Genetic analysis of haemophilia carrier status

DE BRASI CD; ROSSETTI LC; RADIC CP

Glasgow

Simposio; Session "Current Laboratory Issues in Genetic Analysis", World Federation of Hemophilia Congress 2018; 2018

World Federation of Hemophilia (WFH)

As a mediumincome country from Latin-America, laboratory procedures for genetic analysisof haemophilia in Argentina are characterised by its cost-effectiveness,covering virtually all requirements to offer genetic counselling to affectedfamilies that include reliable carrier diagnosis. The aim of this section is todemonstrate our set of applied molecular approaches for genotyping the F8 andF9 genes, achieving carrier diagnosis in haemophilia A (HA) and haemophilia B(HB), respectively; additionally, to discuss the technical approaches toanalyse and understand the molecular basis of haemophilia symptoms expressed incarriers and in those having genetic mosaicisms. In families with at least acase with severe-HA, our laboratory algorithm for F8-analysis starts with theinvestigation of haemophilia inversion hotspots, the F8 intron 22 inversion(Inv22) and intron 1 inversion (Inv1) causing 35-50% and 2-5% of severe-HAcases, respectively. Analysis of Inv22 and Inv1 are performed by inverseshifting-PCR (IS-PCR) and includes simultaneous detection of allint22h-associated rearrangements including deletions (Del22), duplications(Dup22) and BclI-related variants (1) (IS-PCR version 2016). Non-inversionint22h-mediated rearrangements (i.e., Dup22 and Del22) are rarely, or not,detected in a Haemophilia Gene Testing Laboratory as none of them expresseshaemophilia. Hemizygous Del22 associates with in utero lethality whileheterozygous females show extremely skewed-XCI (X-chromosome inactivation) andrecurrent abortions of male foetuses. Dup22, comprises a structurally normalcopy of F8, associates with recognisable facial features and mild cognitivedeficiency in hemizygous males while heterozygous Dup22 females show extremelyskewed-XCI (2). Thereafter, the screening F8 or F9 large deletions areperformed in a sample from the hemizygous patient by applying PCR amplificationschemes including 38 and 12 amplimers, respectively, and sets of genomic tagsup-and downstream of each gene until defining the limits of sequence loss.Carrier detection of large deletions may be achieved by either analyzing adeletion-specific PCR product across its 5′ and 3′ breakpoint junctions (3), orby a gene dosage approach using quantitative real time-PCR (qPCR) using anX/autosome amplification ratio and samples from control males and females toassess deletion carrier probabilities (4). Small F8/F9 mutations includinginsertions or deletions of <10 bp and nucleotide substitutions are primarilydetected by high-resolution conformation-sensitive gel electrophoresis (CSGE)and characterised by Sanger sequencing (5, 6). Finally, the significance andpotential phenotypic impact of the observed F8/F9 genotype in the hemizygous orheterozygous state should be critically investigated and provided in thegenetic report. A crucial topic to understand haemophilia expression in carrierfemales relates to the phenomenon of XCI. The assessment of XCI takes advantageof the epigenetic methylation marks associated with the transcriptionalsilencing of genes AR (on Xq12) (7) and RP2 loci (on Xp11.3) (8). Although inmost cases XCI is a random process, roughly resulting in an average of half ofthe cells having paternal (or alternatively maternal) X-inactive; in rarecases, for different complex causes (discussed in Puck & Willard (9)), XCImay be skewed, shutting down expression of the targeted X chromosome in allcells from a tissue under analysis. In haemophilia carrier females, the formerscenario associates with about a half of normal factor VIII activity (FVIII:C)levels while the latter (carriers with skewed-XCI) may associate with eithernormal FVIII:C levels or with significantly reduced FVIII:C, which may causehaemophilia symptoms, depending on the gamete phase between the F8-mutation andthe preferentially active-X. In contrast to this theoretical framework, anunexpected absence of correlation between FVIII:C and XCI levels in bloodleucocytes from haemophilia carriers was published (10). In order to addressthis correlation, we designed a linear binary model (V-model) taking intoaccount, the experimental and biological variability of FVIII:C existing innon-carriers in which XCI (either skewed or random) does not play any role(11). Applying the V-model, Radic et al (2015) analysed the FVIII:C-XCI correlationin a series of 38 heterozygous carriers of severe F8 mutations versus 20non-carrier controls and concluded that the theoretically natural correlation(i.e., extreme XCI-extreme FVIII:C) cannot be statistically excluded as theunderlying mechanism of haemophilia expression in carriers (11). The tissue tointerrogate for XCI to infer its state in hepatocytes (FVIII and FIX secretingcells) is of crucial importance. In our experience, the ideal tissue to achievethis indirect estimation is through studying cells collected from urine(desquamated from bladder and ureter inner epithelia), as they derive fromendoderm (the same embryonic layer as liver derives from) and there are noarguments to predict a significant departure from its original XCI state. Onthe other hand, peripheral blood leucocyte samples hardly resemble the relevantliver?s XCI mosaic level as they are derived from embryonic mesoderm and aresubjected to selected clonal expansions and retractions associated with immunesystem activity, which raise the XCI skewing rate with the female?s age (12).The molecular tools necessary for detection and assessment of geneticmosaicisms from suitable tissue samples as surrogate for inferring the state ofthe relevant tissues in haemophilia patients and carriers are of importance toestimate the haemophilia-phenotype expression and the variant heritabilityassociated with the gonads. In addition, we have reported an interesting caseof a combined somatic-gonadal mosaic involving the maternal grandfather of asevere-HA affected patient with a 2 kb F8-promoter deletion in whom thepercentage of mosaic cells measured in urine and peripheral blood closely alignwith FVIII:C levels and the ratio of carrier to non-carrier offspring,respectively (13). In conclusion, carrier diagnosis in haemophilia can besuccessfully achieved in developing countries covering the provision of all crucialmolecular information required to offer accurate genetic counselling.