Proof-of-principle rapid noninvasive prenatal diagnosis of autosomal recessive founder mutations
David A. Zeevi, … , Arndt Rolfs, Ari Zimran
David A. Zeevi, … , Arndt Rolfs, Ari Zimran
Published August 31, 2015
Citation Information: J Clin Invest. 2015;125(10):3757-3765. https://doi.org/10.1172/JCI79322.
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Clinical Research and Public Health Genetics

Proof-of-principle rapid noninvasive prenatal diagnosis of autosomal recessive founder mutations

Abstract

BACKGROUND. Noninvasive prenatal testing can be used to accurately detect chromosomal aneuploidies in circulating fetal DNA; however, the necessity of parental haplotype construction is a primary drawback to noninvasive prenatal diagnosis (NIPD) of monogenic disease. Family-specific haplotype assembly is essential for accurate diagnosis of minuscule amounts of circulating cell-free fetal DNA; however, current haplotyping techniques are too time-consuming and laborious to be carried out within the limited time constraints of prenatal testing, hampering practical application of NIPD in the clinic. Here, we have addressed this pitfall and devised a universal strategy for rapid NIPD of a prevalent mutation in the Ashkenazi Jewish (AJ) population.

METHODS. Pregnant AJ couples, carrying mutation(s) in GBA, which encodes acid β-glucosidase, were recruited at the SZMC Gaucher Clinic. Targeted next-generation sequencing of GBA-flanking SNPs was performed on peripheral blood samples from each couple, relevant mutation carrier family members, and unrelated individuals who are homozygotes for an AJ founder mutation. Allele-specific haplotypes were constructed based on linkage, and a consensus Gaucher disease–associated founder mutation–flanking haplotype was fine mapped. Together, these haplotypes were used for NIPD. All test results were validated by conventional prenatal or postnatal diagnostic methods.

RESULTS. Ten parental alleles in eight unrelated fetuses were diagnosed successfully based on the noninvasive method developed in this study. The consensus mutation–flanking haplotype aided diagnosis for 6 of 9 founder mutation alleles.

CONCLUSIONS. The founder NIPD method developed and described here is rapid, economical, and readily adaptable for prenatal testing of prevalent autosomal recessive disease-causing mutations in an assortment of worldwide populations.

FUNDING. SZMC, Protalix Biotherapeutics Inc., and Centogene AG.

Authors

David A. Zeevi, Gheona Altarescu, Ariella Weinberg-Shukron, Fouad Zahdeh, Tama Dinur, Gaya Chicco, Yair Herskovitz, Paul Renbaum, Deborah Elstein, Ephrat Levy-Lahad, Arndt Rolfs, Ari Zimran

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Figure 4

Extended fine mapping of the consensus and near-consensus AJ N370S founder haplotype region as a tool for phasing fetal haplotypes.

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Extended fine mapping of the consensus and near-consensus AJ N370S found...
Illustration depicting the GBA locus (±2 Mb) and thousands of SNPs that were deep sequenced for the construction and typing of fetal alleles according to the analytical pipeline (as indicated in the key). (A) An extended deep-sequencing panel was used to better fine map the conserved N370S founder haplotype, as in Figure 2. Accordingly, a 301-SNP haplotype (termed “full-consensus N370S haplotype”) was identified in all N370S chromosomes in this study (28 chromosomes altogether). In addition, the consensus haplotype was found to extend 500 kb further downstream of GBA (620 additional SNPs) in 15 of 16 chromosomes from 8 N370S homozygotes. Furthermore, in all N370S homozygotes (but not all N370S carriers), the consensus haplotype was found to extend another 120 kb upstream of GBA (100 additional SNPs). Altogether, these extended haplotypes were termed “near-consensus N370S haplotypes.” (B) The N370S haplotype from each N370S carrier parent in the study was carefully mapped according to homozygous regions and family-based linkage analysis. After comparison to the near-consensus haplotype in A, new parent-specific 5′ and/or 3′ demarcations of the N370S near-consensus haplotype were set (this haplotype was termed the “parent-specific consensus N370S haplotype”). (C) In this example, deep sequencing of the GBA-flanking region in a fetus identified stretches of a linkage-based parental N370S haplotype that resided outside of the consensus N370S region. In addition, some stretches of fetal sequence could not be phased according to family-based linkage. (D) When unphased fetal sequence, such as in C, fell within the parent-specific consensus N370S haplotype (as determined in B), the consensus information was used to phase the fetus (here, with the N370S-linked haplotype), thereby increasing confidence in the diagnostic test result.