遗传性平滑肌瘤病与肾细胞癌（HLRCC）是一种常染色体显性遗传病，其特征是皮肤和子宫平滑肌瘤的发展，并且存在发育一种侵袭性乳头状肾细胞癌的风险。HLRCC由富马酸水合酶（FH）基因中的胚系失活致病变异引起，该基因编码催化富马酸和L-苹果酸互转的酶。我们利用酶活性和蛋白质活性分析评估了一个患有HLRCC且FH基因中已知病理性变异阴性的患者队列中的FH酶。尽管qPCR测得的FH mRNA表达水平正常，但在家族三位成员中，FH酶活性和蛋白质水平下降了50%或更多。利用直接纳米孔RNA测序技术，我们发现这些患者的多聚腺苷酸化FH mRNA的9号外显子和10号外显子之间保留了57个碱基对的内含子序列，导致了截断的FH蛋白质。基因组测序揭示了FH基因（chr1: 241498239 T/C）中的杂合内含子改变，导致在多嘧啶链附近形成剪接受体位点，并且从患者获得的子宫肌瘤显示了在此位点的等位基因丧失。在一个与此无关的患者中，同样发现了相同内含子FH变异，并且该患者也表现出HLRCC的临床表型。这些数据表明，仔细的临床评估以及对FH酶活性、蛋白质表达、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，能够鉴定FH基因编码区域之外的致病变异。由牛津大学出版社于2023年出版。

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 qPCR. 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.Published by Oxford University Press 2023.