Abstract
In a proportion of patients presenting mismatch repair (MMR)-deficient tumors, no germline MMR mutations are identified, the socalled Lynch-like syndrome (LLS). Recently, MMR-deficient tumors have been associated with germline mutations in POLE and MUTYH or double somatic MMR events. Our aim was to elucidate the molecular basis of MSH2-deficient LS-suspected cases using a comprehensive analysis of colorectal cancer (CRC)-associated genes at germline and somatic level. Fifty-eight probands harboring MSH2-deficient tumors were included. Germline mutational analysis of MSH2 (including EPCAM deletions) and MSH6 was performed. Pathogenicity of MSH2 variants was assessed by RNA analysis and multifactorial likelihood calculations. MSH2 cDNA and methylation of MSH2 and MSH6 promoters were studied. Matched blood and tumor DNA were analyzed using a customized next generation sequencing panel. Thirty-five individuals were carriers of pathogenic or probably pathogenic variants in MSH2 and EPCAM. Five patients harbored 4 different MSH2 variants of unknown significance (VUS) and one had 2 novel MSH6 promoter VUS. Pathogenicity assessment allowed the reclassification of the 4 MSH2 VUS and 6 probably pathogenic variants as pathogenic mutations, enabling a total of 40 LS diagnostics. Predicted pathogenic germline variants in BUB1, SETD2, FAN1 and MUTYH were identified in 5 cases. Three patients had double somatic hits in MSH2 or MSH6, and another 2 had somatic alterations in other MMR genes and/or proofreading polymerases. In conclusion, our comprehensive strategy combining germline and somatic mutational status of CRC-associated genes by means of a subexome panel allows the elucidation of up to 86% of MSH2-deficient suspected LS tumors.

Lynch syndrome (LS) is an inherited autosomal dominant cancer syndrome that accounts for 2-4% of all newly diagnosed colorectal and endometrial cancers. [1] [2] [3] It is caused by defective mismatch repair (MMR) activity due to germline (epi)mutations in MMR genes (MLH1, MSH2, MSH6 and PMS2). The diagnostic algorithm of LS is based on the identification of microsatellite instability (MSI) and/or loss of expression of MMR proteins by immunohistochemistry (IHC) in tumors. After identification of MMR deficiency (in the absence of MLH1 promoter methylation and/or BRAF p.V600E mutation), germline MMR testing is performed. However, about 55% of patients with MMR-deficient colorectal and endometrial tumors lack identified pathogenic mutations by conventional analyses, thus hampering appropriate clinical management and risk assessment in these socalled Lynch-like syndrome (LLS) patients. 4 LLS patients together with their first-degree relatives are considered to have an intermediate risk of developing CRC. 5 Recently, somatic double hits in DNA repair genes have been detected in a variable proportion (30-82%) of LLS. [6] [7] [8] [9] While somatic MLH1 promoter hypermethylation is common in MSI tumors, 10, 11 the relative contribution of somatic methylation in other MMR gene promoters in LLS has been poorly studied. 12, 13 Limitations in the molecular analysis techniques utilized could be responsible for the lack of detection of germline MMR mutations, due to false-positive IHC/MSI results, falsenegative results in MMR mutational analysis due to complex or cryptic mutations [14] [15] [16] [17] [18] or lack of sensitivity (i.e. in mosaic cases). 9 Moreover, up to 30% MMR variants are classified as variants of unknown significance (VUS), in which their clinical significance is not evident. 19 Moreover, germline mutations in genes other than MMR genes (biallelic MUTYH and POLE) have been reported rarely in patients with MMRdeficient tumors, 7, [20] [21] [22] [23] reinforcing the need to implement NGS gene panels (either commercially available or custommade) in the routine setting. 24, 25 In this work, we aimed at elucidating the molecular basis underlying tumorigenesis in a cohort of 58 LS-suspected patients harboring MSH2-deficient tumors using a comprehensive strategy. Sequencing of a panel of CRC-associated genes in germline and tumor formalin-fixed paraffin-embedded (FFPE) samples was used to complement the germline MMR gene (epi)mutation testing.
Material and Methods

Patients
Mutational screening of MSH2 was performed in a cohort of 58 probands with LS-associated tumors showing loss of MSH2 protein expression by IHC (Supporting Information, Table S1 ). Patients were assessed at Cancer Genetic Counseling Units at the Catalan Institute of Oncology from 1998 to 2012. Twenty patients fulfilled Amsterdam criteria, 36 revised Bethesda criteria and the remaining 2 were referred to the Genetic Counseling Unit for showing histological features suggestive of MMR-deficiency and loss of MSH2 expression. Clinical and pathological information of affected individuals was recorded. DNA samples from controls of a hospital based CRC case-control study were used to analyze the frequency of the detected MSH2 VUS. 26 Informed consent was obtained from all individuals enrolled, and internal Ethics Committees of participant hospitals approved this study. Of note, three patients initially classified as LLS were excluded from this cohort due to the detection of biallelic MUTYH mutations as reported.
20,27
Isolation of genomic DNA Peripheral blood leukocyte (PBL) DNA was extracted using FlexiGene DNA kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. For each available specimen of formalin-fixed paraffin-embedded (FFPE) tissue, 10-20 3 10-lm FFPE sections were cut from a single representative block per case, using macrodissection with a scalpel as needed to enrich for tumor cells. After deparaffinization with 480 ll of Deparaffinization Solution (Qiagen, Hilden, Germany), DNA isolation was performed using either the DNAeasy Tissue Kit or QIAmp DNA FFPE Tissue Kit (Qiagen) according to the manufacturer's instructions.
Mismatch repair genes mutational analysis
Mutational analysis of coding regions of MSH2 and MSH6
genes. Point mutation analysis of MSH2 (NM_000251.2, NG_007110.1) and MSH6 (NM_000179.2; NG_007111.1) was performed by PCR amplification of exonic regions and exonintron boundaries followed by Sanger sequencing (primers and conditions available upon request). Genomic rearrangements in MMR genes were analyzed by multiplex ligation dependent probe amplification using SALSA-MLH1/MSH2 P003-B1 and MSH6 P072 kits (MRC-Holland), which include 
Pathogenicity assessment of MSH2 variants
Variant frequency and co-segregation analysis. Global population frequency of the identified MSH2 variants was retrieved from the Exome Aggregation Consortium (ExAC; http://exac.broadinstitute.org/) and NHLBI Exome Sequencing Project (ESP; http://evs.gs.washington.edu/EVS) databases and Spanish population frequency was screened by Sanger sequencing in a cohort of 246 healthy controls.
26 MSH2 variants were also screened in DNA samples from family relatives by Sanger sequencing.
In silico prediction of the functional impact. DNA sequences containing the identified MSH2 variants were analyzed using several bioinformatic tools to evaluate their impact at the RNA and protein level, as previously reported.
28,29 PROMO computational tool was used to analyze the predicted impact of promoter variants. Protein stability predictions were obtained by applying PoPMuSic (http://dezyme.com/), CUPSAT (http://cupsat.tu-bs.de), ERIS (http://troll.med.unc. edu/eris/), I-Mutant 3.0 (http://gpcr2.biocomp.unibo.it/cgi/ predictors/I-Mutant3.0/I-Mutant3.0.cgi) and FoldX 4 (http:// foldxsuite.crg.eu). For the structural analysis, the structure of the DNA lesion recognition complex (PDB code: 2O8B) was used, which includes human MSH2, MSH6 and a DNA substrate. PyMOL Molecular Graphics System v171.132.149.166 (Schr€ odinger, LLC) was used to visualize structures. The disease-related variants in the close vicinity of the MSH2 variants identified in this study were calculated using StructurePPi. 30 The 3 D clustering of missense variants is often used as a supporting evidence for the involvement of those variants in the disease or as a basis for functional hypotheses about the clustered mutations.
Multifactorial likelihood analysis. Multifactorial likelihood analysis was based on estimated prior probabilities of pathogenicity and likelihood ratios for segregation and tumor characteristics as described. 19 For variants without available prior probabilities (exonic variants altering splicing), a prior probability of 0.5 was used (no prior assumptions). Variants were classified according to the 5 class IARC scheme, 31 based on the calculated posterior probability.
mRNA splicing analysis and allele-specific expression analysis. Human lymphocytes from variant carriers were cultured and total RNA was extracted as described above. Subsequently cDNA was synthesized as described. 28 Amplification of MSH2 coding region containing the variants was performed using specific primers (Supporting Information, Table S2 conditions available upon request). Sequences of carriers' transcripts were compared with transcripts from three control lymphocyte cultures. Allele-specific expression (ASE) was analyzed by SNuPE 28 (Supporting Information, Table S2 ; conditions available upon request). ASE was calculated by dividing the ratio of variant/wildtype allele in cDNA by the ratio of variant/wildtype allele in gDNA. We used 0.5 as a threshold value for ASE definition. Experiments were performed in quadruplicate.

Targeted next generation sequencing
Agilent SureDesign web-based application (Agilent Technologies, USA) was used to design DNA capture probes of 509 target regions, including the coding exons plus 10 flanking bases of 26 genes associated to CRC, and their promoter regions (comprising 650 bases upstream their TSS) (Supporting Information, Table S3 ). Regions containing somatic hotspot mutations in 12 actionable target genes and MSI CRC-associated loci of SETD2, SETD1B and SETDB2 were also included 32 (Supporting Information, Table S3 ). Design was optimized for FFPE samples. Final design was composed of 11,012 amplicons covering 99.61% of the submitted target regions, in a total sequenceable design size of 319,653 kb.
DNA quality was tested using NanoDrop ND 1000 Spectrophotometer (Thermo Fischer Scientific), by electrophoresis in agarose gel and by Qubit Fluorometer using dsDNA BR Assay (Invitrogen, Carlsbad, CA, USA). To assess FFPEderived DNA integrity, a PCR amplifying two GAPDH products was performed and the products were visualized using High Sensitivity DNA chips in a Bioanalyzer (Agilent Technologies). Capture of the target regions was performed using HaloPlex Target Enrichment kit 1-500 kb (Agilent Technologies), according to the HaloPlex Target Enrichment System-Fast Protocol Version B. Briefly, the protocol consists of four steps: (1) digestion of genomic DNA using eight different restriction reactions; (2) hybridization of restricted fragments to probes whose ends are complementary to the target fragments, circularization of fragments and incorporation of sequencing motifs including index sequences; (3) capture of target DNA using streptavidin beads and ligation of circularized fragments; (4) PCR amplification of captured target libraries. Quality control and dilution estimates of libraries were performed using High Sensitivity DNA chips in a Bioanalyzer. Library concentrations were normalized to 0.44 nM. Pooled libraries were sequenced in a MiSeq (Illumina) with paired-end 250 bp reads plus an 8-base index read, using MiSeq Reagent Kit v3.
Agilent SureCall application was used to trim, align and call variants. Variant filtering was performed based on Phred quality 30, alternative allele ratio 0.05, read depth 383 in PBL samples and 103 in FFPE samples. Identified variants were then filtered against common single-nucleotide polymorphisms (MAF>1 according to ExAC and ESP databases). Predicted pathogenic germline rare variants and MSH2 double somatic hits were further confirmed by Sanger sequencing using independent DNA samples.
Loss of heterozygosity analysis
Loss of heterozygosity (LOH) was assessed in FFPE tumor DNA by analyzing the alternative allele ratio of germline heterozygous MSH2 SNPs genotyped by NGS and three microsatellite markers (D2S2328, D2S288 and D2S378) spreading over 17 Mb around MSH2 (Supporting Information, Table  S4 ).
Methylation analysis of MSH2 and MSH6 genes
Methylation was evaluated by Methylation Specific-Melting Curve Analysis (MS-MCA), consisting of a real-time PCR followed by temperature dissociation of bisulfite-treated DNA, using the EZ DNA Methylation-Gold Kit (Zymo Research, USA). Each promoter region was preamplified using 2 ll of external primers at 2 lM, 1 ll of bisulfite-treated DNA and 5 ll of Double MegaMix solution (Microzone Ltd., UK). Heminested PCRs of both promoter regions were carried out in a LightCycler 480 II (Roche, Germany) using 1 ll of a 1:10 dilution of preamplified fragments in 9 ll of Light Cycler 480 SYBR Green I (Roche) containing 0.5 lM of each internal primer. Primer sequences are listed in Supporting Information, Table  S2 . The amplified region of MSH2 and MSH6 promoters covered 13 and 18 CpGs, respectively. In vitro methylated DNA from CpG methylated Jurkatt Genomic DNA (New England Biolabs, MA, USA) and a CRC sample from an EPCAM deletion carrier were used as methylated controls in these experiments. Analytical sensitivity of the method to detect methylation was assessed using serial dilutions of methylated Jurkatt DNA and lymphocyte DNA from a healthy patient (after bisulfite sequencing corroboration of unmethylation). Analytical sensitivities of 10 and 25% were achieved in the analysis of MSH2 and MSH6 promoters, respectively (Supporting Information, Fig. S1 ).
Results

Clinical characteristics of patients with MSH2-deficient tumors
We identified 58 probands diagnosed with LS-associated tumors showing loss of MSH2 expression (Supporting Information, Table S1A ). Accordingly, MSI was evident in all the informative tumors available (n 5 28). DNA mutational analysis allowed the identification of 25 patients harboring bona fide germline pathogenic variants (IARC class 5) in MSH2 and 2 in EPCAM and 8 harboring MSH2 likely pathogenic variants (class 4) according to the InSiGHT classification rules 19 ( Table   1 ). In all, 35 of 58 patients were classified as LS. The remaining 23 were categorized as Lynch-like syndrome (LLS), 5 of them being carriers of MSH2 variants of unknown significance (VUS; class 3).
In the identified LS patients, the mean age at first LSassociated-tumor diagnosis was of 45.8 years, while it was of 49.2 years in LLS cases (Supporting Information, Table S1B ). Concerning clinical criteria fulfillment, 49% of LS cases met Bethesda criteria being this proportion higher (82.6%) in LLS. Fifty-seven percent of LS cases and 25% of LLS patients (n 5 20 and n 5 6, respectively) presented multiple LS-associated tumors. (Tables 1 and 2 ). None of them was described in ExAC and NHLBI ESP Databases nor identified in Spanish cohorts of control individuals (Table 2 ). In silico predictions are shown in Supporting Information, Table S5. cDNA splicing evaluation was performed in carriers of 7 variants (the 4 VUS, c.211G > C, c.989T > C and c.1276 G > A), with available lymphocytes. In four of them, aberrant transcripts were identified (Supporting Information, Table S5 and Fig. S2 ): (i) MSH2 c.211G > C (Case 234) results in a partial deletion of exon 1 (r.195_211del), which is predicted to generate a truncated protein (p.Tyr66Serfs*10); (ii) MSH2 c.1276G > A (Case 258) leads to a partial deletion of exon 7 by activation of a cryptic donor site (r.1230_1277del), which is predicted to generate an in-frame deletion of 16 amino acids (p.Ile411_Gly426del) in a highly conserved MSH2 domain (Supporting Information , Fig. S3 ); (iii) the duplication of exon 11 (Case 264) causes its duplication in tandem (r.1662_1759dup; p.Gly587Alafs*3); and (iv) the duplication of exons 11-16 identified (Case 120) generates a longer transcript (r.1662_*23dup) containing a tandem duplication of exons 11-16 and 23 nucleotides of the 3 0 UTR downstream the stop codon. Therefore, three MSH2 variants (c.211G > C, c.1276G > A and exon 11 duplication) were reclassified as pathogenic based on the generation of aberrant transcripts leading to premature stop codons or in frame-deletions disrupting functional domains 19 (Table 1) . Although the 
Cancer Genetics and Epigenetics
Vargas-Parra et al. duplication of exons 11-16 leads to the generation of an aberrant transcript, as the duplicated region is inserted after the stop codon, its pathogenic effect at the protein level cannot be unequivocally demonstrated. The remaining MSH2 variants (c.518T > G, c.989T > C and c.2069A > G) analyzed had no apparent effect on mRNA splicing and stability (Supporting Information, Table S5 ). Clinicopathological data from all the families carrying class 3 and 4 variants were used in multifactorial calculations (Supporting Information, Fig. S4 and Table 2 ). As variant MSH2 c.518 T > G was further identified in two additional families from other centers ( Supporting Information, Fig. S4C ), their data were also included in this analysis. Posterior probability of pathogenicity resulted >0. (Tables 1 and 2 ). Therefore, multifactorial analyses allowed the classification as pathogenic mutations of the 7 variants not previously classified as pathogenic by cDNA analysis. Accordingly, the 4 missense variants (c.518T > G, c.989T > C, c.2069A > G, c.2074G > C) were in silico predicted as functionally damaging and destabilizing at the protein level (Supporting Information, Table S5 ), being involved in a network of interactions with other disease-associated variants ( Supporting  Information, Fig. S3 ).
In-depth germline analysis of LLS cases
To rule out having missed RNA-affecting mutations, we resequenced the whole MSH2 transcript in 10 PBL samples. A splicing alteration was detected in one patient (Case 109). In absence of puromycin, a deletion of almost all the first exon was identified (r.216_211del; p.?) ( Fig. 1 and Table 1) . Moreover, in presence of puromycin, an in-frame deletion of 16 bases (r.195_211del; p.Tyr66Serfs*10) was also detected. Further NGS analysis (see below) revealed a mutation in the last nucleotide of the first MSH2 exon (c.211G > C), previously missed by Sanger sequencing due to primer design.
In the analysis of MSH2 promoter region and the 3 0 UTR of EPCAM only known polymorphisms were detected (Table  1) . Unfortunately, the low prevalence of heterozygous SNPs in these regions prevented the analysis of the presence of germline allelic imbalance (data not shown). Interestingly, 2 variants (c.225C > T and c.2204C > G) were detected in MSH6 promoter in Case 102. The MSH6 variant c.225 C > T is predicted to produce a premature out-of-frame start codon. In silico, variants c.225C > T and c.2204C > G are predicted to affect FOXP3 binding, and binding of TFII-I, STAT4, NFkappaB1, c-Ets-1, RelA and Elk-1, respectively.
Next, 17 PBL samples from LLS patients and 4 samples from reclassified MSH2 variants carriers were analyzed with our CRC associated genes NGS custom panel (Tables 1 and  3 ). The MSH2 c.211G > C variant was identified in Case 109, which is responsible for the splicing defect previously observed (Fig. 1) . In LLS cases, germline missense variants predicted as pathogenic by at least 3 functional in silico tools were found in distinct CRC genes: one in BUB1, three in the H3K36 trimethyltransferase SETD2, 1 in FAN1 and two in MUTYH (monoallelic). Of note, probably pathogenic variants in SETD2 and FAN1 coexisted in one of the heterozygous MUTYH carriers (Case 105).
In all, germline and functional characterization classified 5 additional cases as LS, 4 harboring 3 reclassified MMR VUS and the missed MSH2 mutation (Table 1) . Also, predicted pathogenic variants were identified in other genes in 5 additional cases.
Molecular analysis of LLS tumors
Next, we explored whether combined germline and somatic testing could help in elucidating the molecular basis of the remaining cases. Somatic hits in DNA repair genes were found in 5 tumors from the 4 LLS individuals tested (Table  4) : double somatic hits in MSH2 and MSH6 (cases 111 and 114), apparent MSH2 loss of heterozygosity (Case 108 C1/ C2) and coexistence of double somatic mutations in other MMR genes and/or proof-reading polymerases (POLD1 and POLE) (cases 108 C1/C2 and 121). Also, somatic mutations in other cancer genes (APC, AXIN2, BMPR1A, PTEN or BUB1B) and in CRC actionable genes coexisted with the aforementioned alterations (Tables 4 and 5 ). Interestingly, the two colorectal tumors from Case 108 showed completely different profiles: the MSI tumor (cancer 1) mainly harbored deletions at homopolymeric sequences, whereas the MSS tumor (cancer 2) harbored substitutions.
Somatic methylation did not account for any other case as promoter methylation in MSH2 (0/8 tumors) or MSH6 (0/11 tumors) was not detected (Supporting Information, Fig. S1 and Table 1 ). As previous studies have reported somatic methylation at the MSH2 promoter in LS MSH2 mutation carriers, 12 8 additional tumor samples from LS MSH2 mutation carriers from our LS series were studied, none of which were methylated (data not shown).
Discussion
A comprehensive germline and somatic mutational analysis allowed the molecular characterization of a high proportion of MSH2-deficient tumors in a series of LS suspected patients. The reclassification as pathogenic of 4 MSH2 VUS and the identification of a new MSH2 splicing mutation yielded a 71% (41/58) mutation detection rate. Furthermore, predicted pathogenic germline variants in DNA repair and genomic instability genes BUB1, SETD2, FAN1 and MSH6 were identified in 5 patients. Finally, the presence of double MMR or combined MMR/polymerase somatic hits in tumors from the informative LLS individuals analyzed may increase this yield up to 86% (50/58). The obtained results further evidence the great heterogeneity present in this subset of cases, as previously reported, 7, 8, [20] [21] [22] 27 and reinforce the notion that negative germline DNA and RNA testing should be complemented with somatic analysis. RNA analyses allowed classification of three MSH2 variants as pathogenic mutations affecting mRNA processing. Splicing analysis in combination with multifactorial likelihood calculations offered a good performance, allowing reclassification of the 10 variants analyzed (6 class 4 variants and 4 class 3 variants) as disease causing mutations. These results highlight the benefit of applying quantitative and qualitative analyses for variant interpretation and classification. Moreover they showed the usefulness of the implementation of RNA analyses (either splicing or allelic imbalance) in the diagnostic routine, as previously also demonstrated for the identification of cryptic variants in MMR genes. 18, 33 The MSH2 variant c.211G > C, identified in two patients, illustrates the complexity of variant classification and the challenge associated with functional characterization. Splicing analysis of the whole transcript in Case 109 identified two aberrant transcripts (r.216_211del and r.195_211del). In contrast, in Case 234, the splicing analysis performed encompassing a smaller region containing the variant (from exon 1-nucleotide c.85-to exon 4) identified only the r.195_211del transcript. The variant c.211G > C was finally classified as pathogenic based on the generation of aberrant transcripts. 19 The germline mutational analysis of selected CRCassociated genes has yielded promising results in this set of MSH2-deficient LLS cases. Germline biallelic MUTYH mutation carriers were detected and excluded prior to this analysis. 20, 27 The identification of a predicted pathogenic alteration in FAN1 reinforces the notion that FAN1 is a CRC predisposing gene. 34 To the best of our knowledge, this is the first report of a germline predicted pathogenic BUB1 35 variant in a patient with breast and endometrial cancers, which has been recently associated with early onset and familial CRC. 36 Moreover, 3 LLS patients diagnosed with CRC before age 50 harbored germline predicted pathogenic variants in SETD2, an H3K36 trimethyltransferase, which was included in our customized NGS panel for being frequently reported mutated in MSI CRC. 32 Its depletion results in MSI and elevated mutation rates in vivo, as H3K36me3 activity is necessary for recruiting MSH2/MSH6 to chromatin. 37 With the identification of rare and potentially pathogenic variants, FAN1, BUB1 and SETD2 are emerging as candidate genes responsible for LLS. Functional and cosegregation analysis are needed to elucidate the pathogenicity of the identified variants, and further collaborative efforts should be made to confirm their involvement in the inherited predisposition to cancer. Moreover, it must be borne in mind that both undetected germline alterations in the MMR genes -complex mutations, structural variations and The number of "C" in tumor tested corresponds to the Cancer number of Table 1. ( ) See Supporting Information, Table 5 .
Abbreviations: B, benign; D, damaging; PrD, probably damaging; PsD, possibly damaging. Brown-colored, frameshift and predicted probably pathogenic variants. The number of "C" in tumor tested corresponds to the cancer number of Table 1 . Abbreviations: B, benign; D, damaging; PrD, probably damaging; PsD, possibly damaging. Brown-colored, frameshift and predicted probably pathogenic variants.
variants in regulatory regions-and alterations in genes not analyzed in this study could be having a role in LLS. The accumulation of somatic alterations in DNA repair genes can certainly mimic germline associated phenotypes. Subexome analysis at a high coverage has shown to be useful for the identification and characterization of these cases. Indeed, somatic double hits in MMR genes were evidenced in two of 5 tumors, confirming previous observations. [6] [7] [8] [9] In the remaining three, putative loss of heterozygosity and double heterozygous MMR genes and/or proofreading polymerases were identified in accordance with a recent report. 38 The limited number of cases analyzed precludes drawing conclusions on these findings although it must be considered that pediatric tumors arising in CMMRD cases strongly associate with mutations in the exonuclease domain of proofreading polymerases. 39 In line with previous reports, our observations reinforce the notion that somatic variants in MSH2 or MSH6 may be a frequent event in LLS cases, while somatic promoter hypermethylation does not play a significant role.
The lack of detection of MSH2 methylation in LLS MSH2-deficient tumors is in agreement with the low proportion of methylated tumors in MSH2-deficient LLS patients (1 of 46) reported in two previous series 12, 13 . When testing for methylation, the dependability of the technique is critical. MS-MCA is a robust technique that could simultaneously analyze several CpGs. 40 The use of methylation-independent primers further increases its consistency validated by the inclusion of adequate positive and negative controls in each run. Moreover, none of the 8 available tumors from MSH2 mutated LS cases were methylated, in contrast to a previous report. 12 It must be emphasized that they analyzed an upstream region not included in our amplicon and, in consequence, the results could not be directly compared.
Our study highlights the importance of an in-depth strategy, combining germline and somatic mutational analysis by parallel high-throughput deep sequencing and characterization of variants identified. The yield of subexome testing is directly related to the selection of genes, the sample type analyzed and the quality and depth of the analysis. With a mean coverage of 12003, we have probably ruled out most germline mosaicisms with a 5% cutoff value in PBLs. While mean coverage was similar for PBL and FFPE DNA, it was highly variable in FFPE samples depending upon the amplicon chosen. The combined germline-somatic analysis allows for a bona fide identification of somatic variants. However, the better the quality of FFPE DNA, the higher the yield of the analysis. The Haloplex technology partially bypasses possible artifacts related to sample processing by using many probes of different lengths at distinct regions minimizing lack of hybridization due to DNA fragmentation.
Our study also shows some limitations. The identified somatic mutations have been detected in amplicons with good coverage (14003) making our findings dependable, although variability may have led to the loss of other relevant findings. The lack of available tumor sample has precluded the identification of second hits within the tumor in the majority of cases. Of note, the prevalence of double somatic MMR mutations in the analyzed tumors is similar to other series. 6, 9 It must be acknowledged that our custom-made subexome panel can be improved by including novel putative CRC predisposition genes. Also, the inclusion of homopolymers as target regions could help in ascertaining MSI in tumor samples, as recently reported. 41 Finally, the yield observed in MSH2-deficient tumors needs to be confirmed when loss of other MMR proteins is observed. In all, comprehensive germline and somatic analysis has proved useful in the elucidation of the underlying molecular basis of suspected LS in MSH2-deficient cases. Subexome analysis opens the scope of the genes underlying the development of these tumors, expanding the spectrum of overlapping phenotypes in these selected cases. Further studies of larger series and more indepth functional characterization of variants detected are mandatory to establish the true clinical validity of the proposed algorithm. Our approach further illustrates the relevance of germline and somatic testing when deciphering the genetic basis of LLS or other CRC predisposition syndromes.

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