High‐resolution combined linkage physical map of short tandem repeat loci on human chromosome band Xq28 for indirect haemophilia A carrier detection

Abstract

Haemophilia A, the most common severe hereditary bleeding disorder in humans, is chiefly caused by mutations in the coagulation factor VIII F8 gene, which maps on chromosome band Xq28. Linkage analysis with F8 intragenic and/or closely located extragenic short tandem repeat (STR) elements is an effective method for indirect tracing of F8 pathogenic allelic variants in at-risk families. STR profiling is currently limited to 14 markers, 11 of which are dinucleotide elements. The aim of this study was to define novel polymorphic STR loci for haemophilia A carrier screening. The combined linkage physical map was restricted to a 2.4-Mb region on Xq28 that hosts 81 annotated genes, F8 inclusive. The inventory was in silico through comparative analyses with three X chromosome reference sequences, using microsatellite mining and validation computer software. Genetic distances for unmapped markers were interpolated on the Rutgers map of the human genome. The effort yielded 94 STR loci: 53 extragenic and 41 intragenic (14 STR elements map on the F8 gene; the other 27 on 19 further genes). The distribution per repeat period size was 61.7% di-, 5.3% tri-, 26.6% tetra- and 6.4% pentanucleotide loci. The success rate of validation of polymorphism for the new STR loci was 56.3%. For STR elements 0.78 Mb equidistant of the F8 gene, the interpolated downstream genetic length is 3.27 times the upstream genetic length. The inventory represents a 5.7-fold increase in polymorphic STR loci useful in carrier detection. Genotyping with the upstream extragenic tetra- and pentanucleotide markers is thus apprized.

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

“…The seven markers typed in this study mapped to a 0.23 cM interval (physical interval of 0.813 Mb) to minimize misdiagnosis due to recombination between markers [5]. The tetranucleotide marker border limits are REN90833 and HEMA154507.3, which map 0.01 and 0.15 cM distant from the end and start points of the F8 gene, respectively.…”

“…The markers included in this study were from the comprehensive high‐resolution combined linkage map of microsatellites located on human chromosome band Xq28, reported earlier [5] and 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), HEMA154507.3 ( F8 extragenic tetranucleotide). Information about the STRs loci, primer sequences, 5′ end modifications, and observed allele range comprising the heptaplex fluorescent PCR assay is in Table S1.…”

“…To improve distribution of the PCR products within the 120–450 bp range of analysis we re‐designed the reported REN90833 and TMLHE Int5 primer sequences (Table S1). All other primers sequences were as reported [5]. We are aware of the fact that the re‐designed REN90833 primers also anneal to a non‐STR locus on chromosome 10, yielding a non‐polymorphic PCR product of 106 bp with DNA from both males and females.…”

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.…”

Single-Tube Dodecaplex PCR Panel of Polymorphic Microsatellite Markers Closely Linked to the DMPK CTG Repeat for Preimplantation Genetic Diagnosis of Myotonic Dystrophy Type 1

“…The Tandem Repeats Finder (TRF) (19 ) DNA analysis program was used to identify all microsatellite markers within each DNA segment. Markers were filtered according to previously described criteria (20 ) and primer pairs flanking each selected microsatellite marker were designed. Markers genotypes were determined from 13-16 lymphoblastoid cell line DNAs, and those with low heterozygosity values and poor capillary electrophoretic peak patterns were excluded.…”