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

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.

SUBMITTER: Abelleyro MM

PROVIDER: S-EPMC6460640 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Project description:In different species, embryonic aneuploidies and genome-wide errors are a major cause of developmental failure. The increasing number of equine embryos being produced worldwide provides the opportunity to characterize and rank or select embryos based on their genetic profile prior to transfer. Here, we explored the possibility of generic, genome-wide preimplantation genetic testing concurrently for aneuploidies (PGT-A) and monogenic (PGT-M) traits and diseases in the horse, meanwhile assessing the incidence and spectrum of chromosomal and genome-wide errors in in vitro-produced equine embryos. To this end, over 70,000 single nucleotide polymorphism (SNP) positions were genotyped in 14 trophectoderm biopsies and corresponding biopsied blastocysts, and in 26 individual blastomeres from six arrested cleavage-stage embryos. Subsequently, concurrent genome-wide copy number detection and haplotyping by haplarithmisis was performed and the presence of aneuploidies and genome-wide errors and the inherited parental haplotypes for four common disease-associated genes with high carrier frequency in different horse breeds (GBE1, PLOD1, B3GALNT2, MUTYH), and for one color coat-associated gene (STX17) were compared in biopsy-blastocyst combinations. The euploid (n = 12) or fully aneuploid (n = 2) state and the inherited parental haplotypes for 42/45 loci of interest of the biopsied blastocysts were predicted by the biopsy samples in all successfully analyzed biopsy-blastocyst combinations (n = 9). Two biopsies showed a loss of maternal chromosome 28 and 31, respectively, which were confirmed in the corresponding blastocysts. In one of those biopsies, additional complex aneuploidies not present in the blastocyst were found. Five out of six arrested embryos contained chromosomal and/or genome-wide errors in most of their blastomeres, demonstrating their contribution to equine embryonic arrest in vitro. The application of the described PGT strategy would allow to select equine embryos devoid of genetic errors and pathogenetic variants, and with the variants of interest, which will improve foaling rate and horse quality. We believe this approach will be a gamechanger in horse breeding.

Project description:BACKGROUND:Preimplantation genetic testing (PGT) has already been applied in chromosomally balanced translocation carriers to improve the clinical outcome of assisted reproduction. However, traditional methods could not further distinguish embryos carrying a translocation from those with a normal karyotype prior to implantation. METHODS:To solve this problem, we developed a method named "Chromosomal Phasing on Base level" (BasePhasing), which based on Infinium Asian Screening Array-24 v1.0 (ASA) and a specially phasing pipeline. Firstly, by comparing the number of single nucleotide polymorphism (SNP) loci in different minor allele frequencies (MAFs) and in 2Mbp continuous windows of ASA chip and karyomap-12 chip, we verified whether ASA could be adopted for genome-wide haplotype linkage analysis. Besides, the whole gene amplification (WGA) of 3-10 cells of GM16457 cell line was used to verify whether ASA chip could be used for testing of WGA products. Finally, two balanced translocation families were utilized to carry out BasePhasing and to validate the feasibility of its clinical application. RESULTS:The average number of SNP loci in each window of ASA (473.2) was twice of that of Karyomap-12 (201.2). The coincidence rate of SNP loci in genomic DNA and WGA products was about 97%. The 5.3Mbp deletion was detected positively in cell line GM16457 of both genomic DNA and WGA products, and haplotype linkage analysis was performed in genome wide successfully. In the two balanced translocation families, 18 blastocysts were analyzed, in which 8 were unbalanced and the other 10 were balanced or normal chromosomes. Two embryos were transferred back to the patients successfully, and prenatal cytogenetic analysis of amniotic fluid was performed in the second trimester. The results predicted by BasePhasing and prenatal diagnosis were totally consistent. CONCLUSIONS:Infinium ASA bead chip based BasePhasing pipeline shows good performance in balanced translocation carrier testing. With the characteristics of simple operation procedure and accurate results, we demonstrate that BasePhasing is one of the most suitable methods to distinguish between balanced and structurally normal chromosome embryos from translocation carriers in PGT at present.

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

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