Inverse PCR to perform long-distance haplotyping: main applications to improve preimplantation genetic diagnosis in hemophilia

Eur J Hum Genet. 2019 Apr;27(4):603-611. doi: 10.1038/s41431-018-0334-9. Epub 2019 Jan 9.

Abstract

Among other applications of long-distance haplotype phasing in clinical genetics, determination of linked DNA markers as surrogate for problematic structural variants (e.g., repeat-mediated rearrangements) is essential to perform diagnosis from low-quality DNA samples. We describe a next-of-kin-independent (physical) phasing approach based on inverse-PCR (iPCR) paired-end amplification (PI). This method enables typing the multialleles of the short tandem repeat (STR) F8Int21[CA]n at the F8-intron 21, as a surrogate DNA marker for the F8-intron 22 inversion (Inv22), the hemophilia A-causative hotspot, within the transmitted haplotype in informative carriers. We provide proof-of-concept by blindly validating the PI approach in 15 carrier mother/affected-son duos. Every F8Int21[CA]n STR allele determined in phase with the Inv22 allele in the female carriers from the informative duos was confirmed in the hemizygous proband (P = 0.00003). A second surrogate STR locus at the F8-IVS22 was obtained by the PI approach improving severe-HA preimplantation genetic diagnosis by augmenting heterozygosity in Inv22 carriers bypassing the requirement for family linkage analysis. The ability of the PI-assay to combine other marker pairs was demonstrated by haplotyping a SNV (F8:c.6118T > C) with a >28kb-distant F8-IVS22 STR. The PI approach has proven flexibility to target different marker pairs and has potential for multiplex characterization of iPCR products by massively parallel sequencing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Female
  • Genetic Markers / genetics
  • Genotype
  • Haplotypes / genetics
  • Hemophilia A / diagnosis
  • Hemophilia A / genetics*
  • Hemophilia A / physiopathology
  • Humans
  • Introns
  • Male
  • Microsatellite Repeats / genetics*
  • Polymerase Chain Reaction / methods*
  • Pregnancy
  • Preimplantation Diagnosis*

Substances

  • Genetic Markers