The index case was a 56-year-old male who presented with hematochezia and tenesmus for 5 months prior to a surgical consultation in November 2017. The patient had a history of stage-I squamous cell carcinoma at the lower lip which had been removed by a wide excision 6 years before. He also had essential hypertension and dyslipidemia. On colonoscopy, a rectal tumor was seen at 7 cm from the anal verge, from which the rectum had nearly complete obstruction. A tissue biopsy lead to a diagnosis of adenocarcinoma of the rectum of poor differentiation. The immunohistochemistry panel for MMR proteins showed diffusely strong positivity for MLH1 and PMS2 expression and weak positivity for MSH2 and MSH6 (Fig. 1). A computerized tomography study showed no distant metastasis. Magnetic resonance imaging of the rectum revealed an asymmetrical circumferential rectal mass of 1.1 cm thickness and 6.0 cm length. The muscularis propria hyposignal T2W was lost, suggestive of perirectal fat invasion and stage T3N0M0. No colonic polyps were seen. The patient was treated with upfront chemoradiation therapy before undergoing a laparoscopic low anterior resection. Histology of the surgical specimens showed no residual tumor at the rectum and no metastasis in 9 lymph nodes (pT0pN0cM0). As indicated by the histopathology, post-operative chemotherapy (Mayo regimen) was given. On follow-up, the patient was tumor-free at 3 years after the surgery.
Three relatives of the patient also had had colorectal cancer: his mother, his elder brother and his daughter. His mother was diagnosed with CRC at 55 years of age and died from cerebrovascular disease in the same year. The brother was diagnosed with rectal cancer at 55 years and the daughter at 20 years. Histopathology of the rectal cancer in the brother indicated poorly differentiated adenocarcinoma without polyps. The daughter was diagnosed with colonic adenocarcinoma together with ovarian cancer which was treated surgically at another hospital. Unfortunately, we have no histopathological details of the cancer in the daughter or the mother of the patient. Apart from the daughter, the patient also had two others sons, whose colonoscopies were negative. According to the pedigree of this family (Fig. 2), the mode of inheritance was likely autosomal dominant.
Blood specimens were collected from the index case, his brother, his wife, and all 3 of their children. Collection of the biospecimens and the genetic studies were all performed following informed consent. The study was approved by the Human Research Ethics Committee, Faculty of Medicine, Prince of Songkhla University (REC.65-084-10-1). For the genomic studies, DNA was prepared from a peripheral blood sample using a High Pure PCR Template Preparation Kit (Roche, Berlin, Germany). The extracted DNA was qualified and quantified by Bioanalyzer (Agilent Technologies, Santa Clara, California, United States) and Nanodrop (Thermo Scientific, Delaware, United States). Whole exome studies were performed in the index case, the wife, 2 brother and one daughter. Coding regions were captured and enriched using an Agilent Sure Select XT Human All Exon V6 machine. Paired-end exome sequencing was done using an Illumina HiSeq-2000 platform (Illumina, California, United States). The FASTQ samples were qualified by FastQC and quality control was verified using a Trimmomatic machine following the criteria of an average base quality score of more than Q30 and a read length of at least 50 bp. Sequences were mapped with the human reference genome GRCh38 by Burrows-Wheeler Aligner (BWA-MEM) then pre-processed (sort, add read group, mark duplication, base quality score recalibration) with SAMtools, Picard and Genome Analysis Toolkit (GATK), then the mapping statistics were calculated for the aligned sequences, with coverage at 40x of more than 90%. Variants were searched for using a GATK-Haplotypecaller with intermediate genotype (gVCF) mode individually, then representative samples including the index case, his brother, his daughter and his wife were merged together and called for genotype, separately. Variants were filtered out by machine learning using a gaussian mixture model with 99% truth sensitivity for selecting true variants by GATK-Variant Quality Score Recalibration (VQSR). For passed variants, functional annotation was interpreted by SnpEff and Variant Effect Predictor (VEP). Possible pathogenic variants among this family were prioritized, when they were non-synonymous variants found only in the CRC cases (index patient, the daughter and the brother, but not found in the wife), known pathogenic genes reported in colorectal cancer and/or familial cancer, allele calling depth of more than 25%, total read depth of more than 40x, and minor allele frequencies of less than 0.05 in the East Asian population. Supporting genotype-phenotype correlation data of the selected variants used the Human Gene Mutation Database, NCBI: ClinVar and Online Mendelian Inheritance in Man (OMIM). Beginning with well-known MMR genes (MLH1, PMS2, MSH2 and MSH6), we found no remarkable variants in these genes, nor in other genes reported in hereditary colorectal cancer(12). Two pathogenic variants passes the filter criteria: 1 non-synonymous MET variant located on chromosome 7 at position 7:116700208 (c.1124A > G), resulting in a missense mutation (p. Asn375Ser), and a non-synonymous variant in PTCH1 located on chromosome 9 at position 9:95447211 (c.4045C > T), resulting in a missense mutation (p.Pro1349Ser). The heterozygous MET and PTCH1 mutations were confirmed in all CRC cases in this family by direct nucleotide sequencing method (Fig. 3). Both sons’ DNA also were made by direct nucleotide sequencing method. We found only PTCH1 mutation, no MET mutation was detected in both sons.