Targeted linked-read sequencing for direct haplotype phasing of parental GJB2/SLC26A4 alleles: A universal and dependable noninvasive prenatal diagnosis method applied to autosomal recessive nonsyndromic hearing loss in at-risk families

Abstract

Abstract Background Noninvasive prenatal diagnosis (NIPD) for autosomal recessive nonsyndromic hearing loss (ARNSHL) was rarely reported until recent years. However, previous method would be unavailable without the proband genotype or challenging genome loci(e.g. CNV, deletions, inversions, or gene recombinants). Here, We assess the performance of relative haplotype dosage analysis (RHDO) - based NIPD for identifying fetal genotyping of pregnancies at risk of ARNSHL. Methods Fifty couples carrying pathogenic variants associated with ARNSHL in either GJB2 or SLC26A4 were recruited. The fetal cell-free DNA (cfDNA) of 49 families that met the quality control standard was genotyped using RHDO-based targeted linked-read sequencing. Fetal amniocyte samples were genotyped by invasive prenatal diagnosis (IPD) to evaluate the performance of NIPD. Results The 49 fetal genotypes were identified as 14 normal homozygotes, 17 heterozygotes, and 18 affected homozygotes or compound heterozygotes by NIPD, which were all concordant with those diagnosed by IPD. The concordance rate was 100% for the GJB2 (31/31) as well as SLC26A4 (18/18) genotyping. The sensitivity and specificity were both 100%. Conclusions Sufficient specific informative SNPs for haplotyping, the fetal cfDNA concentration as well as the sequencing depth are prerequisites for the RHDO-based NIPD, which has the merits of being proband-free and qualified for copy number variation analysis, thus it is suitable for universal application. It also has clinical potential as an alternative to traditional IPD for ARNSHL.