Abstract：
OBJECTIVE: Analysis of the F8 gene helps predict the risk of developing factor VIII (FVIII) inhibitors and the depth of phenotype in haemophilia A (HA) patients. Since data in Southeast Asian countries remain scarce, we aim to study F8 variation correlated with HA phenotypes in Thailand.
METHODS: Thai patients with HA were enrolled from seven haemophilia treatment centres during 2022-2023. Using peripheral blood DNA, inverse shifting-polymerase chain reaction (IS-PCR) for F8-intron 22 inversion (Inv22) and F8-intron 1 inversion (Inv1) was performed. Whole exome sequencing (WES) was explored in cases without Inv22/Inv1.
RESULTS: Of 124 patients with HA, 91.9% were detected with a causative F8 variant, including Inv22 (30.6%), Inv1 (1.6%), missense (23.4%), nonsense (16.9%) and small insertion/deletion (16.1%) mutations. Inv22, small insertion/deletion and nonsense were associated with severe HA, compared with missense variants, by the ORs of 13.9 (95% CI, 4.2 to 56.7), 14.7 (95% CI, 3.4 to 104.7) and 15.6 (95% CI, 3.6 to 110.2), respectively. While nonsense variants affecting the light chain increased the risk of developing FVIII inhibitors (OR, 6.8; 95% CI, 1.5 to 32.6) compared with the low-risk (small insertion/deletion, missense and splice-site) variants. Twelve patients (9.7%) harboured novel F8 variants, comprising five missense (p.Pro540Leu, p.Ser564Pro, p.Leu668Pro, p.Ala1721Glu, p.His2024Pro), five small insertion/deletion (p.Val502SerfsTer13, p.Ile522PhefsTer13, p.Phe992LysfsTer11, p.Leu1223PhefsTer18, c.6427_6429+3delATGGTA) and one nonsense mutations (p.Glu1292Ter).
CONCLUSIONS: IS-PCR followed by WES successfully assesses F8 alterations in most HA cases. With several unique variants, severe HA in Thailand is considerably caused by Inv22, small insertion/deletion and nonsense, whereas missense variants are more responsible for nonsevere HA phenotypes.
METHODS: Thai patients with HA were enrolled from seven haemophilia treatment centres during 2022-2023. Using peripheral blood DNA, inverse shifting-polymerase chain reaction (IS-PCR) for F8-intron 22 inversion (Inv22) and F8-intron 1 inversion (Inv1) was performed. Whole exome sequencing (WES) was explored in cases without Inv22/Inv1.
RESULTS: Of 124 patients with HA, 91.9% were detected with a causative F8 variant, including Inv22 (30.6%), Inv1 (1.6%), missense (23.4%), nonsense (16.9%) and small insertion/deletion (16.1%) mutations. Inv22, small insertion/deletion and nonsense were associated with severe HA, compared with missense variants, by the ORs of 13.9 (95% CI, 4.2 to 56.7), 14.7 (95% CI, 3.4 to 104.7) and 15.6 (95% CI, 3.6 to 110.2), respectively. While nonsense variants affecting the light chain increased the risk of developing FVIII inhibitors (OR, 6.8; 95% CI, 1.5 to 32.6) compared with the low-risk (small insertion/deletion, missense and splice-site) variants. Twelve patients (9.7%) harboured novel F8 variants, comprising five missense (p.Pro540Leu, p.Ser564Pro, p.Leu668Pro, p.Ala1721Glu, p.His2024Pro), five small insertion/deletion (p.Val502SerfsTer13, p.Ile522PhefsTer13, p.Phe992LysfsTer11, p.Leu1223PhefsTer18, c.6427_6429+3delATGGTA) and one nonsense mutations (p.Glu1292Ter).
CONCLUSIONS: IS-PCR followed by WES successfully assesses F8 alterations in most HA cases. With several unique variants, severe HA in Thailand is considerably caused by Inv22, small insertion/deletion and nonsense, whereas missense variants are more responsible for nonsevere HA phenotypes.
摘要：
目的：对F8基因的分析有助于预测A型血友病（HA）患者发展因子VIII（FVIII）抑制剂的风险和表型深度。由于东南亚国家的数据仍然很少，我们旨在研究泰国与HA表型相关的F8变异。
方法：在2022-2023年期间，从七个血友病治疗中心招募了泰国HA患者。使用外周血DNA，进行F8-内含子22反转(Inv22)和F8-内含子1反转(Inv1)的反向移位-聚合酶链反应(IS-PCR)。在没有Inv22/Inv1的情况下探索全外显子组测序(WES)。
结果：在124例HA患者中，91.9%被检测到有致病F8变异，包括Inv22（30.6%），Inv1（1.6%），错觉(23.4%)，无义(16.9%)和小插入/缺失(16.1%)突变。Inv22，小的插入/缺失和无义与严重的HA有关，与错义变体相比，通过13.9的OR(95%CI，4.2至56.7)，14.7（95%CI，3.4至104.7）和15.6（95%CI，3.6至110.2），分别。虽然影响轻链的无义变体增加了开发FVIII抑制剂的风险（OR，6.8；95%CI，1.5至32.6）与低风险（小插入/缺失，错义和剪接位点)变体。12名患者（9.7%）携带新的F8变异体，包括五个错觉(p。Pro540Leu,p.Ser564Pro,p.Leu668Pro,p.Ala1721Glu,p.His2024Pro)，五个小的插入/删除(p.Val502SerfsTer13，p.Ile522PhefsTer13，p.Phe992LysfsTer11，p.Leu1223PhefsTer18，c.6427_64293delATGGTA）和一个无义突变（p。Glu1292Ter）。
结论：IS-PCR和WES在大多数HA病例中成功评估了F8改变。有几个独特的变体，泰国的严重HA是由Inv22、小插入/删除和无意义引起的，而错义变异是非严重HA表型的主要原因。
方法：在2022-2023年期间，从七个血友病治疗中心招募了泰国HA患者。使用外周血DNA，进行F8-内含子22反转(Inv22)和F8-内含子1反转(Inv1)的反向移位-聚合酶链反应(IS-PCR)。在没有Inv22/Inv1的情况下探索全外显子组测序(WES)。
结果：在124例HA患者中，91.9%被检测到有致病F8变异，包括Inv22（30.6%），Inv1（1.6%），错觉(23.4%)，无义(16.9%)和小插入/缺失(16.1%)突变。Inv22，小的插入/缺失和无义与严重的HA有关，与错义变体相比，通过13.9的OR(95%CI，4.2至56.7)，14.7（95%CI，3.4至104.7）和15.6（95%CI，3.6至110.2），分别。虽然影响轻链的无义变体增加了开发FVIII抑制剂的风险（OR，6.8；95%CI，1.5至32.6）与低风险（小插入/缺失，错义和剪接位点)变体。12名患者（9.7%）携带新的F8变异体，包括五个错觉(p。Pro540Leu,p.Ser564Pro,p.Leu668Pro,p.Ala1721Glu,p.His2024Pro)，五个小的插入/删除(p.Val502SerfsTer13，p.Ile522PhefsTer13，p.Phe992LysfsTer11，p.Leu1223PhefsTer18，c.6427_64293delATGGTA）和一个无义突变（p。Glu1292Ter）。
结论：IS-PCR和WES在大多数HA病例中成功评估了F8改变。有几个独特的变体，泰国的严重HA是由Inv22、小插入/删除和无意义引起的，而错义变异是非严重HA表型的主要原因。
