Revisiting Exome Data Identified Missed Splice Site Variant of the Asparagine Synthetase ( ASNS ) Gene

Abstract

Next-generation sequencing, such as whole-exome sequencing (WES), is increasingly used in the study of Mendelian disorders, yet many are reported as "negative." Inappropriate variant annotation and filtering steps are reasons for missing the molecular diagnosis. Noncoding variants, including splicing mutations, are examples of variants that can be overlooked. Herein, we report a family of four affected newborns, and all presented with severe congenital microcephaly. Initial research WES analysis identified a damaging homozygous variant in NME1 gene as a possible cause of primary microcephaly phenotype in these patients. However, reanalysis of the exome data uncovered a biallelic splice site variant in asparagine synthetase gene which seems to be the possible cause of the phenotype in these patients. This study highlights the importance of revisiting the exome data and the issue of "negative" exome and the afterward approaches to identify and prove new candidate genes.

Keywords: ASNS; NME1; candidate genes; microcephaly; whole-exome sequencing.

Fig. 1

Omani family pedigree and the ASNS genetic variant effect findings. ( A ) Pedigree of the Omani family with four affected offspring with primary microcephaly. Shaded symbols indicate affected individuals. The genotype for NME1 and ASNS genes (determined by Sanger sequencing) is shown for tested individuals. These genes are at two different chromosomes. ( B ) cDNA amplification of sample III-6 with expected PCR product with a size of 292 bp, using RT-PCR. This indicates that the aberrant transcript is stable and does not undergo NMD pathway. ( C ) Sanger sequencing of wild-type and mutant cDNA extracted from control and the affected patient, respectively. Sequencing analysis showed that four base pair insertion occurred in the mutant ASNS cDNA. ( D ) Immunoblot of whole cell lysate from peripheral lymphocytes extracted from wild-type control and III-6; two types of antibodies were used to detect ASNS protein in the samples. GAPDH (37 kDa) was used as a loading control. The ASNS protein is absent in the patient's sample. All experiments were repeated at least three times. Abbreviations: ASNS, asparagine synthetase; NMD, nonsense-mediated decay; RT-PCR, reverse transcription polymerase chain reaction.

Fig. 2

Sequence chromatograms of the identified variants and schematic representation of ASNS genetic variants. ( A ) Chromatograms show the NME1 and ASNS variants. ( B ) Schematic illustration for the typical sequence of the ASNS gene showing the variant. ( C ) Amino acids sequence of the wild-type and mutant protein as predicted by in silico tools. It was predicted that the insertion would change a glycine residue at position 260 by arginine and subsequent change of amino acids from this point until reaching a premature stop codon after amino acid 300 (p.Gly260Argfs*41). ( D ) Graphic representation for ASNS wild-type protein (P08243) showing two domains within this protein and the location of the predicted variant in the ASNS domain. Abbreviation: ASNS, asparagine synthetase.