目的·针对1例临床表现疑似甲基丙二酸血症（methylmalonic acidemia，MMA）但外显子组测序（whole exome sequencing，WES）为阴性的罕见疑难病例，利用多组学测序分析其遗传学致病原因。方法·提取先证者及父母的外周血DNA及RNA样本，先应用MMA相关基因Panel测序和WES，再利用RNA测序（RNA sequencing，RNA-seq）和全基因组测序（whole genome sequencing，WGS）综合分析该患儿基因变异、来源及可能的遗传方式。结果·MMA相关基因Panel测序及WES常规分析均未发现与患儿表型相关的致病性变异。对WES的拓展分析提示患儿可能存在4号染色体单亲二体；WGS发现患儿MMAA（metabolism of cobalamin associated A）基因非编码区域c.-66+2T>C纯合剪接变异。该变异位于5'非翻译区（5' untranslated region，5'UTR），具体位置为第1个外显子的剪接供体位点下游第2个碱基处（参考序列：NM_172250）；在基因组坐标（hg19）中，该变异的位置为4号染色体第146540561个碱基（chr4：146540561）。Sanger测序验证发现其母亲携带该位点杂合变异，父亲未携带；RNA-seq发现该患儿4号染色体上的MMAA基因表达量几乎为0，反转录实时定量PCR进一步证实该基因mRNA几乎不表达，提示MMAA非编码区剪接变异影响转录水平表达。结论·位于WES范围之外的MMAA基因非编码区c.-66+2T>C纯合剪接变异是该患儿可能的致病原因，推测由4号染色体母源单亲二体所致。

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.