MMAA基因非编码区变异叠加单亲二体所致甲基丙二酸血症的多组学分析

doi:10.3969/j.issn.1674-8115.2025.06.016

上海交通大学学报（医学版） ›› 2025, Vol. 45 ›› Issue (6): 800-806.doi: 10.3969/j.issn.1674-8115.2025.06.016

• 短篇论著 • 上一篇

MMAA基因非编码区变异叠加单亲二体所致甲基丙二酸血症的多组学分析

霍晓燕¹, 罗小梅¹, 叶贤涛², 孙昱¹, 余永国¹, 梁黎黎¹(), 范燕洁¹()

^1.上海交通大学医学院附属新华医院儿遗传内分泌科，上海市儿科医学研究所，上海 200092
^2.江西省妇幼保健院医学遗传中心，南昌 330000

收稿日期:2024-11-06 接受日期:2025-03-05 出版日期:2025-06-23 发布日期:2025-06-23
通讯作者: 梁黎黎，副主任医师，博士；电子信箱：lianglili@xinhuamed.com.cn
范燕洁，副研究员，博士；电子信箱：fanyanjie@shsmu.edu.cn。
作者简介:霍晓燕（1999—），女，硕士生；电子信箱：huoxiaoyan@sjtu.edu.cn。
基金资助:
国家重点研发计划(2022YFC2703405);国家自然科学基金(82171165)

Multi-omics analysis of methylmalonic acidemia caused by a non-coding region variant in MMAA gene combined with uniparental disomy

HUO Xiaoyan¹, LUO Xiaomei¹, YE Xiantao², SUN Yu¹, YU Yongguo¹, LIANG Lili¹(), FAN Yanjie¹()

^1.Department of Pediatric Genetics and Endocrinology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine; Shanghai Institute of Pediatric Research, Shanghai 200092, China
^2.Medical Genetics Center, Jiangxi Maternal and Child Health Hospital, Nanchang 330000, China

Received:2024-11-06 Accepted:2025-03-05 Online:2025-06-23 Published:2025-06-23
Contact: LIANG Lili, E-mail: lianglili@xinhuamed.com.cn
FAN Yanjie, E-mail: fanyanjie@shsmu.edu.cn.
Supported by:
National Key R&D Program of China(2022YFC2703405);National Natural Science Foundation of China(82171165)

摘要/Abstract

摘要：

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。

关键词: 非编码区变异, 基因组测序, 转录组测序, 甲基丙二酸血症

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)

中图分类号:

R596.3

霍晓燕, 罗小梅, 叶贤涛, 孙昱, 余永国, 梁黎黎, 范燕洁. MMAA基因非编码区变异叠加单亲二体所致甲基丙二酸血症的多组学分析[J]. 上海交通大学学报（医学版）, 2025, 45(6): 800-806.

HUO Xiaoyan, LUO Xiaomei, YE Xiantao, SUN Yu, YU Yongguo, LIANG Lili, FAN Yanjie. Multi-omics analysis of methylmalonic acidemia caused by a non-coding region variant in MMAA gene combined with uniparental disomy[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(6): 800-806.

图/表 3

图1 先证者的家系图及测序结果Note： A. Family pedigree of the proband. B. Extended analysis of WES: run of ROH analysis. Blue strips represent regions of homozygosity. UPD—uniparental disomy. C. MMAA gene Sanger sequencing of the family. Ⅰ-1—father of the proband; Ⅰ-2—mother of the proband; Ⅱ-2—the proband. D. Schematic diagram of the MMAA gene mutation. WT—wild type; MUT—mutation; UTR—untranslated region; E1‒7—exon 1‒7; ATG—start codon; CDS—coding sequence.

Fig 1 Proband's pedigree and sequencing results

图2 先证者 MMAA 基因mRNA表达Note： A. mRNA expression of the MMAA gene in the proband compared with healthy controls in the detection of RNA-seq. B. RT-qPCR results of exon 3‒4 of the MMAA gene in the family members.

Fig 2 mRNA expression of the proband's MMAA gene

图3 Puffin软件预测chr4:146540561在转录起始中的作用Note： Prediction of base pair resolution strand-specific transcription initiation signal at chr4:146540561. The x-axis indicates the position relative to the annotated transcription start site and the y-axis is shown on a Log10 scale. Motif activation: motif activation stands for base pair motif contribution scores to motif activation, and motif contribution score represents the amount of contribution from a motif type to any position or window in the sequence. Motif effects: effects of the U1 small nuclear ribonucleoprotein (U1 snRNP) motif. Prediction: TSS prediction. Solid arrow: this site was located within the binding domain for the U1 small nuclear RNA (U1 snRNA). Hollow arrow: Puffin software predicted this site to be the transcription initiation site of the MMAA gene.

Fig 3 Prediction of the role of chr4:146540561 in transcription initiation using Puffin software

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MMAA基因非编码区变异叠加单亲二体所致甲基丙二酸血症的多组学分析

Multi-omics analysis of methylmalonic acidemia caused by a non-coding region variant in MMAA gene combined with uniparental disomy

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