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.
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Acknowledgements
This study was supported by grants from the National Research Council (CONICET), National Agency for Scientific and Technological Promotion (ANPCyT) and the World Federation of Hemophilia. The authors thank Miguel de Tezanos Pinto (National Academy of Medicine and National Hemophilia Foundation) and Judith Mincman and Roberto Cocco (Fecunditas Clinic) for their help in different phases of the work.
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All authors have contributed to the intellectual content of this paper and have met the following three requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the manuscript.
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The Ethics Committee of the Institutes of the National Academy of Medicine of Buenos Aires, Argentina, approved the project (date: 13/06/2012). Peripheral blood samples from participating control, carrier, and hemophilia subjects in nuclear families were collected with written informed consent.
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Abelleyro, M.M., Marchione, V.D., Palmitelli, M. et al. Inverse PCR to perform long-distance haplotyping: main applications to improve preimplantation genetic diagnosis in hemophilia. Eur J Hum Genet 27, 603–611 (2019). https://doi.org/10.1038/s41431-018-0334-9
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DOI: https://doi.org/10.1038/s41431-018-0334-9
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