Accessibility and visibility of genetic testing for haemophilia across Europe: Where do we stand?

INTRODUCTION: Haemophilia is characterized by bleeding complications resulting from clotting factor VIII (FVIII) or IX (FIX) deficiency. Identifying the causal pathogenic genetic variant denotes a vital aspect of haemophilia management. AIM: This study evaluated the accessibility and performances of genetic testing for haemophilia across Europe. The types of genetic analyses, techniques used, turn-around time (TAT) and costs were collected and analysed, as were data updating and quality control. METHODS: Reported data were retrieved from open access resources, including international databases, Google, laboratory websites, PubMed and government organizations. RESULTS: Overall, 51 genetic laboratories across 15 European countries providing recently updated molecular haemophilia testing were identified. Gene sequencing for small variants of both F8 and F9 genes was provided in most surveyed laboratories. Almost two-thirds of them offer analysis for inversions using a polymerase chain reaction (PCR) method and detection of copy number variation (CNV) using multiplex ligation-dependent probe amplification (MLPA). Cost and TAT were found to vary considerably. In total, 74% of laboratories exhibited a last modified date or change history. The same percentage of laboratories was in possession of an ISO 15189 standard accreditation, whereas only few of them recently performed external quality assessment schemes (EQA) for haemophilia. CONCLUSION: Despite several initiatives to improve access to genetic testing for haemophilia, such access must still be improved. Our study similarly revealed the large heterogeneity of the variants tested, techniques employed, TAT, cost and quality among the surveyed laboratories.

Genetic mosaicism in haemophilia: A practical review to help evaluate the risk of transmitting the disease.

Approximately 70% of patients with haemophilia exhibit a clear inheritance pattern, while for the remaining 30%, patients are the first to be diagnosed in their family and are considered sporadic cases. In such a setting, the determination of carrier status and the risk estimation of disease transmission to another child are major challenges for genetic counselling. Large studies have suggested that genetic testing reveals 70% of sporadic patients' mothers are carriers. In the remaining 30%, in some apparently non-carrier mothers, the pathogenic variant can be detected as low somatic and gonosomal mosaicism. The significance of mosaic pathogenic variants has thus far been underestimated, since conventional Sanger sequencing and other technology are not sufficiently sensitive. The study of various tissue samples and recent extra-sensitive molecular methods have now made it easier to detect both single-nucleotide variants (SNVs) and copy-number variants (CNVs), at a mosaic level in parents, and to predict the probability of disease recurrence. This review seeks to examine various kinds of mosaicism in haemophilia, including the mechanisms by which they arise and the risk of passing these variants on to the next generation. In addition, we focus on the selection of cell tissues and methods to detect these mosaic variants in the haemophilia setting. Taking into account the high rate of mosaicism in mothers of sporadic cases, we propose a diagnostic flow chart that could facilitate better evaluation of the risk of transmitting haemophilia in genetic and prenatal counselling.

Inhibitor epidemiology and genetic-related risk factors in people with haemophilia from Côte d'Ivoire.

INTRODUCTION: In Sub-Saharan Africa, inhibitor prevalence data in people with haemophilia (PWH) are scarce, as are data on genetic or treatment-related risk factors. AIMS AND METHODS: We performed a prospective study on PWH from Côte d'Ivoire to collect data into inhibitor prevalence, create a database of haemophilia genotypes, establish correlations between inhibitor presence and genetic variants identified amongst Ivoirian PWHs and evaluate exposure to CFCs. RESULTS: The study included 54 unrelated participants (43 severe, four moderate, two mild haemophilia A and five severe haemophilia B). PWH were treated on-demand with various product types for short periods, non-intensively, and using low-dose regimens. We reported similar distributions of intron 22 inversions (39.5%), point pathogenic variants (32.6%) and rearrangements in Ivoirian severe haemophilia A patients versus non-African ethnic groups. The haplotypes H1 (29.6%), H2 (36.3%) and H3 (34.1%) frequencies in haemophilia A were consistent with results published on African populations. We identified eight new causal variants. An inhibitor was found in 12% of haemophilia A patients previously exposed to replacement therapies. Among PWH with inhibitors, 66.7% had a positive intron 22 inversion and 50% the H1 haplotype. CONCLUSION: This study provides original data on molecular diagnosis of haemophilia, inhibitor prevalence and risk factors for inhibitor development previously associated with inhibitors in Côte d'Ivoire. The low inhibitor prevalence likely reflects the limited exposure to replacement therapy in Côte d'Ivoire. Further larger, multicentric and international studies are needed to gain more insight on inhibitor incidence and risk factors in African PWH.

Functional assessment of TSC2 variants identified in individuals with tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in the TSC1 or TSC2 genes. The TSC1 and TSC2 gene products, TSC1 and TSC2, form a complex that inhibits the mammalian target of rapamycin (mTOR) complex 1 (TORC1). Here, we investigate the effects of 78 TSC2 variants identified in individuals suspected of TSC, on the function of the TSC1-TSC2 complex. According to our functional assessment, 40 variants disrupted the TSC1-TSC2-dependent inhibition of TORC1. We classified 34 of these as pathogenic, three as probably pathogenic and three as possibly pathogenic. In one case, a likely effect on splicing as well as an effect on function was noted. In 15 cases, our functional assessment did not agree with the predictions of the SIFT amino acid substitution analysis software. Our data support the notion that different, nonterminating TSC2 mutations can have distinct effects on TSC1-TSC2 function, and therefore, on TSC pathology.

Intron 22 homologous regions are implicated in exons 1-22 duplications of the F8 gene.

The intron 22 inversion found in up to 50% of severe hemophilia A patients results from a recombination between three intron 22 homologous copies (int22h). This study evaluated the implication of these copies in the formation of extended duplications comprising exons 1-22 of the factor 8 (F8) gene and their association with hemophilia and mental retardation. Two hemophilic patients with moderate and severe phenotypes and a third nonhemophilic patient with developmental delay were studied. All exhibited a duplication of F8 gene exons 1-22 identified by multiplex ligation-dependent probe amplification along with abnormal patterns on Southern blotting and unexpected long-range PCR amplification. Breakpoint analysis using array comparative genomic hybridization was performed to delimit the extent of these rearrangements. These duplications were bounded on one side by the F8 intragenic int22h-1 repeat and on the other side by extragenic int22h-2 or int22h-3 copies. However, the simultaneous identification of a second duplication containing F8 gene exons 2-14 for the moderate patient and the classical intron 22 inversion for the severe patient are considered in this study as the genetic causal defects of hemophilia. This study shows that the well-known int22h copies are involved in extended duplications comprising F8 gene exons 1-22. These specific duplications are probably not responsible for hemophilia and intellectual disability, but should be carefully considered in genetic counseling, while continuing to investigate the causal mutation of hemophilia.

CF gene and cystic fibrosis transmembrane conductance regulator expression in autosomal dominant polycystic kidney disease.

Disease-modifying genes might participate in the significant intrafamilial variability of the renal phenotype in autosomal dominant polycystic kidney disease (ADPKD). Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is a chloride channel that promotes intracystic fluid secretion, and thus cyst progression, in ADPKD. The hypothesis that mutations of the CF gene, which encodes CFTR, might be associated with a milder renal phenotype in ADPKD was tested. A series of 117 unrelated ADPKD probands and 136 unaffected control subjects were screened for the 12 most common mutations and the frequency of the alleles of the intron 8 polymorphic TN: locus of CF. The prevalence of CF mutations was not significantly different in the ADPKD (1.7%, n = 2) and control (3.7%, n = 5) groups. The CF mutation was DeltaF508 in all cases, except for one control subject (1717-1G A). The frequencies of the 5T, 7T, and 9T intron 8 alleles were also similar in the ADPKD and control groups. Two additional patients with ADPKD and the DeltaF508 mutation were detected in the families of the two probands with CF mutations. Kidney volumes and renal function levels were similar for these four patients with ADPKD and DeltaF508 CFTR (heterozygous for three and homozygous for one) and for control patients with ADPKD collected in the University of Colorado Health Sciences Center database. The absence of a renal protective effect of the homozygous DeltaF508 mutation might be related to the lack of a renal phenotype in CF and the variable, tissue-specific expression of DeltaF508 CFTR. Immunohistochemical analysis of a kidney from the patient with ADPKD who was homozygous for the DeltaF508 mutation substantiated that hypothesis, because CFTR expression was detected in 75% of cysts (compared with <50% in control ADPKD kidneys) and at least partly in the apical membrane area of cyst-lining cells. These data do not exclude a potential protective role of some CFTR mutations in ADPKD but suggest that it might be related to the nature of the mutation and renal expression of the mutated CFTR.