develop inhibitors during the course of their treatment.^10-14

A basic coagulation profile is crucial in specific diagnosis and assessing the severity of hemophilia. Isolated prolongation of Activated Partial Thromboplastin, correction studies and specific factor assay are done. Mutation analysis of these patients enables us to understand the pathogenetic mechanisms of their clinical manifestations and predisposing factors for inhibitor formation.15 In the present study, clinical and haematological manifestations have been analysed and their correlation with molecular mutations in patients with haemophilia A (PwHA) has been tested.

Materials and Methods

Sample collection

This study was undertaken in the Department of Pathology, JJM Medical College, Davangere, under Rajiv Gandhi University Health Sciences, Advanced Research Project support (2017- 2018), and was approved by the Institutional Ethical Committee (Ref No: JJMMC/ IEC/23-2017). Informed written consent was obtained from all patients diagnosed with Hemophilia A for the collection of samples. Under aseptic conditions, venous blood was drawn into 3.2% Sodium citrate vacutainer in 1:9 proportion for basic coagulation workup. For molecular analysis, 5 mL of venous blood was collected in an EDTA vacutainer and stored at 4°C till transported to the genetic laboratory (Karnataka Institute for DNA Research [KIDNAR], Dharwad). A total of 90 Hemophilia A (HA) patients were included in this study, which was followed up at Karnataka Hemophilia Society, Davangere. A detailed clinical history was recorded along with assessment of socio-economic status as measured by modified Kuppuswamy scale and thorough medical examination was conducted.

Basic coagulation procedure

Plasma coagulation test prothrombin time (PT), partial thromboplastin time (APTT) and correction study, factor VIII assay, inhibitors screening tests were done for all patients and quantification tests (Modified Bethesda method) were performed for those positive for inhibitors.

Molecular analysis

All the 90 HA patients were included in the mutation analysis. Genomic DNA was extracted using the QIAamp DNA blood mini kit [QIAGEN, Germany]. The quantity and quality of extracted DNA was analysed by using a nanodrop UV spectrophotometer and 0.8% agarose gel electrophoresis. Further, exonic region of F8 gene was amplified using standard PCR master mix and cycling condition. Sanger sequencing analysis was performed for the exonic region of the F8 gene using the ABI 3500 Sanger sequencing platform using the BigDye Terminator v3.1 Cycle Sequencing Kit [Thermoscientic, USA]. DNA sequence analysis software v5.4 was used for the result analysis. Mutation analysis was conducted to evaluate the molecular alteration in F8 gene only on exon.

Statistical analysis

The obtained data were tabulated and analysed using SPSS version 15.0 (SPSS Inc., Chicago, IL, USA). Data were presented as mean ± SD.

Results

Demographic profile of patients with Hemophilia A

The present study included 90 Hemophilia A patients, aged between 0-44 years with mean age of 22 years. Out of 90 Hemophilia A cases, 3 [3.3%] were diagnosed as mild, 15 [16.6%] as moderate and 72 [80%] were diagnosed as severe hemophilic cases. Positive family history was observed in 62 (68.8%) of the studied HA patients. In the present study, 35 (38.8%) of the HA patients were born to consanguineously married parents.

Socio-economic status was graded according to Kuppuswamy scale for all included HA patients which revealed that 7 (7.7%) were grade-I, 23 (25.5%) were grade-II, 20 (22.2%) were grade–III, 34 (37.7%) were grade IV and 6 (6.6%) were grade-V. The detailed demographic characteristics are summarized in Table 1.

In the present study, joint bleeding was recorded in 80 (88.8%) cases with knee joint being the most commonly affected site, followed by ankle, elbow and shoulder (Table 1). Apart from joint bleeding, mucocutaneous bleeding (66.6%), dental bleeding (48.8%), genitourinary bleeding (16.6%), gastrointestinal bleeding (15.5%), muscle bleeding (14.4%) (in which Iliopsoas were most commonly affected followed by Gastrocnemius muscle) were recorded and a rare occurrence of intracranial bleeding (10%) was also noted (Table 1). Around 13 (14.4%) HA patients developed inhibitors, among which five were of low titer and eight were of high titer (Figure 1).

Mutation analysis of 90 HA patients revealed a total of 21 mutations including 11 (52.4%) missense mutations, 5 (23.8%) nonsense mutations, 5 (23.8%) frameshift mutations (3; frameshift deletion, 2; frameshift duplication). All the mutations observed were previously recorded mutations (Table 2). No novel mutation was observed in the present study cohort. Particularly c.3637 delA, c.4825 dupA, and c.1203 G>A were recorded only in severe cases with high frequency. 46.2% of the inhibitor positive HA patients recorded pathogenic nonsense and frameshift mutations [c.1203G>A, c.1628C>A, c5953C>T, c.6869G>A, c.2645-2646 DelTG, c.3637DelA and c.5293-5295DelCCC], followed by 30.7% with missense mutations [c.857A>C, c.980T>C, c.1702G>A, c.5387A>G and c.5573C>T]. Around 23.1% of inhibitor positive HA patients did not record any mutation in exonic region Detailed genotypic and phenotypic associations of the mutations recorded in the study population are summarised in Table 3.

Discussion

In the present study, an attempt was made to correlate the clinical-hematological manifestations with molecular basis in PwHA from Karnataka, India. In the present study, a higher percentage of severe hemophilia cases 72 (80%) were observed as compared to moderate and mild hemophilia.^16-21 This could be because patients with mild disease lack the symptoms in contrast to severe hemophilic patients presenting with spontaneous bleeds.

The most common age of Haemophilia A patients observed in the present study was between 19–44 years, constituting 43 (47.7%) cases, correlating with the Annual Global Report 2020 of WFH.⁴ According to the national average as per the Census report, the median age is 27.6 years, while we observed the lower median age to be 21.5 years, corresponding to a study conducted by John M J et al. ^5,22,23

In the present study, the median age of onset of the first bleed was noted as two years and this correlates with the reports of other studies including the Annual Global Report.^4,24,25 Early onset of bleeding episodes may be attributed to the severity of factor deficiency, spontaneous nature of bleed and hyperactivity in the early age group.²⁶ As this is an inherited bleeding disorder, a detailed family history including consanguinity was recorded. Among 90 patients, 62 (68.8%) had a family history of bleeding, which is similar to various studies referred.^25,20 As consanguinity is widely practiced in our region, its impact on genetic transmission among families was studied.²⁷ Around 35 (38.8%) patients were born to consanguineously married couples, indicating a potential increase in the transmission of genetic traits among the families. The literature review supports the implication of consanguinity in increasing the incidence of children born with hemophilia, as also evident in studies conducted by MM Uddin et al. ^28,29

Hemophilia is popularly known as a Royal family disease, because it was seen in the family of Queen Victoria of England, but the fact, particularly in developing countries like India is different. Most of the patients diagnosed are from the lower income groups. In our study, modified Kuppuswamy scale was applied for classification of patients according to socioeconomic status and was found that 34 patients (37.7%) belonged to upper or lower socioeconomic status (SES). This is an important factor contributing to poor quality of life because of unaffordability of expensive treatment.^30-32

Approximately 90% of bleeding episodes involved musculoskeletal system, and in 80% of cases, the joints were particularly affected.¹⁰ In this study, the most common manifestation was acute hemarthrosis of the knee (88.8%), correlating with the findings of Parthiban et al.,³³ Agarwal et al.,³⁴ Sharma et al.³⁵ and Karim et al.,⁸ followed by mucocutaneous bleeds, dental bleeding, muscle bleeds and other types. Acute hemarthrosis is more common in severe disease than in moderate and mild PwHA. However, only two cases with mild form had joint bleeds which was due to trauma. Mucocutaneous bleeds were generally caused by injuries, predominantly among moderate to severe forms of disease and dental bleeds were observed in about 48.8% of patients as most of them had poor oral hygiene resulting in carious tooth necessitating extraction under factor coverage. Hence, education of children with hemophilia about maintaining oral hygiene must be emphasized.³⁶ A considerable number of patients also had hematuria and gastrointestinal bleeds,³⁷ and most of them had severe forms of the disease. The occurrence of hematuria was spontaneous and most of the cases were treated by reassurance and conservative management. Gastrointestinal bleeds were caused because of dietary patterns with spicy food and long-standing use of pain-relieving medicines causing erosion and ulcers.³⁸ Intracranial haemorrhage (ICH) is the most serious event, but uncommon in hemophilia. We found around nine (10%) cases with ICH in our study and all belonged to severe forms of disease.^39,40

The lifetime risk to develop inhibitors is 25%–30% in severe hemophilia A.⁴¹ Our study showed much lower number with 13 (14.4%) severe hemophilia A patients being inhibitor positive. The study conducted by Wight J et al., also showed lower percentage of inhibitor positive cases in severe HA.⁴² Inhibitors with 5.0 NBU/BU are called as “low titer” inhibitors, whereas greater than 5.0 NBU/BU are called “high titer” inhibitors. Out of 13 inhibitor positive cases, eight were high titer cases and five were low titer cases. We closely studied the possible predisposing factors because it involves multifactorial pathogenetic mechanisms. It was observed that most patients with severe hemophilia A received episodic replacement therapy, majority with plasma-derived factors and wet blood products for bleeding episodes. We also observed the role of intensive replacement therapy in the development of inhibitors. In our study, only one patient with CNS and GIT bleeds who required long term treatment developed inhibitors.^43,44

In view of discrepancies between phenotypic and genotypic variations, an attempt was made to evaluate all the 90 patients for mutational analysis. Coding region mutations are typically thought to result in altered protein biosynthesis or dysfunction. Therefore, it is generally accepted that mutations altering the amino acid composition (missense mutations) may affect the protein activity in addition to reducing biosynthesis, whereas null mutations (such as the intron 22 inversion, large gene deletions, stop codons in haemophilia A etc.) are thought to reduce or abolish the synthesis and/or release of the protein.⁴⁵ All the mutations recorded in the present study were coding sequence mutations which included missense, frameshift, and nonsense mutations. The mutation on exon detection rate in severely affected patients was 81.9%, in moderately affected patients was 86.6% and was 66.6% in mildly affected individuals. The rest did not show any mutation in the exonic region of the F8 gene. No novel mutation was observed in our study. The majority of mutations were associated only with severe HA.

Frameshift mutation followed by point mutation which lead to stop codons were commonly associated with severe HA cases. In the present study, frameshift mutations and non-sense mutations were also recorded in high frequency among severe HA cases. Particularly c.3637 delA, c.4825 dupA, and c.1203 G>A were recorded only in severe cases with high frequency. Missense mutations occurred in less than 20% of the severe HA cases in previous studies but in the majority, they were associated with mild and moderate HA cases.¹⁵ But in slight contrast to earlier studies, missense mutations viz, c.403 G>T, c.857 A>C, c.980 T>C and c.1702 G>A were recorded in severe cases with high frequency as compared to moderate cases in our study cohort. Apart from two missense mutations viz, c.5387 A>G and c.5573 C>T, none of the mutations were recorded in mild HA cases. Previous studies recorded null mutations to be responsible for 80%, 15%, and less than 1% of cases of severe, moderate, or mild hemophilia A, respectively.¹⁵

Severe hemophilia cases develop inhibitors more commonly than moderate/mild cases. In our study, all 13 inhibitor positive HA patients belonged to severe group . Mutations which are expected to be associated with high risk inhibitor production were frameshift mutations and nonsense mutations. Mutations which were associated with partial production of FVIII protein showed a low risk for inhibitor production.^46-49 It was recorded that severe HA patients with pathogenic mutations developed inhibitors more frequently (22% to 67%) than those with missense mutations (5%). As opposed to this, 46.2% of the inhibitor positive HA patients recorded pathogenic nonsense and frameshift mutations [c.1203G>A, c.1628C>A, c5953C>T, c.6869G>A, c.2645-2646 DelTG, c.3637DelA and c.5293-5295DelCCC], followed by 30.7% missense mutations [c.857A>C, c.980T>C, c.1702G>A, c.5387A>G and c.5573C>T] and 23.1% of inhibitor positive HA patients did not record any mutation in exonic region. Negative selection of anti-FVIII-specific T and B cells does not occur, and regulatory T cells that are specific to FVIII cannot be produced. Then, lymphocytes that are specifically anti-FVIII enter the periphery and may react against the infused FVIII product.¹⁵ Patients with these mutations have a higher chance of acquiring inhibitors because they completely lack any production of circulating FVIII polypeptides due to large deletions, nonsense mutations, and intron 1 and 22 inversions. In case of missense mutation risk of inhibitor, production is high when the mutation is located in the light chain [A3, C1 or C2 domain] than when it is located outside of the light chain (A1, A2 or B] of FVIII protein.⁵⁰ Interestingly, present study recorded 45.5% of missense mutations [c.403G>T, c.857A>C, c.980T>C, c.1702G>A and c.1809 C>G] in inhibitor positive HA patients being located in A1 and A2 chains. Unfortunately, circumstances are not simple and clear. Patients with identical mutations may or may not consistently produce inhibitors. Other aspects like genetic and non-genetic factors [age at first exposure, type of product used, mode of delivery, the intensity of replacement, and treatment modality] are undoubtedly involved in the inhibitor development.^51,52

Conclusion

The current study points at the fact that frameshift mutations and nonsense mutations play an important role in the development of inhibitors in severe HA cases and missense mutations in the light chain of FVIII also increases the risk for inhibitors. This result is a slight deviation from the reports of earlier Indian studies which could be due to difference in geographic populations, ethnicity and other factors. Hence detailed genotype and phenotype screening for Hemophilia A in relation to severity and development of inhibitors is very much essential for timely monitoring and appropriate treatment.

Conflict of interest

None

Acknowledgement

We thank all hemophilia A patients for agreeing to participate in the study. We thank Department of Pathology, JJM Medical College, Davangere, and Karnataka Institute for DNA Research, Dharwad and Karnataka Hemophilia Society, Davangere for their constant support throughout the research. Special thanks to Rajiv Gandhi University Health Sciences, Advanced Research Project for the support and encouragement.