Authors: Gopinath Padmavathi , Ravi Sudesh , Avaniyapuram Kannan Murugan , Arasambattu Kannan Munirajan ,

Abstract

Background: Recurrent fetal loss can arise from a wide range of genetic abnormalities, many of which remain undetected in the absence of comprehensive molecular testing. Trio-based clinical exome sequencing enables the systematic identification of candidate mutations and variants of uncertain significance (VUS) that underlie fetal loss and supports accurate diagnosis and informed genetic counselling for affected families.

Methods: Genomic DNA was extracted from fetal tissue and peripheral blood leukocytes of the couple, siblings, and grandparents. Trio-clinical exome sequencing was performed, followed by Sanger sequencing to confirm candidate variants. The variants were evaluated for pathogenicity and the pattern of inheritance.

Results: Trio-clinical exome sequencing identified two homozygous nonsynonymous variants in the NEB gene, one in exon 6 (NM_001164507.2: c.367C>T; p.Arg123Cys) and another in exon 133 (NM_001164507.2: c.20279A>G; p.Asp6760Gly) in the proband. In addition, a hemizygous nonsynonymous variant in exon 10 of the X-linked OFD1 gene (NM_003611.3: c.1035G>T; p.Lys345Asn) and a homozygous nonsynonymous variant in exon 8 of the SLCO2A1 gene (NM_005630.3: c.1105G>T; p.Gly369Cys) were observed in the proband. The NEB and SLCO2A1 variants were heterozygous in the couple, and only the mother was heterozygous for the OFD1 variant.

Conclusion:  This study identified two candidate NEB gene variants (classified as variants of uncertain significance, VUS) as the most likely candidate contributors to recurrent fetal loss. The findings provide the basis for informed genetic counselling and reproductive risk assessment.

Keywords: Clinical exome sequencing, fetal tissue, NEB, recurrent pregnancy loss, variants of uncertain significance, consanguinity.