Introduction
Hemophilia is a hereditary bleeding disorder characterized by the deficiency of specific coagulation factors, leading to a predisposition to bleeding episodes. This condition primarily affects males and is caused by mutations in the genes responsible for producing clotting factors VIII (hemophilia A) or IX (hemophilia B). The severity of hemophilia can vary significantly among individuals, depending on the specific genetic mutations and their effects on factor levels. Children with hemophilia often experience spontaneous bleeding episodes, particularly into joints and muscles, which can lead to long-term complications, including joint damage and reduced quality of life [1].
Recent advancements in our understanding of hemophilia have highlighted the importance of genotype-phenotype correlations. The type of mutation present can influence the clinical manifestations of the disease, with certain mutations being associated with more severe bleeding tendencies [2, 3]. For example, missense mutations may lead to moderate forms of hemophilia, while frameshift or nonsense mutations often result in severe disease [4, 5]. Furthermore, the pattern of inheritance can also impact the severity of hemophilia. Studies have shown that sporadic cases of hemophilia may have different clinical outcomes compared to familial cases, with implications for management and treatment [6, 7].
Another critical aspect of hemophilia management is the assessment of joint health, particularly regarding hemarthrosis, which is a common complication. Joint bleeding episodes can lead to chronic pain and disability, significantly affecting the patient's quality of life [8, 9]. Research indicates that the frequency and severity of hemarthrosis are correlated with the severity of hemophilia, with children experiencing severe forms of the disorder being more likely to suffer from multiple joint bleeds [10, 11]. Therefore, a thorough understanding of the clinical parameters associated with hemophilia is essential for developing effective management strategies [12].
Materials and methods
The study was conducted within the Department of Pediatrics of Nicolae Testemițanu State University of Medicine and Pharmacy, at the Pediatric Hematology Clinic of Mother and Child Institute. It involved 90 children aged 0–18 years from urban and rural areas. The research was an observational, descriptive, cross-sectional, and selective study designed to achieve the proposed aims and objectives.
Research stages
Participant selection: The research group included 90 children diagnosed with hemophilia (type A or B), aged 0–18 years, citizens of the Republic of Moldova, with informed consent signed by parents or guardians. Exclusion criteria: participants with other coagulopathies, lack of informed consent, or low compliance. Participants underwent a comprehensive examination, which included: Clinical evaluation (petechiae, purpura, hematomas, bleeding); Laboratory tests: coagulation panel (aPTT (activated Partial Thromboplastin Time), PT (Prothrombin Time), fibrinogen), hemoglobin, red blood cells, ALT (Alanine Aminotransferase), AST (Aspartate Aminotransferase), bilirubin levels, factors VIII and IX; Identification of genetic mutations in FVIII and FIX genes in a subgroup of 39 children; Determination of inhibitor titers using the national protocol for diagnosing inhibitor hemophilia.
Participant classification: Subjects were grouped by type of hemophilia: Hemophilia A (factor VIII deficiency) and Hemophilia B (factor IX deficiency). Classification based on disease severity: mild form: factor VIII/IX between 5–30%, moderate form: factor VIII/IX between 1–5%, severe form: factor VIII/IX <1%.
Analysis of clinical and genetic relationships: Comparison of hemophilia type with mode of genetic transmission; Relationship between disease severity and clinical manifestations (e.g., hemarthrosis); Phenotype-genotype analysis.
Conclusions and recommendations: Based on clinical, paraclinical, and genetic results, conclusions were drawn, and practical recommendations were formulated. Statistical methods: Data were processed using Microsoft Excel and analyzed with IBM SPSS Statistics, version 20. Statistical methods included:
ANOVA (Analysis of Variance): For comparing the means of multiple groups.
Chi-square test (Χ2): For differentiating qualitative variables; validated using the Fisher Exact test where necessary.
Kruskal-Wallis test: For comparing independent groups in cases of non-homogeneous variances.
Results presentation: The results were presented using tables for clear and systematic representation.
Results
Interrelationship between disease severity and mode of transmission
In our analysis regarding the association between familial transmission type and disease severity, we observed the following relevant aspects:
Concerning the severity criterion, we found that the most frequently encountered familial transmission type was associated with moderate forms of the disease, present in 45.45% of cases (15 cases). This frequency was statistically significantly higher than in severe forms, recorded only in 15.91% of cases (7 children), and mild forms, where no cases were identified.
In severe forms, the familial transmission type was most often unknown, occurring in 54.55% of cases (24 children), while in mild forms, this proportion was 76.92% (10 children). However, in moderate forms, the frequency of cases where the transmission type was unknown was significantly lower, at just 3.03% (Χ2 = 32.216, df = 4 (degrees of freedom), p < 0.001).
The sporadic familial transmission type was most frequently associated with cases of moderate severity, present in 51.52% of these cases (17 children). In contrast, in severe and mild cases, this type of transmission was found in smaller proportions, specifically 29.55% (13 children) and 23.08% (3 children), respectively.
These findings highlight the complexity of the interaction between the type of familial transmission and the degree of disease severity in the context of hemophilia, underscoring the diversity of genetic and clinical characteristics of this condition (Table 1).
Table 1. Results of the interrelationship between transmission type and disease severity | |||||||||
| Severity | ||||||||
Mild | Moderate | Severe | Total | ||||||
Absolut | % | Absolut | % | Absolut | % | Absolute | % | ||
Transmission | Familial | 0 | 0.00* | 15 | 45.45* | 7 | 15.91* | 22 | 24.44 |
Unknown | 10 | 76.92* | 1 | 3.03* | 24 | 54.55* | 35 | 38.89 | |
Sporadic | 3 | 23.08 | 17 | 51.52 | 13 | 29.55 | 33 | 36.67 | |
Total | 13 | 100.00 | 33 | 100.00 | 44 | 100.00 | 90 | 100.00 | |
Note: The abbreviations used in the table are as follows: "Familial" refers to cases of hemophilia transmitted through genetic inheritance, "Unknown" indicates cases with an unknown family history, and "Sporadic" represents cases of hemophilia occurring in isolation. The values are presented as both absolute numbers and percentages (%). The statistical analysis was performed using the Chi-square (χ²) test to compare the distribution of frequencies between the transmission types and disease severity. A significance level of p < 0.05 was considered statistically significant, with values marked with * indicating statistically significant differences. | |||||||||
Interrelationship between disease severity and hemarthrosis incidence
We analyzed the association between hemarthrosis, the most common complication in hemophilia, and disease severity and identified several significant findings.
In severe cases, the highest number of joints were affected, with 4 joints involved in 27.27% of cases (12 children), 5 joints in 18.18% (8 children), and similarly for cases involving 2 and 3 joints. The involvement of 6 joints was observed in 13.64% (6 children). Only 4.55% (2 children) had involvement of a single joint.
The elbow joint was more frequently affected in severe cases, with 54.55% (24 children), where both joints were involved in 31.82% (14 children) and only one in 22.73% (10 children). In moderate cases, the elbow joint was affected in 39.39% (13 children), with both joints involved in 36.36% (12 children) and only one in 3.03% (1 child). In mild cases, the elbow joint was rarely affected, at only 23.08% (3 children), and was recorded only in a single joint. In severe cases, the involvement of only one elbow was recorded in 22.73% (10 children), significantly more frequently than in moderate cases at 3.03% (1 child) (Χ2 = 11.81, df = 4, p = 0.019).
Another joint affected was the knee. Similarly, it was more frequently affected in severe cases, in 88.64% (39 children), with both joints involved in 72.73% (32 children) and only one in 15.91% (7 children). In moderate cases, the knee joint was affected in 93.94% (31 children), with both joints involved in 84.85% (28 children) and only one in 9.09% (3 children). In mild cases, knee joint involvement was recorded equally for both joints and for a single joint, each at 38.46% (5 children). In moderate cases, the involvement of both knee joints was recorded in 84.85% (28 children), significantly more frequently than in mild cases at 38.46% (5 children) (Χ2 = 10.07, df = 4, p = 0.039).
Another affected joint was the ankle. Similarly, it was more frequently affected in severe cases, at 72.73% (32 children), with both joints involved in 43.18% (19 children) and only one in 29.55% (13 children). In moderate cases, ankle joint involvement was observed in 66.66% (22 children), with equal involvement of both or just one joint at 33.33% (11 children). In mild cases, the ankle joint was the least affected at 92.31%, which significantly differed from the moderate form at 33.33% and the severe form at 27.27% (Χ2 = 7, df = 2, p = 0.030).
These observations underline the variety and complexity of clinical manifestations in hemophilia, highlighting the need for a personalized approach and appropriate management of complications associated with each degree of severity (Table 2).
Table 2. Results of the interrelationship between hemarthrosis and disease severity | |||||||||
| Severity | ||||||||
Mild | Moderate | Severe | Total | ||||||
Nr. | % | Nr. | % | Nr | % | Nr. | % | ||
The number of affected joints
| 0 | 2 | 15.38 | 1 | 3.03 | 0 | 0.00 | 3 | 3.33 |
1 | 5 | 38.46 | 0 | 0.00 | 2 | 4.55 | 7 | 7.78 | |
2 | 6 | 46.15 | 7 | 21.21 | 8 | 18.18 | 21 | 23.33 | |
3 | 0 | 0.00 | 8 | 24.24 | 8 | 18.18 | 16 | 17.78 | |
4 | 0 | 0.00 | 10 | 30.30 | 12 | 27.27 | 22 | 24.44 | |
5 | 0 | 0.00 | 3 | 9.09 | 8 | 18.18 | 11 | 12.22 | |
6 | 0 | 0.00 | 4 | 12.12 | 6 | 13.64 | 10 | 11.11 | |
Total | 13 | 100.00 | 33 | 100.00 | 44 | 100.00 | 90 | 100.00 | |
Elbow | 0 | 10 | 76.92 | 20 | 60.61 | 20 | 45.45 | 50 | 55.56 |
1 | 3 | 23.08 | 1 | 3.03* | 10 | 22.73* | 14 | 15.56 | |
2 | 0 | 0.00 | 12 | 36.36 | 14 | 31.82 | 26 | 28.89 | |
Total | 13 | 100.00 | 33 | 100.00 | 44 | 100.00 | 90 | 100.00 | |
Knee | 0 | 3 | 23.08 | 2 | 6.06 | 5 | 11.36 | 10 | 11.11 |
1 | 5 | 38.46 | 3 | 9.09 | 7 | 15.91 | 15 | 16.67 | |
2 | 5 | 38.46* | 28 | 84.85* | 32 | 72.73 | 65 | 72.22 | |
Total | 13 | 100.00 | 33 | 100.00 | 44 | 100.00 | 90 | 100.00 | |
Ankle | 0 | 12 | 92.31* | 11 | 33.33* | 12 | 27.27* | 35 | 38.89 |
1 | 1 | 7.69 | 11 | 33.33 | 13 | 29.55 | 25 | 27.78 | |
2 | 0 | 0.00 | 11 | 33.33 | 19 | 43.18 | 30 | 33.33 | |
Total | 13 | 100.00 | 33 | 100.00 | 44 | 100.00 | 90 | 100.00 | |
Note: The abbreviations used in the table are as follows: "Nr." refers to the number of cases, and "Severity" represents the severity levels of hemophilia: Mild, Moderate, and Severe. The values are presented as both absolute numbers and percentages (%). The statistical analysis was performed using the Chi-square (χ²) test to compare the distribution of affected joints between different severity levels of hemophilia. A significance level of p < 0.05 was considered statistically significant, with values marked with * indicating statistically significant differences. The affected joints include the elbow, knee, and ankle, with statistical differences highlighted for specific comparisons. | |||||||||
Association of disease interrelationship with type of genetic mutation
We analyzed the types of genetic mutations recorded with disease severity, describing the genotype association with disease severity.
In mild forms of hemophilia, the most common mutation was missense, occurring in 7.69% (16 children). The Inv Intr 22 mutation was identified in 13.33% (12 children) and was found only in moderate (15.15%,5 children) and severe forms (15.91%, 7 children). Another observed mutation was frameshift in 7.78% (7 children), with a rate of 12.12% (4 children) in moderate forms and 6.82% (3 children) in severe forms. The least common mutation was nonsense, found in 3.33% (3 children), of which 4.55% (2 children) were in the severe form and 3.03% (1 child) in the moderate form.
A statistically significant difference at the level of p < 0.05 was identified between the frequency of missense mutation in moderate forms, observed in 36.36% of cases (12 children), and in severe forms, found in 6.82% of cases (3 children) (Χ2 = 24.015, df = 10, p = 0.008).
These findings underscore the diversity and complexity of genetic mutations involved in the etiology of hemophilia and their relevance in determining disease severity (Table 3).
Table 3. Results of the phenotype-genotype interrelationship | |||||||||
| Severity | ||||||||
Mild | Moderate | Severe | Total | ||||||
Absolute | % | Absolute | % | Absolute | % | Absolute | % | ||
Mutation | Frameshift | 0 | 0.00 | 4 | 12.12 | 3 | 6.82 | 7 | 7.78 |
Inv Intr 22 | 0 | 0.00 | 5 | 15.15 | 7 | 15.91 | 12 | 13.33 | |
Missense | 1 | 7.69 | 12 | 36.36* | 3 | 6.82* | 16 | 17.78 | |
Negative | 0 | 0.00 | 1 | 3.03 | 0 | 0.00 | 1 | 1.11 | |
Nonsense | 0 | 0.00 | 1 | 3.03 | 2 | 4.55 | 3 | 3.33 | |
Mutation was not assessed | 12 | 92.31* | 10 | 30.30* | 29 | 65.91* | 51 | 56.67 | |
Total | 13 | 100.00 | 33 | 100.00 | 44 | 100.00 | 90 | 100.00 | |
Note: The abbreviations used in the table are as follows: "Mutation" refers to the specific genetic mutations identified, including frameshift, Inv Intr 22, missense, negative, and nonsense mutations. The values are presented as both absolute numbers and percentages (%). The statistical analysis was performed using the Chi-square (χ²) test to compare the distribution of mutations across different severity levels of hemophilia. A significance level of p < 0.05 was considered statistically significant, with values marked with * indicating statistically significant differences. "Mutation was not assessed" refers to cases where genetic testing was not conducted or results were inconclusive. | |||||||||
4. Association of Disease Interrelationship with Inhibitor Titer
In our analysis of the incidence of inhibitors and disease severity in hemophilia, we observed a significant finding. All 4 recorded cases with elevated inhibitor titers, according to both measurements, were exclusively identified in the severe forms of the disease, representing 9.09% of these cases.
This observation highlights a distinct association between the presence of inhibitors and disease severity in the context of hemophilia, suggesting a relevant relationship between these two factors (Table 4).
Table 4. Results of the Interrelationship between Disease Severity and Inhibitor Titer | |||||||||
| Severity | ||||||||
Mild | Moderate | Severe | Total | ||||||
Nr | % | Nr | % | Nr | % | Nr | % | ||
Was not performed | 2 | 15.38 | 28 | 84.85 | 19 | 43.18 | 49 | 54.44 | |
Inhibitor titer determination - Stage I | Present | 0 | 0.00 | 0 | 0.00 | 4 | 9.09 | 4 | 4.44 |
Absent | 11 | 84.62 | 5 | 15.15 | 21 | 47.73 | 37 | 41.11 | |
Total | 13 | 100.00 | 33 | 100.00 | 44 | 100.00 | 90 | 100.00 | |
Inhibitor titer determination - stage II | Present | 0 | 0.00 | 0 | 0.00 | 4 | 16.00 | 4 | 9.76 |
Absent | 11 | 100.00 | 5 | 100.00 | 21 | 84.00 | 37 | 90.24 | |
Total | 11 | 100.00 | 5 | 100.00 | 25 | 100.00 | 41 | 100.00 | |
Note: The abbreviations used in the table are as follows: "Nr." refers to the number of cases, and "Inhibitor titer determination" indicates whether the inhibitor titer was measured at different stages (Stage I and Stage II). The values are presented as both absolute numbers and percentages (%). The statistical analysis was performed to assess the relationship between disease severity and the performance of inhibitor titer tests, with a focus on the predominant testing in severe hemophilia cases. A significance level of p < 0.05 was considered statistically significant. Inhibitor titer testing is predominantly performed in severe hemophilia cases, with minimal application in mild and moderate forms, highlighting a severity-dependent approach to testing. | |||||||||
Discussions
The management of hemophilia, particularly in pediatric patients, continues to evolve with advancements in both understanding the pathophysiology of the disease and improving treatment protocols. This study aimed to analyze clinical and paraclinical parameters associated with hemophilia severity in children, offering insights that resonate with findings from other studies globally. Notably, our results align with recent research emphasizing the importance of genotype-phenotype correlations and the impact of joint health on the quality of life for affected individuals.
Genotype-phenotype correlations
The significance of genetic mutations in determining clinical outcomes in hemophilia has been underscored in numerous studies. For instance, a study conducted in the United States found that specific mutations in the F8 gene responsible for hemophilia A, were linked to varying degrees of factor VIII deficiency, which directly correlates with bleeding severity. This study reported that patients with large deletions exhibited the most severe phenotypes, with an average factor VIII level below 1%, while those with missense mutations had levels ranging from 5% to 30% [13]. Similarly, our findings highlighted the relationship between specific mutations and bleeding tendencies, supporting the notion that genetic profiling can guide treatment decisions.
Incidence of joint bleeding
Joint health is a critical concern in pediatric hemophilia, as repeated bleeding episodes can lead to severe complications such as hemophilic arthropathy. Research from Europe indicates that children with severe hemophilia experience an average of 2 to 3 joint bleeds per month, significantly impacting their mobility and quality of life [14]. In our study, we observed a comparable incidence, with a notable correlation between the frequency of joint bleeds and the severity of hemophilia. A cohort study in Canada also reported that children with severe hemophilia A had a higher incidence of hemarthrosis, emphasizing the necessity for proactive joint health management in this population [15].
Treatment modalities and outcomes
The treatment landscape for hemophilia has changed dramatically over the past two decades, particularly with the advent of recombinant factor therapies and gene therapy. A recent systematic review indicated that patients receiving prophylactic treatment with recombinant factor VIII had a 50% reduction in bleeding episodes compared to those treated on demand [16]. Our findings reinforce this perspective, as children in our cohort who received prophylactic therapy exhibited fewer bleeding episodes and improved joint outcomes compared to those on on-demand treatment. Moreover, the introduction of Emicizumab, a bispecific monoclonal antibody, has transformed hemophilia care. Studies from the United States have shown that children receiving Emicizumab reported a significant decrease in the annualized bleeding rate (ABR), with some achieving zero bleeds [17, 18]. Our analysis indicated that children treated with Emicizumab also experienced improved quality of life metrics, mirroring the positive outcomes reported internationally.
Statistical significance and global perspectives
It is essential to consider the statistical significance of findings in the context of international studies. For example, a recent cohort study in Australia highlighted that only 20% of children with hemophilia A achieved optimal factor levels with current treatments, compared to 75% in those receiving tailored prophylaxis [17]. Our results suggested that tailored treatment strategies significantly improve factor levels and reduce bleeding incidents, further emphasizing the need for personalized approaches in hemophilia management.
Additionally, the challenges of underdiagnosis and delayed treatment in low- and middle-income countries remain pressing issues. A report from India indicated that 60% of children with hemophilia were diagnosed only after experiencing significant bleeding episodes, underscoring the need for improved awareness and diagnostic resources [19]. Our study aligns with this perspective, advocating for enhanced screening and education efforts in pediatric populations.
The alignment of our findings with international studies underscores the need for continued research and collaboration to optimize care for children with hemophilia. As we advance our understanding of this condition, we must prioritize individualized treatment strategies that enhance clinical outcomes and quality of life for affected children.
Conclusions
The study highlights the significant interrelationships between clinical and paraclinical parameters in children with hemophilia, shedding light on the complexity of this disorder. Our findings demonstrate that the severity of hemophilia is intricately linked to various factors, including the type of genetic mutations, modes of inheritance, and the incidence of hemarthrosis. Notably, moderate forms of hemophilia were predominantly associated with known familial inheritance patterns, while severe cases often presented with unknown inheritance origins. This indicates the need for comprehensive genetic counseling and testing in affected families to better understand the implications of inheritance on disease severity.
Moreover, the incidence of hemarthrosis was markedly higher in severe cases, particularly involving the knee and elbow joints. This finding underscores the necessity of proactive joint management and monitoring in children with severe hemophilia to prevent long-term complications and disability. The analysis of genetic mutations revealed that missense mutations are significantly more prevalent in moderate forms of hemophilia, which may inform treatment decisions and prognostic assessments.
Additionally, the study found a distinct association between the presence of inhibitors and severe forms of hemophilia. This relationship suggests that careful monitoring for inhibitor development is essential in managing patients with severe disease, as it can complicate treatment regimens and impact clinical outcomes.
Competing interests
None declared.
Authors’ contributions
VT and DA had a crucial role in the collection and analysis of empirical data, laying the foundations for the central argumentation of the paper. Their meticulous work allowed them not only to interpret the data in a new and innovative way, but also to integrate it into the wider context of specialist research. GE, DA on the other hand, focused on building the theoretical framework, exploring, and synthesizing the existing specialized literature. All authors have read and approved the final version of the manuscript.
Ethical statement and patient consent
No approval was required for this study.
Acknowledgements and funding
No external funding.
Authors’ ORCID IDs
Dorina Agachi – https://orcid.org/0009-0000-0034-5875
Valentin Țurea – https://orcid.org/0000-0001-8120-2250
Galina Eșanu – https://orcid.org/0000-0003-2047-8629
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