Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, 10 Center Drive, Bethesda, MD 20892, United States.
RNA Metabolism and Epitranscriptomics Unit, Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, 9000 Rockville Pike, Bethesda, MD 20892, United States.
Hum Mol Genet. 2023 Nov 3;32(22):3135-3145. doi: 10.1093/hmg/ddad131.
Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an autosomal dominant condition characterized by the development of cutaneous and uterine leiomyomas and risk for development of an aggressive form of papillary renal cell cancer. HLRCC is caused by germline inactivating pathogenic variants in the fumarate hydratase (FH) gene, which encodes the enzyme that catalyzes the interconversion of fumarate and L-malate. We utilized enzyme and protein mobility assays to evaluate the FH enzyme in a cohort of patients who showed clinical manifestations of HLRCC but were negative for known pathogenic FH gene variants. FH enzyme activity and protein levels were decreased by 50% or greater in three family members, despite normal FH mRNA expression levels as measured by quantitative PCR. Direct Nanopore RNA sequencing demonstrated 57 base pairs of retained intron sequence between exons 9 and 10 of polyadenylated FH mRNA in these patients, resulting in a truncated FH protein. Genomic sequencing revealed a heterozygous intronic alteration of the FH gene (chr1: 241498239 T/C) resulting in formation of a splice acceptor site near a polypyrimidine tract, and a uterine fibroid obtained from a patient showed loss of heterozygosity at this site. The same intronic FH variant was identified in an unrelated patient who also showed a clinical phenotype of HLRCC. These data demonstrate that careful clinical assessment as well as biochemical characterization of FH enzyme activity, protein expression, direct RNA sequencing, and genomic DNA sequencing of patient-derived cells can identify pathogenic variants outside of the protein coding regions of the FH gene.
遗传性平滑肌瘤病和肾细胞癌（HLRCC）是一种常染色体显性遗传疾病，其特征是皮肤和子宫平滑肌瘤的发展，以及发生侵袭性乳头状肾细胞癌的风险。HLRCC 是由富马酸水合酶（FH）基因的种系失活致病性变异引起的，该基因编码催化富马酸和 L-苹果酸相互转化的酶。我们利用酶和蛋白质迁移率测定法，评估了 FH 酶在一组表现出 HLRCC 临床症状但 FH 基因已知致病性变异为阴性的患者中的活性。尽管通过定量 PCR 测量 FH mRNA 表达水平正常，但在三个家族成员中，FH 酶活性和蛋白水平降低了 50%或更多。直接 Nanopore RNA 测序显示，这些患者的多聚腺苷酸化 FH mRNA 的外显子 9 和 10 之间存在 57 个碱基对的内含子序列保留，导致 FH 蛋白截短。基因组测序显示 FH 基因的杂合性内含子改变（chr1:241498239 T/C），导致在多嘧啶区附近形成剪接受体位点，并且从患者获得的子宫纤维瘤显示该位点的杂合性丢失。在另一位也表现出 HLRCC 临床表型的无关患者中也发现了相同的内含子 FH 变体。这些数据表明，仔细的临床评估以及 FH 酶活性、蛋白表达、直接 RNA 测序和患者来源细胞的基因组 DNA 测序的生化特征分析，可以确定 FH 基因蛋白编码区以外的致病性变异。
