Abstract:

Objective ·To investigate the genetic etiology of a rare and complex case clinically suspected to be methylmalonic acidemia (MMA), but with negative whole exome sequencing (WES) results, using a multi-omics sequencing approach. Methods ·DNA and RNA samples were extracted from the peripheral blood of the proband and both parents. Targeted MMA-related gene Panel sequencing and WES were first performed. Subsequently, RNA sequencing (RNA-seq) and whole genome sequencing (WGS) were conducted to comprehensively analyze the child's genetic variants, their origins and potential inheritance patterns. Results ·No pathogenic variants associated with the patient's phenotype were identified through the MMA Panel or standard WES analysis. Extended analysis of WES suggested the possibility of uniparental disomy (UPD) of chromosome 4. WGS revealed a homozygous splice-site variant (c.-66+2T>C) in the non-coding region of the metabolism of cobalamin associated A (MMAA) gene. The variant was located in the 5' untranslated region (5'UTR), specifically at the second base downstream of the splice donor site of exon 1 (reference sequence: NM_172250). In genomic coordinates (hg19), the variant was located at base 146540561 on chromosome 4 (chr4:146540561). Sanger sequencing confirmed that the mother was heterozygous for this variant, while the father did not carry it. RNA-seq showed no detectable expression of the MMAA gene on chromosome 4 in the patient. This was further confirmed by reverse transcription real time quantitative PCR, indicating nearly absent mRNA expression, suggesting that the non-coding splice-site variant affected transcriptional expression. Conclusion ·A homozygous splice-site variant (c.-66+2T>C) in the non-coding region of the MMAA gene—outside the coverage of WES—is likely the pathogenic cause in this case, presumably resulting from maternal UPD of chromosome 4.

Key words: non-coding variant, genomic sequencing, transcriptome sequencing, methylmalonic acidemia (MMA)