Improved criterion‐referenced assessment in indirect tracking of haemophilia A using a 0.23 cM‐resolution dense polymorphic marker set

Abstract

In haemophilia A, linkage analysis with coagulation factor VIII (F8) intragenic and/or neighbouring extragenic short tandem repeats (STRs) enables indirect tracking F8 pathogenic allelic variant-carriers. Even where DNA sequencing is available, linkage analysis still has a role if no causative or candidate mutation is unveiled. The cumulative heterozygosity rate of the available multiplexed STRs haplotyping assays rarely reaches 100%. This means that in a proportion of women these loci are uninformative. The norm-referenced assessment is based on at least one informative marker criterion. We reasoned that by typing a dense market set, spanning a small fraction of recombination, we should be able to improve assessment. The aim of this study was to improve criterion-referenced assessment in polymorphism segregation analyses using a low-recombination fraction and dense informative STRs set. The multiplex quantitative fluorescence PCR assay comprises four novel tetranucleotide and pentanucleotide STRs distant < or = 0.15 cM from the F8 gene, and three F8 intragenic dinucleotide STRs, mapped to a 0.23 cM interval spanning the F8 on human chromosome band Xq28. We determined heterozygosity rates and allele frequencies from 100 unrelated healthy females. To investigate about segregation stability, we typed 50 true trios (mother, daughter and father) and 50 true mother-and-son duos from the general population. The heterozygosity rates for the extragenic markers ranged 0.49-0.76. The 0.23 cM-resolution heptaplex rendered a cumulative heterozygosity of 0.89 for a minimum of two informative markers, with at least one F8 intragenic. The heptaplex assay enabled improving the criterion-referenced assessment in indirect carrier-detection.

5meCpG Epigenetic Marks Neighboring a Primate-Conserved Core Promoter Short Tandem Repeat Indicate X-Chromosome Inactivation

“…Matching these criteria should improve the base-calling precision of templates and the measurement of true alleles by effectively limiting Taq polymerase stuttering (the magnitude of stuttering decreases as the repeat unit length increases [28], [29]), and allow to achieve the power of informativeness of the AR disease-linked CAG repeat assay regarding the methylation statuses of X-chromosomes [12] ( AR does not escape XCI [8], and the informativeness of repeats on X correlates with the number of perfect tandem repeat units [29], [30]). The real power of this combined approach for predicting highly polymorphic STR loci in promoter regions is its direct applicability to available X-chromosome sequences of any mammalian species.…”

Interlocus non‐random association of multiallelic polymorphisms spanning the coagulation factor VIII gene on human chromosome distalmost Xq28

“…higher the mean rates for biallelic markers) will be expected to identify >99% of X chromosomes in HEMA at‐risk families. On the other hand, frequent and intense GD between loci comprised in a limited recombination fraction considerably diminishes the chances of observing concurrent mutation and/or recombination events in two or more physically linked loci [17], which ultimately will translate in assay robustness. It follows that the use of the information on GD presented here will prevent errors in HEMA carrier detection and distortion of haplotypes in linkage analysis.…”

“…Physical position, primer sequences, 5′ end modifications and observed allele range, comprising the heptaplex fluorescent PCR assay for indirect tracking of HEMA were as described [17], and are summarized in Table S3. The seven loci are REN90833 ( F8 extragenic tetranucleotide), F8 Int25.2 ( F8 intragenic dinucleotide), F8 Int22 ( F8 intragenic dinucleotide), F8 Int13.2 ( F8 intragenic dinucleotide), HEMA154311.3 ( F8 extragenic pentanucleotide), TMLHE Int5 ( F8 extragenic pentanucleotide) and HEMA154507.3 ( F8 extragenic tetranucleotide), all representing perfect STR (i.e.…”

Informativeness of a novel multiallelic marker-set comprising an F8 intron 21 and three tightly linked loci for haemophilia A carriership analysis

“…The heterozygosity rates and allele frequencies were determined from 100 unrelated females (herein, unphased diplotypes) from the population of the Northern Region of the State of Rio de Janeiro, Brazil, undergoing kinship analysis. To determine the impact on informativeness of the new markers, we further genotyped the 11 females reported earlier [18], who did not meet the criterion of a ‘minimum two informative markers, with at least one F8 ‐intragenic marker’. To exemplify linkage and segregation analyses, we genotyped: (i) a two‐generation kindred, with two severe HEMA‐affected full siblings, unscreened for the causative mutation, their non‐affected full sibling and the non‐affected maternal half sibling; (ii) ten unrelated males screened positive for Inv22‐1; and (iii) ten unrelated males screened positive for Inv22‐2 type mutations, and whose inversion genotypes had been determined using inverse shifting PCR [9].…”

“…We recently proposed a minimum of two informative STR markers, with at least one F8 ‐intragenic as criterion‐referenced assessment in indirect HEMA carrier detection [18]. We reasoned that typing with a denser set of informative F8 ‐intragenic and extragenic and non‐recombining loci that exhibit heterozygosity rates >0.4 (i.e.…”