The gene panel was applied to 91 patients, previously tested negative for mutations in the polyposis genes (APC, MUTYH, BMPR1A, SMAD4, STK11) and/or a combination of different MMR genes (MLH1, MSH2, MSH6, PMS2,) depending on the primary indication when the referral was issued. The patients were sub-grouped based on their clinical characteristics (Supplementary Table 1). Sequencing was performed over the entire gene regions as described and all coding regions were covered at least 30 × except for CDH1 ex1, EPCAM ex1, MSH3, ex1 and MLH1 ex12 which in five samples were covered at least 25x. For the whole targeted region the mean coverage was 417x in all 91 samples. The analyses of variants included the coding region and ± 20 bp of intronic sequences. The CNV analysis was based on the entire covered gene regions.
In total 8 pathogenic class 5 and 8 likely pathogenic class 4 variants were found (Tables
1,
2). This gives a mutation detection frequency of 8.8 % (8/91) for the class 5 variants only and a frequency of 17.6 % (16/91) when also class 4 variants are included. These results are in concordance with the results obtained in other studies of similar gene panels [
6,
18]. Two pathogenic variants in
PMS2 in patients I:26 and I:50 were missed in the initial analyses of the MMR genes performed in an external laboratory. Thirty-two missense variants, all of them found in a heterozygote state, with MAF ≤1 %, according to the filtration criteria, were analyzed and classified manually or according to the InSiGHT database [
46] in the case the variant was included in this database. The results are presented in Table
2 and include four class 5 pathogenic variants, two likely pathogenic class 4 variants and 26 class 3 variants of unknown clinical significance (VUS). The
APC variant, c.1902 T > G, was recently found to have a major splicing effect on exon 14 resulting in loss of this exon [
10]. The variant was found in a patient (III:61) with unexplained familial adenomatous polyposis (1–100 polyps), this patient also had a VUS,
APC c.4472T > A, p.Phe1491Tyr. Both of these variants segregated with affected individuals and neither of them were found among healthy individuals from the family. Two class 5 variants were found in
MUTYH, one each in patients III:71 and I:42, respectively. The c.536A > G, p.Tyr179Cys and c.1187G > A, p.Gly396Asp were found in a heterozygote state and are classified as pathogenic if found homozygote or in a compound heterozygote state.
Table 1
Truncating variants among 91 index patients
I:32 | SMAD4 | chr18:g. 48591947-4859195951del | NM_005359: c.1110_1114del | Frameshift | p.His371Aspfs*5 | Not present | 5 |
I:20 | CHEK2 | chr22: g.29130389 | NM_007194: c.319 + 2T > A | Splicing | p.? | Not present | 4 |
I:26 | PMS2 | chr7: g.6026808 | NM_000535: c.1588C > T | Nonsense | p.Gln530* | 0.0017 % | 4 |
I:50 | PMS2 | Chr7:g.6035204_6035207del | NM_000535: c.861_864del | Frameshift | p.Arg287Serfs*19 | 0.0017 % | 5in
|
I:91 | PMS2 | Chr7:g.6031690 | NM_000535:c.904-2A > G | Splicing | p.? | Not present | 4 |
IV:69 | MLH3 | chr14: g.75506621/rs193219754 | NM_001040108: c.3563 C > Gho
| Nonsense | p.Ser1188* | 0.019 % | 4 |
I:55 | AXIN2 | Chr17:g.63554485del | NM_004655.3:c.254del | Frameshift | p.Leu85Tyrfs*24 | Not present | 4 |
II:59 | BMPR1A | chr10: g.88659789 | NM_004329: c.441delT | Frameshift | Phe147Leufs*18 | 0.00082 % | 5 |
IV:76 | BMPR1A | chr10: g.88649983 | NM_004329: c.230 + 2T > C | Splicing | p.? | Not present | 4 |
V:87 | BMPR1A | chr10: g.88679023 | NM_004329: c.969delT | Frameshift | p.Cys323Trpfs*41 | not present | 5 |
Table 2
Classification of missense variants with a MAF < 1 % among 91 index patients
III:61 | APC | Chr5:112170806 | NM_000038:c.1902T > G | Exon 14 skipping | Not present | Deleterious | Probably damaging | 20.9 | 5 |
III:61 | APC | Chr5:112175763 | NM_000038:c.4472T > A | p.Phe1491Tyr | Not present | Deleterious | Probably damaging | 22.8 | 3 |
V:89 | APC | Chr5:112176431/rs148275069 | NM_000038:c.5140G > A | p.Asp1714Asn | 0.020 % | Deleterious | Benign | 13.5 | 3 |
I:1 | APC | Chr5:112177415 | NM_000038:c.6124T > C | p.Cys2042Arg | Not present | Deleterious | Probably damaging | 15.7 | 3 |
I:38 | APC | Chr5:112176196/rs137988845 | NM_000038:c.4905G > A | p.Gly1635Gly | 0.038 % | Not ava | na | 3.2 | 3 |
I:50 | APC | Chr5:112178693 | NM_000038:c.7402T > C | p.Ser2468Pro | 0.002 % | Tolerated | Possibly damaging | 23.5 | 3 |
I:11 | AXIN2 | Chr17:63532528/rs138287857 | NM_004655:c.2051C > T | p.Ala684Val | 0.19 % | Deleterious | Possibly damaging | 22.6 | 3 |
II:58 | BMPR1A | Chr10:88683199/rs199476089 | NM_004329:c.1409T > C | p.Met470Thr | Not present | Deleterious | Probably damaging | 21.6 | 4 |
V:90 | CDH1 | Chr16:68855966/rs35187787 | NM_004360:c.1774G > A | p.Ala592Pro | 0.32 % | Deleterious | Benign | 15.3 | 3 |
V:87 | CHEK2 | Chr22:29130520/rs141568342 | NM_007194:c.190G > A | p.Glu64Lys | 0.016 % | Tolerated | Possibly damaging | 18.2 | 3 |
V:87 | CHEK2 | Chr22:29121087/rs17879961 | NM_007194:c.470T > C | p.Ile157Thr | 0.41 % | Deleterious | Probably damaging | 21.1 | 4 |
III:27 | CTNNB1 | Chr3:41275730 | NM_001904:c.1625G > A | p.Arg542His | 0.00082 % | Deleterious | Benign | 18 | 3 |
I:1 | MET | Chr7:116340039/rs201687037 | NM_001127500:c.901A > G | p.Thr301Ala | 0.022 % | Tolerated | Probably damaging | 11.3 | 3 |
I:57, IV:79 | MLH1 | Chr3:37089130/rs35001569 | NM_000249:c.1852A > G | p.Lys618Glu | 0.34 % | Deleterious | Probably damaging | 27.8 | 3in
|
IV:85 | MLH1 | Chr3:37090050/rs63750109 | NM_000249:c.1939G > A | p.Val647Met | 0.015 % | Deleterious | Benign | 18 | 3in
|
I:41 | MLH1 | Chr3:g.37053590/rs63751711 | NM_000249:c.677G > A | ex 8 skipping | Not present | Deleterious | Probably damaging | 34 | 5in
|
I:8, I:10, I:47 | MLH3 | Chr:75514489/rs28756986 | NM_001040108:c.1870G > C | p.Glu624Gln | 0.73 % | Tolerated | Probably damaging | 17 | 3 |
I:4, I:33 | MLH3 | Chr14:75509146/rs28757008 | NM_001040108:c.3315C > A | p.Asp1105Glu | 0.29 % | Tolerated | Possible damaging | 17.7 | 3 |
II:24 | MLH3 | Chr14:75506718/rs184741686 | NM_001040108:c.3466G > A | p.Val1156Ile | 0.011 % | Tolerated | Benign | 16.8 | 3 |
IV:81 | MLH3 | Chr14:75483796/rs28939071 | NM_001040108:c.4351G > A | p.Glu1451Lys | 0.074 % | Tolerated | Probably damaging | 14. | 3 |
I:12, I:37 | MSH3 | Chr5:80109479/rs41545019 | NM_002439:c.2732T > G | p.Leu911Trp | 0.24 % | Deleterious | Probably damaging | 17.4 | 3 |
I:55 | MSH2 | Chr2:g.47657079/rs63751650 | NM_000251.2:c.1275A > G | p.Glu425Glu | 0.01 % | na | na | 13.6 | 3in
|
I:92 | MSH2 | Chr2:g.47703513/rs587779127 | NM_000251.2:c.2013T > A | p.Asn671Lys | Not present | Deleterious | Probably damaging | 28.2 | 3in
|
IV:79 | MSH6 | Chr2:48027790 | NM_000179:c.2668G > T | p.Val890Phe | Not present | Deleterious | Possibly damaging | 12.9 | 3 |
I:56 | MSH6 | Chr2:g.48030612 | NM_000179:c.3226C > T | p.Arg1076Cys | 0.0091 % | Deleterious | Probably damaging | 32 | 3in
|
III:71 | MUTYH | Chr1:45798475/rs34612342 | NM_001128425:c.536A > G | p.Tyr179Cys | 0.16 % | Deleterious | Probably damaging | 19 | 5ho
|
I:42 | MUTYH | Chr1:g.45797228/rs36053993 | NM_001128425:c.1187G > A | p.Gly396Asp | 0.28 % | Deleterious | Probably damaging | 29.4 | 5ho
|
I:51 | MUTYH | Chr1:g.45797846/rs138089183 | NM_001128425:c.925C > T | p.Arg309Cys | 0.044 % | Tolerated | Benign | 13.9 | 3ho
|
I:33 | PMS1 | Chr2:190660640 | NM_000534:c.278G > A | p.Arg93His | 0.0025 % | Deleterious | Probably damaging | 36 | 3 |
IV:31 | PMS1 | Chr2:190708701/rs143010673 | NM_000534:c.594G > T | p.Trp198Cys | 0.015 % | Deleterious | Probably damaging | 20.8 | 3 |
I:51 | PMS1 | Chr2:g.190660536/rs143323454 | NM_000534:c.174G > T | p.Gly58Gly | 0.33 % | na | na | 9.3 | 3 |
I:48 | STK11 | Chr19:g.1226474/rs199973552 | NM_000455:c.1130C > T | p.Ala377Val | 0.01 % | Tolerated | Benign | 13.7 | 3 |
Nine patients had more than one variant remaining after the filtration, including three with truncating variants in
BMPR1A,
PMS2 and
AXIN2. The
BMPR1A c.969delT variant (Table
1) was found together with one likely pathogenic variant (class 4) in
CHEK2 c.470T > C, p.Ille157Thr (Table
2), in an mixed polyposis case (V:87), additionally this patient carried a
CHEK2 VUS, c.190G > A, p.Glu64Lys (Table
2). A truncating variant in
PMS2 c.861_864del (Table
1) was found together with the VUS
APC c.7402T > C, p.Ser2468Pro (Table
2) in patient I:50. The
AXIN2 c.254del (Table
1) variant and the synonymous VUS
MSH2 c.1275A > G (Table
2) were both found in patient I:55.
Tumor characteristics e.g. MSI and IHC can be of value for interpretation of the VUS. For 52 of these patients we had results from only MSI tests or for both MSI and IHC tests (Supplementary Table 1).When investigating the VUS present among these patients there are some findings. Patient I:47 has a tumor which is MSI-H and present a loss of MLH1/PMS2 proteins, this patient has a VUS in
MLH3, c.1870G > C, p.Glu624Gln. This VUS was also found in two patients with an MSS (I:8, I:10) tumour phenotype. The variant is interpreted differently between the in silico protein predication tools used, it has a low CADD score (17) and is quite common in the ExAc population database (0.73 %). Since tumors from patients with this variant can be both MSI or MSS, it is difficult to conclude the pathogenic effect of the variant. In two patients with a MSI-H tumour phenotype, one
MSH6:c.3226C > T, p.Arg1076Cys (I:56) and one
MSH2:c.2013T > A, p.Asn671Lys (I:92) VUS were found. These variants are predicted damaging by all in silico protein predication tools, exhibit a high CADD score (32 respectively 28.2) and are rare (0.0091 %) or not present in the population database ExAc. Both variants might be predicted to have a likely pathogenic effect. TCGA data (
www.cbioportal.org) shows a high functional impact for the
MSH6 variant. In the patients with an MSS tumor phenotype, eight unique MMR variants were found. The variants exhibit conflicting in silico protein predication results. Combined with a lower CADD score in general, the variants might be predicted to have a likely benign effect, consistent with their MSS phenotype.
Discussion
In this study we show the importance of using multigene panels which allows for a parallel comprehensive screening for CRC syndromes. Mutations in
BMPR1A have been found in an extended phenotypic spectrum beyond juvenile polyposis, including HMPS, AFAP simplex, familial colorectal cancer type X (FCCX) and early onset CRC without familial history and MSI negative tumours [
4,
8,
29,
30]. To this spectrum we add a patient with an atypical polyposis (V:87, this patient also carries two
CHEK2 variants, Table
2) and three patient with unexplained adenomatous polyposis and different number of polyps. Patient IV:76 had a splice-site variant c.230 + 2T > C (class 4), II:59 had a truncating variant, c.441delT, Phe147Leufs*18 (class 5) and the last patient (II:58) had a probable pathogenic (class 4) missense mutation, c.1409 C > T, p.Met470Thr in
BMPR1A. This missense mutation has previously been found in a patient with a juvenile polyposis phenotype and around 300 polyps throughout the entire gastrointestinal tract [
15]. Two patients from Group I, “CRC familial or unknown inheritance not polyposis”, had variants in
AXIN2. In one of these patient with late onset of CRC a truncating
AXIN2 variant was found together with an
MSH2 variant of unknown significance (I:55). The second patient (I:11) presented with an
AXIN2 missense variant c.2051C > T, p.Ala684Val. Variants in
AXIN2 have been reported in patients with CRC and oligodentia and in patients with oligodentia solely [
21,
52]. It is suggested that truncating pathogenic variants in
AXIN2 are more likely to predispose carriers to syndromic oligodontia and colorectal cancer compared to missense variants [
25]. To our knowledge oligodonita was not present in any of our patients.
We found a large deletion in the regulatory region of
SMAD4 in a patient with unexplained adenomatous polyposis (1–100 polyps) (III:65). An insulator element that may act as a barrier to enhancer action is located in the deleted region. Transcription of genes beyond the insulator is not stimulated by the enhancer when the insulator is active. This deletion might therefore have an effect on the expression of the gene. In a recent study two
SMAD4 mutations in patients without juvenile polyps were identified, one with around 20–99 adenomatous polyps and the other one without reported polyps, which further extends the phenotypical spectrum for this gene [
6].
There is a complexity of combinations of possible ligand receptors and downstream effectors in the BMP/TGFR-β signalling pathways and this might explain part of the genotype-phenotype relationship. There might also be a genotype-phenotype correlation depending on where in the gene the mutation is located. Several genes in the BMP/TGFR-β signalling pathway are mutated in hereditary CRC as well as sporadic CRC and possibly inactivation of also other genes in this pathway might predispose carriers to CRC. It seems as if patients with mutations in
APC,
BMPR1A,
SMAD4 and
GREM1 can have similar polyposis phenotypes but carriers of
GREM1 mutation with HMPS might not have the same risk for extra-colonic disease as patients with
BMPR1A mutations and HMPS [
47].
In a recent multigene-panel based CRC study 1.4 % (8/586) had
CHEK2 risk alleles or truncating mutations, two of the patients had the c.470T > C, p.Ile157Thr, variant and four c.1100delC alleles, all had polyps or CRC, none of them had a personal history of breast cancer, but six had at least one family member with breast cancer [
6]. Around 2 % (2/91) of our patients had
CHEK2 variants, V:87 had both c.190G > A, p.Glu64Lys and c.470T > C, p.Ile157Thr and I:20 had the splice variant c.319 + 2T > A, and they did present with polyps or CRC but no breast cancer has been reported in the families as we know of. Variants in
CHEK2 still remain of uncertain clinical relevance as is further emphasized by the fact that V:87 also carried a truncating probably pathogenic variant in the
BMPR1A gene (c.969delT). In a recent study
CHEK2 variants have been found among individuals with various types of cancer, which might be partly due to the high population frequency of the common
CHEK2 variants (c.1100delC and p.Ile157Thr) [
45].
The truncating
MLH3 mutation c.3563 C > G, p.Ser1188* was found in homozygote state in an unexplained polyposis case (IV:69) with duodenal polyps and CRC. The MLH3 protein as well as the PMS1 protein can dimerize with MLH1 and assist in single nucleotide mis-match DNA-repair, but their roles are not well understood [
38]. Variants in the genes have been found in patients without a family history, in some cases also in sporadic patients and/or in healthy controls. Variants have also been found together with other MMR gene variants, suggesting
PMS1 and
MLH3 to be low risk genes in Lynch syndrome [
17]. The clinical significance of the variant we report here, is therefore difficult to estimate. However, recently compound heterozygote loss of function (LoF) germline mutations in the
MSH3 gene were identified in patients with an unexplained adenomatous polyposis. The data presented by Adam et al. strongly support disease causing
MSH3 mutations to follow a recessive mode of inheritance [
2]. A comparable scenario might possibly also be considered for mutations in
MLH3.
When comparing the VUS in the MMR genes to the corresponding results from the MSI and IHC test of the tumours, some conclusion might be drawn concerning the pathogenicity. Two variants, one in MSH6 c.3226C > T, p.Arg1076Cys (I:56) and one in MSH2 c.2013T > A, p.Asn671Lys (I:92), that were identified in patients who presented with a MSI-H phenotype (no IHC results were available), might be predicted to be likely pathogenic. Both of these variants are predicted damaging by all the protein predication tools used, they also have a very high CADD score and are very rare or not present in the population database ExAc. TCGA data shows a high functional impact for the MSH6 variant. It is feasible to predict these variants as presumably likely pathogenic at this point until more functional data is available.
The patient (I:6) with the intronic duplication in CDH1also had breast cancer. It is known that CDH1 mutations can be found in patients with lobular breast cancer and in hereditary diffuse gastric cancer. Although no obvious functional elements are found in this region it cannot be ruled out that the duplication has an effect on the transcription or regulation of the gene.
The search for germ-line mutations in risk individuals have been focused on mutations associated with highly penetrant disease phenotypes, which include a stepwise approach leading to an expensive strategy and underestimation of familial cases [
43]. The increased use of multigene panels have already shown a higher mutation detection rate compared with traditional testing based on clinical criteria [
6,
18], as is also confirmed by this study. The reason for this is probably a large genetic heterogeneity and overlapping clinical presentation of the different CRC syndromes. Limited knowledge of the medical and/or family history or an atypical presentation of the CRC syndromes might lead to an incorrect diagnosis of patients. The possibility of panel-based testing is beneficial not only for the patient but also for time and cost savings. However, there is also a complexity of information that can result from a multigene-panel test. Variants may also be coincidental or explain only part of the clinical phenotype. Segregation analyses could in these cases be used to further understand the clinical significance of variants. In this study, when also structural variants are included, in total 33 % (30/91) of the patients have at least one VUS. When eliminating those with a disease-causing variant already identified 29 % (26/91) of the patients have a VUS of which the majority are located in MMR genes, in concordance also with other reports [
6]. A patient without identified mutation in this study could have mutations in high penetrant recently identified genes, which were not included in this panel. Several candidate genes for both polyposis and non-polyposis syndromes have been identified [
5,
11]. Multigene panels used for detection of pathogenic variants in CRC syndromes frequently include genes for which the cancer risk is not well known and management guidelines are not yet established. Classifying the genes into different categories based on these issues might be advisable [
7]. The implementation of multigene-panel based technology into the clinic implies new opportunities and challenges which might also require introduction of new models for genetic counselling.