一个中国Lynch综合征家系携带的MLH1基因第19号外显子移码突变:一项家系研究

A frameshift mutation in exon 19 of MLH1 in a Chinese Lynch syndrome family: a pedigree study # # 一个中国Lynch综合征家系携带的 MLH1 基因第19号外显子移码突变:一项家系研究 1 3 first-author 1 3 1 3 2 3 1 3 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:panzhzh@sysucc.org.cndingpr@sysucc.org.cn †E-mail:panzhzh@sysucc.org.cndingpr@sysucc.org.cn panzhzh@sysucc.org.cn 1 3 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:panzhzh@sysucc.org.cndingpr@sysucc.org.cn †E-mail:panzhzh@sysucc.org.cndingpr@sysucc.org.cn panzhzh@sysucc.org.cn Department of Colorectal Surgery, Sun Yat-sen University Cancer Center, Guangzhou 510060, China Department of Colorectal Surgery, Sun Yat-sen University Cancer Center, Guangzhou 510060, China Department of Pathology, Sun Yat-sen University Cancer Center, Guangzhou 510060, China Department of Pathology, Sun Yat-sen University Cancer Center, Guangzhou 510060, China State Key Laboratory of Oncology in South China, Guangzhou 510060, China State Key Laboratory of Oncology in South China, Guangzhou 510060, China lynch syndrome (LS), an autosomal dominantly inherited disease previously known as hereditary non-polyposis colorectal cancer (HNPCC), leads to a high risk of colorectal cancer (CRC) as well as malignancy at certain sites including endometrium, ovary, stomach, and small bowel (Hampel et al., 2008 2008 ; Lynch et al., 2009 2009 ). Clinically, LS is considered the most common hereditary CRC-predisposing syndrome, accounting for about 3% of all CRC cases (Popat et al., 2005 2005 ). LS is associated with mutations of DNA mismatch repair (MMR) genes such as MLH1, MSH2, MSH6, PMS2, and EPCAM (Ligtenberg et al., 2009 2009 ; Lynch et al., 2009 2009 ), which can trigger a high frequency of replication errors in both microsatellite regions and repetitive sequences in the coding regions of various cancer-related genes. Immunohistochemistry (IHC) tests followed by genetic analysis of these mutations play a significant role in diagnosis, treatment determination, and therapeutic response prediction of LS (Lynch et al., 2009 2009 ; Alex et al., 2017 2017 ; Ryan et al., 2017 2017 ). Here, we report substitution of one base-pair in exon 1 of MLH3 (c.1397C > A) and a frameshift mutation in exon 19 of MLH1 (c.2250_2251ins AA) in a 43-year-old Chinese male with an LS pedigree. 目的 2 寻找一个Lynch综合征患者所在家系携带的DNA错配修复基因突变,探讨各突变对肿瘤发生发展 的影响. 创新点 2 MLH1 的第19号外显子c.2250_2251insAA移码突变既往被认为是意义未名突变,而我们的研究为明确该突变的致病意义提供了依据.另外,我们首次报道了 MLH3 基因第1号外显子c.1397C > A突变.该突变有可能使Lynch综合征患者的发病年龄提前. 方法 2 运用免疫组织化学技术检测家系先证者肿瘤组织中错配修复基因蛋白的缺失情况,使用二代测序技术通过先证者血标本明确患者所携带的突变.同时运用Sanger法检测家系其他成员该突变的携带情况以明确突变对肿瘤发生发展的影响. 结论 2 我们在患者体内发现 MLH1 基因第19号外显子移码突变(c.2250_2251insAA)以及 MLH3 基因第1号外显子c.1397C > A突变.在患者家系中,我们仅检测到有 MLH1 突变,因此该突变极有可能为致病突变. Lynch syndrome DNA mismatch repair Frameshift mutation Lync综合征 家系 DNA错配修复基因 置换 移码突变 Lynch syndrome (LS), an autosomal dominantly inherited disease previously known as hereditary non-polyposis colorectal cancer (HNPCC), leads to a high risk of colorectal cancer (CRC) as well as malignancy at certain sites including endometrium, ovary, stomach, and small bowel (Hampel et al., 2008 2008 ; Lynch et al., 2009 2009 ). Clinically, LS is considered the most common hereditary CRC-predisposing syndrome, accounting for about 3% of all CRC cases (Popat et al., 2005 2005 ). LS is associated with mutations of DNA mismatch repair (MMR) genes such as MLH1 , MSH2 , MSH6 , PMS2 , and EPCAM (Ligtenberg et al., 2009 2009 ; Lynch et al., 2009 2009 ), which can trigger a high frequency of replication errors in both microsatellite regions and repetitive sequences in the coding regions of various cancer-related genes. Immunohistochemistry (IHC) tests followed by genetic analysis of these mutations play a significant role in diagnosis, treatment determination, and therapeutic response prediction of LS (Lynch et al., 2009 2009 ; Alex et al., 2017 2017 ; Ryan et al., 2017 2017 ). Here, we report substitution of one base-pair in exon 1 of MLH3 (c.1397C > A) and a frameshift mutation in exon 19 of MLH1 (c.2250_2251ins AA) in a 43-year-old Chinese male with an LS pedigree. The patient was diagnosed with ascending colon cancer, receiving a laparoscopic right hemicolectomy at the age of 43 years in our hospital. His mother was diagnosed with endometrial cancer at the age of 58 years and his maternal grandmother suffered from colorectal cancer and died in her 60 years. His maternal uncle was diagnosed with colon cancer at the age of 50 years. As his family history fulfilled the Amsterdam II criteria for LS, we took a sample of his tumor for IHC analysis, which revealed defects in MLH1 and PMS2 proteins (Figs. 1a and 1b 1a and 1b ). IHC examination revealed loss of MLH1 (a) and PMS2 (b) expression in tumor cells, while no loss of MSH2 (c) or MSH6 (d) expression was observed. Scale bar=200 μm. IHC: immunohistochemistry; MMR: mismatch repair We then undertook genetic analysis of genes related to hereditary CRC to further characterize the mutations. Next generation sequencing was performed by polymerase chain reaction (PCR)-direct sequencing analysis, targeting genes including APC , AXIN2 , EPCAM , MLH1 , MLH3 , MSH2 , MSH6 , MUTYH , PMS1 , PMS2 , STK11 , PTEN , SMAD4 , and BMPR1 . Results were compared with the “1000 Genomes” browser (https:// www.ncbi.nlm.nih.gov/variation/tools/1000genomes www.ncbi.nlm.nih.gov/variation/tools/1000genomes ) and the pathogenicity of mutations was classified according to the recommendations of the American College of Medical Genetics and Genomics (ACMG; http://www.acmg.net http://www.acmg.net ). After excluding promoter methylation, the analysis showed a heterozygous substitution of one base-pair (c.1397C > A) in exon 1 of MLH3 , whose transcript variant (p.Ser466Ter) leads to a protein truncated at codon 465 instead of codon 1429 in the wild type. Also, a heterozygous frameshift mutation in exon 19 of MLH1 (c.2250_2251insAA) was detected, which created a new reading frame at Val 752, encountering a premature stop codon at the following 32nd position (p.Val752L-ysfs*32) and eventually a protein of 782 codons instead of 757 in the wild type (Fig. S1 S1 ). Both mutations were found to be dominant. Comparisons with previous findings from the “1000 Genomes” browser and ACMG recommendations showed that the mutation in MLH3 had not been previously reported. Therefore, we considered it to be a suspected pathogenic mutation. Although not previously reported as pathogenic, similar MLH1 nucleotides, detected in a South American LS pedigree (c.2252_2253dup-AA) (Dominguez-Valentin et al., 2013 2013 ) and a Chinese pedigree (Sheng et al., 2008 2008 ), were recognized as variants of unclassified significance (VUS). To confirm the germline mutation in those genes as well as the pathogenic effect of the MLH3 variants, we provided genetic counseling for the patient’s family members via Sanger sequencing, and recommended genetic analysis. The analysis showed that the germline mutation in MLH1 was carried by the patient’s brother, niece, and mother (Fig. S2a S2a ), while the same MLH3 variant was detected only in the patient’s father who was never diagnosed with a tumor (Fig. S2b S2b ). These results suggested that the mutation in MLH1 weighed more in the LS etiology of the proband. Genetic analysis of the maternal uncle with colon cancer was not carried out due to personal refusal. The pedigree chart is shown in Fig. 2 2 . Black symbols indicate individuals diagnosed with cancers, with slashes indicating death. Ages upward mean the current age (years), while ages in the brackets indicate diagnosed age (years). A plus symbol indicates the mutation in the mismatch repair (MMR) genes. The triangle indicates the proband. Square: male; Circle: female Genetic linkage analysis showed that 80% of LS cases are related to germline mutations in MSH2 and MLH1 (Alex et al., 2017 2017 ), whereas the mutation we found in MLH1 , previously reported in only one pedigree of a South American, was predicted to be a VUS by multiple in silico tools (Dominguez-Valentin et al., 2013 2013 ). Germline mutation was revealed in the maternal pedigree, especially in patients with LS involving malignancy, which supports its etiological role according to the law of segregation. Also, deficiencies in both MLH1 and PMS2 were proved by IHC tests in the proband. We believe that an abnormal C-terminus in the MLH1 protein caused by such a mutation could provide an explanation. It was reported that the heterodimer of MLH1 combined with PMS2 serves as a major effector of MMR function in vivo, and the participation of the carboxy terminal homology (CTH) domain (residues 500–756 amino acids in MLH1) in the structure is crucial for its stabilization (Mohd et al., 2006 2006 ). Moreover, mutations truncating or expanding MLH1 peptide are related to the loss of such domains (Kim et al., 2009 2009 ), theoretically triggering the absence of MLH1 and PMS2. For these reasons, the MLH1 variant carried by the proband, extending the peptide and affecting the structure of the CTH domain, was supposed to destroy the stability of the heterodimer, causing MMR deficiency and eventually LS. Nevertheless, previous studies reported that patients with a defective MMR had a mean age of 45 years (Lynch et al., 2009 2009 ), while patients in our study carrying only the MLH1 mutation were diagnosed after their 50 years. Further studies concerning the significance of similar variants are required. We had suspected that the mutation in MLH3 plays an important role in tumorigenicity, but the results from genetic analysis suggested the contrary. The father lived tumor-free as a carrier of MLH3 mutation alone, which did not support individual pathogenicity. Clinically, there have been few reports concerning tumor predisposition with only MLH3 mutations (Hienonen et al., 2003 2003 ; Mohd et al., 2006 2006 ), providing little evidence. Also, Korhonen et al. ( 2008 2008 ) functionally characterized seven missense mutations of MLH3 , and disproved their individual function of interfering with MMR, describing them as low-risk mutations for LS. Although the mutation in MLH1 was considered pathogenic in our case, we still believed that MLH3 variant made a difference. The proband was diagnosed with colon cancer at the age of 43 years, much younger than the other patients in his family. In addition, malignancy has not yet been diagnosed in his 45-year-old brother carrying MLH1 variant. Therefore, it is possible that MLH3 deficiency enhanced the tumorigenicity of MLH1 mutation and accelerated the occurrence of the tumor. According to the study of Chen et al. ( 2005 2005 ), MLH3 , following PMS2 , takes part in tumor suppression in mice with MLH1 . Also, Chen et al. ( 2008 2008 ) showed that a loss of MLH3 accelerates the progression of gastrointestinal tumors on the basis of PMS2 deficiency. We believe that the compound heterozygote of these germline mutations is more likely to trigger LS. Further studies are needed to provide more conclusive evidence, as this is the first time that this phenomenon has been detected. In conclusion, we identified a mutation in exon 19 of MLH1 and a novel germline mutation in exon 1 of MLH3 . The mutation in MLH1 , although described previously as a VUS, was detected in the maternal pedigree and possibly resulted in LS. MLH3 variant was not detected among other patients of the maternal pedigree, but we still believe it may have enhanced tumorigenicity in this case. References AK Alex , S Siqueira , R Coudry , 等 . Response to chemotherapy and prognosis in metastatic colorectal cancer with DNA deficient mismatch repair . Clin Colorectal Cancer , 2017 . 16 ( 3 ): 228 - 239 . 28063788 DOI: 10.1016/j.clcc.2016.11.001 http://doi.org/10.1016/j.clcc.2016.11.001 . 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Mismatch repair gene mutations in Chinese HNPCC patients Compliance with ethics guidelines Qiao-qi SUI, Wu JIANG, Xiao-dan WU, Yi-hong LING, Zhi-zhong PAN, and Pei-rong DING declare that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from all patients for being included in the study. Additional informed consent was obtained from all patients for whom identifying information is included in this article. 10.1631/jzus.B1800105 ,, 2018-02-27 2018-06-26 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2019 2019 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 01 20