Research ArticleFunctional Characterization of a Missense Variant of MLH1Identified in Lynch Syndrome Pedigree

Tahir Zaib ,1,2 Chunhui Zhang ,3 Komal Saleem ,1,2 Lidan Xu ,1,2 Qian Qin ,1,2

Yusi Wang ,1,2 Wei Ji ,1,2 Hanif Khan ,4 Hanfei Yu ,1,2 Siqi Zhu ,1,2 Wei Gao ,1,2

Yun Huang ,1,2 Xueyuan Jia ,1,2 Jie Wu ,1,2 Hongtao Song ,5 Yanqiao Zhang ,3

Wenjing Sun ,1,2 and Songbin Fu 1,2

1Laboratory of Medical Genetics, Harbin Medical University, Harbin 150081, China2Key Laboratory of Preservation of Human Genetic Resources and Disease Control in China (Harbin Medical University),Ministry of Education, China3Department of Gastrointestinal Medical Oncology, Harbin Medical University Cancer Hospital, Harbin 150040, China4Department of Pharmacology, Harbin Medical University, Harbin 150081, China5Department of Pathology, Harbin Medical University Cancer Hospital, Harbin 150040, China

Correspondence should be addressed to Yanqiao Zhang; yanqiaozhang@ems.hrbmu.edu.cn,Wenjing Sun; sunwj@ems.hrbmu.edu.cn, and Songbin Fu; fusb@ems.hrbmu.edu.cn

Received 30 October 2019; Accepted 23 December 2019; Published 29 January 2020

Guest Editor: Zhongjie Shi

Copyright © 2020 Tahir Zaib et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 theunderlying 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 of MLH1 was successfully identified by WES. Sanger sequencing verified the results of WESand also confirmed the cosegregation of the MLH1 missense variant in all affected members of the family including twounaffected 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 identifiedMLH1 missense variant (c.2054C>T:p.S685F) results in reduced expression of both MLH1 and PMS2 proteins. Based on theAmerican College of Medical Genetics and Genomics (ACMG) guidelines, the missense mutation c.2054C>T in MLH1 wasclassified as a “pathogenic” variant. Two unaffected family members were later recommended for colonoscopy and otherimportant cancer diagnostic inspections every 1-2 years as both were at higher risk of LS. In conclusion, our findings widen thegenotypic spectrum of MLH1 mutations responsible for LS. This study increases the phenotypic spectrum of LS which willcertainly help the clinicians in diagnosing LS in multigeneration families. This study also puts emphasis on the importance ofgenetic counselling for the benefit of asymptomatic carriers of MMR gene variants who are at higher risk of LS.

1. Introduction

Lynch syndrome (LS; MIM#120435), also known as heredi-tary nonpolyposis colorectal cancer syndrome (HNPCC), isa hereditary disease that increases the risk of colorectal can-cer (Lynch syndrome 1), as well as several others, such asendometrial cancer, stomach cancer, ovarian cancer, and

cancer of the small intestine or biliary tract (Lynch syndrome2) [1–3]. LS inherits in an autosomal-dominant manner.

The main cause of LS is dysfunctioning of the DNA mis-match repair (MMR) mechanism, which plays a criticalrole in correcting replication errors that escape the proof-reading activity of DNA polymerase [1]. These replicationerrors can be mismatches and small insertions or deletions.

HindawiDisease MarkersVolume 2020, Article ID 8360841, 10 pageshttps://doi.org/10.1155/2020/8360841

There are several genes known to play important roles in theMMR system: MLH1, MSH2, MSH6, PMS2, etc.

Mutation in any of these MMR genes can result in adefective MMR mechanism, which leads to microsatelliteinstability (MSI), which occurs in a high percentage of LStumors [4]. LS patients can carry variants in MLH1 (~50%),MSH2 (~39%), MSH6 (~7%), or PMS2 (~5%) [5]. MLH1and PMS2 proteins bind to form a heterodimer calledMutLα; MSH2 andMSH6 proteins form a heterodimer calledMutSα. The role of MutSα in the MMR mechanism is to rec-ognize mismatch bases along the newly synthesized DNAstrand. MutLα introduces nicks at these sites, and the incor-rect bases are then replaced with the correct bases via DNAreplication machinery [6, 7]. The EPCAM gene, upstreamof MSH2, is also responsible for 3% of LS cases, and muta-tions in this gene can cause epigenetic hypermethylation ofthe MSH2 promoter [8].

To identify the pathogenic causes is the key point forunderstanding and avoiding the recurrence of the inheriteddisease. For that purpose, whole-exome sequencing (WES)was performed on a four-generation family diagnosed withLS, and further cosegregation, bioinformatic tools, andin vitro analyses were performed to evaluate the characteris-tics of the genetic variation.

2. Materials and Methods

2.1. Subjects. The subjects were from the four-generationpedigree of LS from northern China. Comprehensive clinicalpathological analysis of the family revealed 12 membersaffected with the disease. Peripheral blood and clinical infor-mation 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 quali-fied negative-pressure vacuum EDTA anticoagulant tube.The study protocol (HMUIRB20190003) was approved bythe Institutional Research Board of Harbin Medical Univer-sity, 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 animportant role in causing different types of LS-associatedcancer. Genetic alteration in any of these genes could leadto cause any type of LS-associated cancer. To know aboutthe specific gene mutation that caused LS in the four-generation Chinese family, WES was performed. WES ofthe blood sample from patient III-4 was performed by Novo-gene Technology Co. Ltd. (Beijing, China). Briefly, genomicDNA extracted from peripheral blood for each sample wasfragmented to an average size of 180~280 bp, and DNAlibraries were produced using established Illumina paired-end protocols. Agilent SureSelect Human All Exon V6 wasused as the exome capture reagent. The Illumina NovaSeqHiSeq X Ten platform (Illumina Inc., San Diego, CA, USA)was utilized for genomic DNA sequencing to generate150 bp paired-end reads. Base-calling analysis was performedwith bcl2fastq software (version 2.19) (Illumina). The high-quality sequencing data were aligned to the reference humangenome (UCSC hg19) using the Burrows-Wheeler Aligner

(BWA) (version 0.7.8-r455) [9], and duplicate reads weremarked using Sambamba tools (version 0.7.0) [10]. Themean read depth across the target regions was 116.83.Single-nucleotide variants (SNVs) and indels were identifiedwith SAMtools (version 1.0) to generate gVCF [11, 12]. Thecopy number variants (CNVs) fromWES data were detectedusing the SVD-ZRPKM algorithm CoNIFER (version 0.2.2)[13]. Annotation was performed using ANNOVAR (version2017June8) [14].

2.3. Bioinformatic Analysis of Variants. The frequency of var-iants was evaluated in the 1000 Genomes Project (https://www.internationalgenome.org), Exome Aggregation Consor-tium (ExAC), and gnomAD (https://gnomad.broadinstitute.org). The suspected variants that happened in LS-associatedgenes (MLH1, MSH2, MSH6, PMS2, MSH3, EPCAM, FAN1,BRAF, etc.) were further evaluated. Suspected variants with afrequency < 0:01 were considered for analysis.

The significance of variants was assessed to evaluate theirimpact on protein structure and function using differentmutation predictor software programs, i.e., MutationTa-ster (https://www.mutationtaster.org), PROVEAN (https://provean.jcvi.org), SIFT (https://sift.bii.a-star.edu.sg), andPolyPhen-2 (https://genetics.bwh.harvard.edu/pph2/).

The effects of mutations on protein structure wereassessed using SWISS-MODEL (https://www.swissmodel.expasy.org/). Evolutionary conservation of the mutation locuswas patterned through Aminode (https://www.aminode.org).

Standard guidelines and recommendations for theclassification of variants given by the American Collegeof Medical Genetics and Genomics (ACMG) were alsoanalyzed [15].

2.4. DNA Extraction and Amplified PCR. DNA extractionwas performed using the DNeasy Blood & Tissue Kit (Qia-gen, #69506, Dusseldorf, Germany) according to the man-ufacturer’s protocol. Sets of primers targeting exon 18 ofMLH1 (forward primer TAGTCTGTGATCTCCGTTTAand reverse primer TTGTATGAGGTCCTGTCC) weredesigned using Primer Premier5 software. Polymerase chainreaction (PCR) was performed in a total volume of 20μl(10μl 2x GC buffer, 2μl 10x dNTPs, 0.8μl (10 pmol/μl) for-ward primer, 0.8μl (10 pmol/μl) reverse primer, 0.2μl taqDNA polymerase, 4μl (5mol/l) betaine, 2μl DNA, and0.2μl water). The cycling started with an initial 5min dena-turation 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 5minat 72°C. PCR was carried out using an Eppendorf Mastercy-cler nexus GSX1 PCR system.

Amplified PCR products were sequenced by TsingKeBiological Technology (Beijing, China). The results of Sangersequencing were analyzed using different software packages(FinchTV, MegAlign, and EditSeq).

2.5. Expression Vector and Site-Directed Mutagenesis. Toevaluate the effect of the MLH1 missense variant, in vitroexpression analysis was performed. The expression vectorGV141 (pCMV-MCS-3FLAG-pSV40-Neomycin/Amp+)

2 Disease Markers

containing the full open reading frame of MLH1 was con-structed by GeneChem (Shanghai, China). The expressionvector GV141 MLH1-wildtype was used as a template forintroducing the substitution c.2054C>T via site-directedmutagenesis with the Fast Mutagenesis System (TransBio-novo, 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 variantc.2041G>A (p.A681T) of MLH1 as severely pathogenic andc.2146G>A (p.V716M) as a neutral, proposing them for usein comparative analysis of other MLH1 missense variantsof unknown significance [16]. We compared the expressionof the identified MLH1 missense variant (c.2054C>T) toalready classified variants. For that purpose, the expressionvector GV141 MLH1-wildtype was used as a template forintroducing two more substitutions, i.e., c.2041G>A:p.A681T and c.2146G>A:p.V716M. Specific primers were usedto generate these mutations (c.2041G>A: forward, 5′-TCAGTAAAGAATGCACTATGTTCTA-3′, and reverse, 5′-TGCATTCTTTACTGAGGCTTTCAAA-3′; c.2146G>A: for-ward, 5′-CCTGGAAGTGGACTAGGAACACATT-3′, andreverse, 5′-TAGTCCACTTCCAGGAGTTTGGAAT-3′).

2.6. Cell Culture and Transient Transfection. Human embry-onic kidney HEK-293T cells purchased from the AmericanType Culture Collection (ATCC, Manassas, VA) were usedfor expression analysis of the variant MLH1:c.2054C>T, asthe HEK-293T expression system was recently shown tobe a sensitive system for detecting expression of the MLH1protein and stability problems [17]. HEK-293T cells werecultured at 37°C in a humidified 5% CO2 atmosphere in Dul-becco’s modified Eagle’s medium (Invitrogen) supplementedwith 10% fetal bovine serum. The cells were seeded ontopoly-L-lysine-coated 6-well plates at a density of 3:5 × 105cells/well. Then, transient transfection was performed with1μg of DNA and jetPRIME reagent (Polyplus-transfection,Illkirch, France).

2.7. Immunoblot Analysis. Protein expression analysis of themissense variant MLH1:c.2054C>T:p.S685F was performedin parallel with MLH1-wildtype and two other variants(pathogenic and neutral). Protein was extracted from trans-fected cells and used to evaluate the relative expression ofMLH1-wildtype and its variants through immunoblot analy-sis. Interaction between MLH1 and PMS2 proteins was alsoevaluated. For that purpose, protein expression of the endog-enous PMS2 gene was detected in HEK-293T cells.

HEK-293T cells were lysed in ice-cold PBS for immuno-blot analysis. Protein concentrations were determined usingthe BCA assay (Beijing Applygen Technologies, China).Lysates were separated by 7.5% (w/v) SDS-PAGE and trans-ferred to polyvinylidene difluoride (PVDF) membranesfollowed by incubation with the primary anti-MLH1 mono-clonal antibody (Catalog #4C9C7, Invitrogen) at a 1 : 1000dilution and an anti-mouse conjugated secondary antibody(Rockland Immunochemicals, Gilbertsville, PA). To evalu-ate the variation in protein expression levels of the endog-enous PMS2 protein in HEK-293T cells after transfectionwith the MLH1 expression vector, PVDF membranes werealso incubated with a primary anti-PMS2 monoclonal anti-body (Catalog #EPR3947, Abcam) at a 1 : 1000 dilution andan anti-rabbit conjugated secondary antibody at 1 : 10,000(Rockland Immunochemicals, Gilbertsville, PA). The signalwas developed using the Odyssey Imaging System (Li-COR,Lincoln, NE).

3. Results

3.1. Clinical Findings. The proband (III-28) is a 37-year-oldfemale who was diagnosed with colon cancer at the age of31 years. The family medical history was further investigatedfor disease occurrence. The affected family includes 71 indi-viduals in four generations. Overall, 12 members of this fam-ily suffered from autosomal-dominant LS-associated cancers(Figure 1). Descriptive clinical phenotypes of all affectedmembers are shown in Table 1.

The four affectedmembers of the family fromwhombloodsamples were obtained (II-15, III-4, III-28, and III-33) werecarefully examined for LS. II-15 is a 52-year-old female

1 2

1 2 3 4 5 6 7 8 9⁎ 10 11 12 13 14 15⁎ 16

1 2⁎ 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28⁎ 29 30 31 32⁎ 33⁎ 34 35⁎

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

⁎

I

II

III

IV

Figure 1: Pedigree of a Chinese family with autosomal-dominant Lynch syndrome (LS). Notes: symbols filled with black color are affectedfamily members and open symbols represent unaffected family members. The arrow indicates the proband (III-28). The sign (∗) below thesymbols shows family members from whom blood samples were obtained.

3Disease Markers

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 39years of age. III-28 is a 36-year-old female (proband) diag-nosedwithcoloncancer at theageof31years. 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 beaffected 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 genera-tions must be affected, and at least one of the three affectedmembers should be diagnosed before the age of 50 years [18].

3.2. Mutation Analysis Revealed a Missense Variant ofMLH1. To identify the gene responsible for LS, WES was per-

formed on affected family member III-4. Comprehensiveanalysis of the WES data revealed 11,677 synonymous SNVsand 10,832 missense SNVs, 209 nonframeshift deletions and208 nonframeshift insertions, 64 frameshift deletions and 72frameshift insertions, and 83 stop gain variants and 9 stoploss variants in the proband’s genome (Figure 2). We screenedfor pathogenic mutations and included only those variantswith a minor allele frequency (MAF) <0.01 in the 1000Genomes 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).

Eventually, we identified a substitution at chr3:37090459(GRCH37/hg19) causing a missense mutation in MLH1, a

Table 1: Clinical characteristics of the affected members of the LS family.

Family ID Gender Type of cancerAge at diagnosis

(years old)Generation-wise mean ageat 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 = 41:6

Total number of variants 26,649

Pathogenic 20

Likely pathogenic 05

2,485Variant of uncertain significance

Benign 24,139

Variant stat Number ofvariants

Known LS-related gene (MAF<0.01)

Variantfiltration

Rare variant (MAF < 0.01)

Important regions

Dominant model

Damaging

Literature

Variantclassification

Synonymous SNVs = 11,677

Missense SNVs = 10,832

Nonframeshift Del=209

Nonframeshift Ins=208

Frameshift Del=64Frameshift Ins=72

Stop gain=83

Stop loss=9

Unknown=525

MLH1(HZ⁎)

Figure 2: Variant filtration steps followed to isolate the potential causative gene variant (MLH1:c.2054C>T) found in WES data. Note: thisfigure also contains the number of different types of variants found in WES data HZ∗: Heterozygous.

4 Disease Markers

known MMR gene. The missense variant c.2054C>T inexon 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 aminoacid serine (S) with phenylalanine (F) (p.S685F) in the C-terminal domain of the MLH1 protein. We did not followup the other variants listed in Table 2, because most of thevariants had frequency higher than 0.01, or those with lowerfrequency were synonymous (no amino acid change). Themissense variantMLH1:c.2054C>T was absent from all pop-ulation datasets, and it was the most widely reported gene(~50%) for LS in literature. Thus, we considered the missensevariant MLH1:c.2054C>T for further investigation.

To determine whether the mutation in the MLH1 genecosegregates in other family members, targeted DNA frag-ments 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 andthen sequenced by Sanger sequencing. The Sanger sequenc-ing results showed that all patients (II-15, III-4, III-28, andIII-33) did carry the missense variant MLH1:c.2054C>Tincluding two unaffected family members (III-32, III-35),while two other unaffected family members (II-9, III-2) didnot carry the missense variant (Figure 3).

3.3. Bioinformatic Analysis of the Identified MLH1 MissenseVariant Revealed Its Pathogenicity. The missense variantc.2054C>T in exon 18 of the MLH1 gene was not found inthe 1000 Genomes Project, ExAC, or gnomAD. It is absentfrom these population databases.

We used several bioinformatic prediction tools to evalu-ate the identified missense variant of MLH1:c.2054C>T.MutationTaster showed the score of 0.002, PROVEAN pre-dicted it with a score of -3.44, SIFT was with a score of0.002, and PolyPhen-2 showed the score of 1.0 (Figure 4(a)).

A homology model of MLH1-wildtype and mutant pro-teins revealed that the substitution (p.S685F) was located ata linker curve which locally affected the shape of the MLH1protein as structures of both amino acids are different(Figure 4(b)).

Evolutionarily constrained regions (ECRs) of MLH1according to Aminode revealed that the amino acid serine

(S) at position 685 of the MLH1 protein is conserved amongdifferent species, such as Mus musculus and Rattus norvegi-cus (Figure 4(c)).

Table 2: Mutation in LS-associated genes found in WES data.

Gene Sequence cDNA change AA change Mutation typeFrequency in1000 Genomes

Project

Frequencyin 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_002439.5c.178_179insCCGCAG

CGCp.A60delinsAAAP

Nonframeshiftinsertion

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 0.4755

Carrier_III-32

Carrier_III-35

Unaffected_II-9

Unaffected_III-2

MLH1_c.2054C>T

Affected_II-15

Affected_III-4

Affected_III-28

Affected_III-33

Met Phe Ser/ PheTyr Ile Arg Lys

Figure 3: Sanger sequencing results. Missense variant of MLH1:c.2054C>T in heterozygous form was found in four affected familymembers (II-15, III-4, III-28, and III-33) and two unaffected familymembers (III-32 and III-35), while normal sequence was found intwo other unaffected members (II-9 and III-2) of the family.

5Disease Markers

3.4. Loss of Expression of MLH1 and PMS2 in TumorTissues from the Proband (III-28). Hematoxylin and eosin(HE) staining of resected tumor tissues from the probandrevealed malignancy and abnormal growth of cancer cells.Images of the slides were obtained using a Leica microscope(magnification 10 × 20). Furthermore, immunohistochemis-try (IHC) performed on the proband’s tumor tissues showedstrong expression of MSH2 (+) and MSH6 (+) but weakexpression for MLH1 (-) and PMS2 (-) (Figure 5).

3.5. In Vitro Expression Analysis of Identified MLH1MissenseVariant Revealed Its Pathogenicity. Expression analysis ofthe MLH1 missense variant (c.2054C>T) revealed its path-ogenicity. Immunoblot analysis of protein extracts fromtransfected HEK-293T cells showed a reduction in expressionof MLH1-c.2054C>T compared to MLH1-wildtype as wellas the established pathogenic variant MLH1-c.2041G>A.Furthermore, we checked interaction between the MLH1and PMS2 proteins through immunoblot analysis of pro-tein extracts and observed high expression of PMS2 withMLH1-wildtype. However, the lower expression with MLH1-c.2054C>T indicated a high level of interaction betweenthe proteins, which is affected by the missense mutationMLH1:c.2054C>T:p.S685F present in the PMS2-interactiondomain (506-743 aa) of MLH1 (Figure 6).

3.6. ACMG Evaluation for Identified MLH1 MissenseVariant: “Fulfilled the Criteria of a Pathogenic Variant.” Weevaluated the missense variant c.2054C>T in exon 18 ofMLH1 according to the ACMG classification for assessing

the pathogenicity of different variants. The well-establishedin vitro functional study for the variant shows damagingeffect on gene product (PS3); the variation site is in a muta-tion hotspot (PM1), absent in population databases (PM2),and cosegregates in affected members of the family (PP1).Moreover, a missense variant is a commonly reported varia-tion in MLH1 for LS (PP2), and computational evidenceindicates a deleterious effect (PP3), with a highly specific dis-ease phenotype (LS) with a single gene (MLH1) (PP4). Thus,according to the ACMG, there is one strong (PS3), two mod-erate (PM1, PM2), and more than two supportive (PP1, PP2,PP3, and PP4) lines of evidence of the pathogenicity of themissense variant (c.2054C>T) ofMLH1, fulfilling the criteriaof 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 of MLH1in a four-generation Chinese family with LS. Based on clini-cal, cosegregation, in vitro expression analyses, bioinformatictool predictions, and ACMG evaluation, we classified thisvariant MLH1:c.2054C>T:p.S685F as a pathogenic variantand the main cause of LS in this family.

The missense mutation c.2054C>T of MLH1 has notbeen published in the literature for LS or any other MLH1-related diseases. It was registered as a likely benign variantin the International Society for Gastrointestinal Heredi-tary Tumors (InSiGHT) database (http://insight-group.org/

This mulation is predicted to be probably damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00)

0.00 0.20 0.40 0.60 0.80 1.00

(a)

MLH1-wildtype MLH1-p.S685F

p.S685F

Phenylalanine (F)Serine (S)

Serine (S) Phenylalanine (F)

C)

(b)

MLH1-p.S685

(c)

Figure 4: Bioinformatic analysis of the MLH1 missense mutation (c.2054C>T:p.S685F). (a) PolyPhen-2 predicted the MLH1 missensevariant (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 Aminodeshowed that a serine at position 685 of the MLH1 protein is conserved among different species.

6 Disease Markers

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 associateddisorders [19]. Twelve missense mutations in exon 18 ofMLH1 have been classified as class 5 (pathogenic) based onthe 5-tier system proposed by InSiGHT [20]. Similarly, inHGMD, 22 missense/nonsense mutations in exon 18 ofMLH1 have been registered for LS and CRCs. We designedan exon-wise [1 to 19] illustration of currently registered mis-sense/nonsense variants of MLH1 in HGMD, including ourmissense mutation c.2054C>T (Figure 7(a)). The illustrationalso indicates that the substitution variant MLH1:p.S685F wasfound in the PMS2 binding domain of MLH1 (Figure 7(b)).

Complete cosegregation of the variant with the diseasewas evident in this family, which is the most reliableway to evaluate the pathogenicity of a variant [21]. Allpatients (II-15, III-4, III-28, and III-33) carried the MLH1missense variant, including two unaffected members (III-32, III-35). We recommended that both unaffected membersof this family who carry theMLH1:c.2054C>T variant shouldundergo colonoscopy and other important cancer-relateddiagnostic procedures every 1-2 years.

The mean age of LS diagnosis was 41.6 years in thisfamily, while the mean age for each generation was youngerthan that of the previous generation (Table 1). Sui et al.reported a four-generation family with LS, which alsoshowed the similar trend in mean age of LS diagnosis

HE

50𝜇m

(a)

HE

50𝜇m

(b)

MLH1

50𝜇m

(c)

PMS2

50𝜇m

(d)

MSH2

50𝜇m

(e)

MSH6

50𝜇m

(f)

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 normalexpression of MSH2. (f) IHC staining showed normal expression of MSH6.

7Disease Markers

generation-wise [22]. In future, these data can be helpfulfor “on time” genetic counselling to the asymptomatic car-riers of pathogenic MMR gene variants who are at high riskof LS. The phenomenon also suggests that the missensevariant MLH1:c.2054C>T has an efficient genetic effect inthe process of generation evolution.

This study increases the phenotypic spectrum of LS asone member was affected with throat cancer (I-1), onewith 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). Twoindividuals, II-15 and III-13, were affected with multiple

types of cancer, such as kidney, cervical, and ovarian cancers,as well as colon cancer (Table 1). These findings will certainlyhelp the clinicians in the future, by making diagnosing of LSuncomplicated in multigeneration families.

Expression analysis of the identified missense variant ofMLH1:c.2054C>T:p.S685F in the LS family showed reducedexpression compared to that of MLH1-wildtype and theestablished pathogenic variant MLH1:c.2041G>A:p.A681T.It showed that our identified variant is pathogenic, leadingto a defective MMR system and eventually causing LS.Our results are in agreement with previous studies

𝛽-Actin

MLH1

Vect

or

c.205

4C>T

;p.S

685F

Wild

type

c.204

1G>A

;p.A

681T

(pat

hoge

nic v

aria

nt)

c.214

6G>A

;p.V

716M

(neu

tral

var

iant

)

PMS2

MLH1

(a)

⁎⁎⁎⁎⁎⁎

Vect

or

c.205

4C>T

;p.S

685F

Wild

type

c.204

1G>A

;p.A

681T

(pat

hoge

nic v

aria

nt)

c.214

6G>A

;p.V

716M

(neu

tral

var

iant

)

0.0

0.5

1.0

1.5

Rela

tive p

rote

in ex

pres

sion

PMS2MLH1

(b)

Figure 6: Expression levels ofMLH1-wildtype and mutants and changes in expression levels of PMS2 with wildtype and mutantMLH1. (a)Immunoblot results for MLH1 and PMS2. (b) Representative graphic view of the immunoblot results. ∗∗∗P < 0:001, ANOVA followed byBonferroni’s multiple comparison test.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

33 20 23 20 10 5 5 6 12 17 18 31 14 17 11 28 15 22 29

missense/nonsensemutations (HGMD)

Exons

:c.2054C>T

MLH1

MLH1

(a)

ATP binding domain PMS2 binding domain756 aa1 aa

20 aa 147 aa 506 aa 743 aa

p.S685F

MLH1

(b)

Figure 7: Variants of MLH1. (a) Total number of missense/nonsense mutations reported to date in theMLH1 gene exon-wise in HGMD forLS. The missense mutation c.2054C>T found in exon 18 ofMLH1 (b) MLH1 proteins showing the ATP-binding domain and PMS2-bindingdomain. Substitution mutation p.S685F found in the PMS2-binding domain of MLH1.

8 Disease Markers

reporting that missense variants can severely affect expres-sion of MLH1 [6, 16, 21, 23, 24]. Barnetson et al. alsoreported a missense variant (c.2041G>A:p.A681T) in exon18 of MLH1 in a Scottish family with LS [25]. Hinrichsenet al. performed a detailed functional analysis of the missensevariant c.2041G>A:p.A681T, calling it the most pathogenicvariant compared to other variants of MLH1 [16], whichshowed that this site or locus of MLH1 is a mutational hot-spot and very sensitive to substitution. According to Desviatet al., substitutions in certain domains of MLH1 can destabi-lize the protein and consequently reduce expression, alsoproving that the substitution c.2041G>A:p.A681T rendersthe protein clearly less stable and easily degradable [26].

The position of the variant c.2054C>T:p.S685F is alsocrucial because it lies in the PMS2 interaction domain ofMLH1 (aa 506-743) (Figure 7(b)). Both MLH1 and PMS2form a heterodimer called “MutLα,” which is a vital part ofthe human MMR system and involved in the majority ofMMR events [7, 27]. Our interaction analysis of MLH1 andPMS2 through immunoblotting suggests that theMLH1mis-sense variant (c.2054C>T:p.S685F) in the LS family affectedthe interaction between MLH1 and PMS2, as PMS2 showedhigh expression with MLH1-wildtype compared to MLH1-MT (c.2054C>T:p.S685F) and an established pathogenic var-iant (c.2041G>A:p.A681T). Our results are in agreementwith previous studies demonstrating that loss of the PMS2protein in MLH1 mutation carriers is a common phenome-non because PMS2 is stable after binding with MLH1 to formheterodimers in the MMR system and is less stable when itfails to interact with MLH1 [24, 28, 29].

Similarly, IHC staining results for the proband’s tumortissues correspondingly showed loss of expression of bothMLH1 and PMS2. Previously, this subdomain of the MLH1protein was validated as being quite conserved and sensitiveto substitution mutation, which leads to a severely destabi-lized protein [16]. According to the literature, it has beenestablished that variant c.2054C>T:p.S685F is located in thefunctional domain of MLH1 [28]. Biochemical analysis hasshown that the majority of mutations in the PMS2 interactivedomain of MLH1 are pathogenic [30].

With this variant (c.2054C>T:p.S685F), serine (S) isreplaced with phenylalanine (F) at position 685 aa of theMLH1 protein. Serine is a nonaromatic amino acid, whereasphenylalanine is aromatic. Serine is also smaller in size thanphenylalanine, which may affect the ability of the mutantresidue to fit into the core domain of the protein, whichin turn may affect the structure and binding of this partic-ular domain with other proteins, e.g., PMS2. Moreover, ser-ine is hydrophilic, whereas phenylalanine is not, and serineforms hydrogen bonds with other amino acids (threonineand serine at positions 553 and 556, respectively), whichmay not be formed in the case of phenylalanine due tothe different structure orientation [31]. The chemical prop-erties and structure of both amino acids are entirely differ-ent, which can affect the structure and stability of theMLH1 protein.

Peltomaki and Vasen stated that a missense mutation canbe called a pathogenic mutation if (i) the chemical propertiesof amino acids are changed, (ii) the amino acid is evolution-

arily conserved, (iii) the mutation is absent in the normalpopulation, (iv) the mutation cosegregates with the disease,and (v) MSI is high with an absence of IHC staining for thatparticular MMR protein [32]. In our case, all five points weremet, confirming the pathogenicity of the identified missensevariant (c.2054C>T) of MLH1.

5. Conclusions

We successfully identified a missense variant (c.2054C>T:p.S685F) in exon 18 of MLH1 (NM_000249.3). Based onclinical data, IHC staining, cosegregation analysis, in silicopredictions, and in vitro functional analysis, we classifiedthe MLH1 variant (c.2054C>T) as pathogenic and the maincause 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 inspec-tions every 1-2 years were recommended for both. Ourresults increase the genotypic spectrum of MLH1 mutationsthat cause LS. This study also emphasizes the significanceof genetic counselling for carriers of pathogenic MMR genevariants who are at high risk of LS.

Data Availability

Data is available upon request.

Conflicts of Interest

The authors declare that they have no competing interests.

Authors’ Contributions

TZ, CZ, KS, WS, and SF were involved in all aspects of thisstudy. TZ did literature review and drafted this manuscript.SF, WS, and KS critically reviewed this manuscript. TZ andKS conducted experiments. TZ and XJ performed WESdata analyses. TZ, QQ, YW, WJ, HK, HY, SZ, WG, YH,and JW conducted bioinformatics study and analyses. CZ,LX, HS, and YZ performed clinical analyses. All authorsparticipated in manuscript formation by providing com-ments and suggestions. All authors read and approved thefinal manuscript. Tahir Zaib and Chunhui Zhang contrib-uted equally to this work.

Acknowledgments

This work was supported by the National Key Research andDevelopment Program (grant number 2016YFC1000504, toSF). We would like to thank all family members for theirparticipation and cooperation.

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