Analysis of Genetic Test Results of 47 Hemophiliacs in Anhui and Nearby Areas in China
Authors: Cheng Zhengyi , Zhang Ziyue , Zhou Li , Zhai Zhimin 
Published: December 2024

Objective To observe 47 cases of hemophiliacs from Anhui Province and nearby areas, analyze and summarize the types of gene mutations in the patients, and explore the pathogenesis of hemophilia.
Methods Whole genome sequencing was used to detect the periphery blood of hemophilia patients. At the same time, long-chain PCR (LD-PCR) technology + gene sequencing + MLPA technology was used to detect F8 gene: long-chain PCR (LD-PCR) technology was used to detect intron 1 and intron 22 inversion mutations of F8 gene. After DNA extraction from the samples, PCR-free sequencing libraries were constructed. High-throughput sequencing was performed on the Illumina sequencing platform, covering coding, non-coding, and gene regulatory regions in the whole genome. The bi-directional sequences obtained were assembled and aligned according to the reference sequence of GRCh37/hg19 from the human genome. The average sequencing depth of the whole genome was more than 30X, with 90% of the regions sequenced at a depth of more than 20X. Potential clinically significant variants were confirmed by Sanger first-generation sequencing. A report of the molecular genetic testing was obtained. The molecular genetic test reports of 47 patients were systematically compiled to identify the causative genes and analyze the causes of the disease in each patient.
Results Of the 47 hemophiliacs who underwent genetic testing, 45 were male and 2 were female. There were patients with hemophilia A(HA), 6 patients with hemophilia B(HB), and 1 patient with an undetermined type of hemophilia. Mutations were detected in all 47 patients with positive detection rate of 100%. Among the 40 HA patients, 10 had intron 22 inversion mutations in the coagulation factor VIII gene, one of which was associated with a pathogenic variant of the CD36 gene with an inversion in intron 22 of F8. 1 had heterozygous intron 22 inversion mutation in the coagulation factor VIII gene. 24 mutation types in the FⅧ gene were detected in 24 HA patients, one of which combined the mutation with a duplication of the Xq28 region of the chromosome. Of the remaining 5 HA patients, 2 had hemizygous deletion variants in the Xq28 region of the chromosome, 1 had mutations in both the VWF gene and the FGA gene, and 2 had no detectable variants that could explain the patient's phenotype. 5 FⅨ mutation types were detected in 6 HB patients. In total, the study found five suspected causative genes including c.6273G&gt;A (p.Lys2091=), c.901C&gt;T (p.Arg301Cys), c.1316del (p.Asn439Thrfs*5), c.1163A&gt;T (p.Gln388Leu), c.6429+9948G&gt;A, c.2039 del (p.Asp680Valfs*4), three mutations of unknown clinical significance including c.1069T&gt;C (p.Tyr357His), c.6429+9948G&gt;A, c.5564C&gt;A (p.Ala1855Asp), and seven unreported variants including c.532T&gt;C (p.Cys178Arg), c.206T&gt;C (p.Leu69Pro), c.601+5 G&gt;A, c.60_70del (p.Thr21Leufs*15), c.2159G&gt;C (p.Gly720Ala), c.7033T&gt;G (p.Cys2345Gly) (p.Asn439Thrfs*5), c.1316del (p.Asn439Thrfs*5).
Conclusion Genetic analysis of mutations in 47 patients identified hemophilia-causing mutations. Whole genome sequencing combined with long-chain PCR (LD-PCR) technology+ MLPA technology can more accurately detect the causative genes of hemophiliacs and the causative genes of other diseases in combination. It is of great significance in the study of hemophilia-related gene diagnosis and pathogenesis, and is conducive to enriching the database of hemophilia-related gene mutations, which is helpful for clinical decision-making.