Systematic molecular analysis of hemophilia A patients from Colombia

Abstract

Hemophilia A (HA) is an X-linked recessive disorder and the second most common coagulation disorder with an incidence of 1 in 5,000 live born males. Worldwide, there are 178,500 affected individuals, 60% with the severe form of the disease. Intron 22 and 1 inversions (Inv22 and Inv1) are the most frequent molecular alterations found in severe HA patients with a frequency of 45-50% and 0.5-5%, respectively. We have implemented a systematic cost-effective strategy for the identification of the molecular alteration in HA patients using Inverse shifting-PCR for Inv22 and Inv1, followed by the analysis of the F8 gene coding region by means of high resolution melting (HRM) PCR and Sanger sequencing in Inv22 and Inv1 negative patients. A total of 33 male HA patients and 6 women were analyzed. Inversion 22 was detected in 14/33 male patients (42.4%), 3/33 (9.1%) had Inv1, 3/33 (9.1%) had large structural variants, and 11/33 (33.3%) single nucleotide/ small frameshift variants. No genetic variant was found in 2/33 patients (6%). With this systematic approach we detected pathogenic variants in 31 out of 33 male affected individuals (94%) tested for the first time.in a cohort of patients from Colombia.

Genetic and Bioinformatic Strategies to Improve Diagnosis in Three Inherited Bleeding Disorders in Bogotá, Colombia

“…The intron 22 and 1 inversions (Inv22 and Inv1) are the most frequent molecular alterations found in severe HA patients with a frequency of 45-50% and 0.5-5%, respectively. However, in a study carried out in Colombia by Yunis et al (2018), inversion 22 was detected in 14/33 male patients (42.4%), and inversion 1 was detected in 3/33 [10]. These results are consistent with our study since it must be considered that, in total, we had 11 patients with hemophilia A, from which four were men classified as severe, three men classified as mild, and four carriers.…”

“…Table 3 summarizes and classifies the variations found in the population under study, according to their effect on the protein and their report in the literature. To our knowledge, these new variants have not been reported in other previous studies carried out in the Colombian population [10]. For each domain, the start and end of the melting region were found by finding the minimum points of the curve that enclosed the peak.…”

“…In these cases, domain analysis allowed us to improve the previously reported sensitivity of 93% [11] to 95% at a lower cost. Unlike the methodology used by other studies where 52 reactions per patient were needed to carry out the gene analysis [10,11], we only used 35 reactions per patient. We propose a protocol for molecular analysis using HRM that supports the clinical diagnosis of patients with hemophilia, shown in Figure 7.…”

Molecular study of a large cohort of 109 haemophilia patients from Cuba using a gene panel with next generation sequencing‐based technology

“…In addition, the spectrum of variants in moderate HA did not correlate with those in previous reports, as most of them (72%) led to a null allele in the Cuban cohort, including 12 Inv22 and 4 Inv1. 7,24,[34][35][36][37] Both these discrepancies can be attributed to variability affecting FVIII:C determination, used to classify the patients. These could be due to the two different methods used and the intrinsic variability of each technique.…”

Condições clí­nicas de pacientes com hemofilia assistidos em um hemocentro regional: um estudo transversal

“…No perfil traçado por esse estudo, a gravidade da hemofilia, mostrou-se grave, corroborando com estudos realizados em outras localidades, como no Hemocentro Regional de Pelotas-HE-MOPEL 7 . Em uma pesquisa realizada na Colômbia obteve-se que em uma amostra de 33 pacientes hemofílicos, a severidade da doença no nível grave representou 82% 12 .…”