Filling the gap: A thorough investigation for the genetic diagnosis of unsolved polyposis patients with monoallelic MUTYH pathogenic variants.

BACKGROUNDS: MUTYH-associated polyposis (MAP) is an autosomal recessive disease caused by biallelic pathogenic variants (PV) of the MUTYH gene. The aim of this study was to investigate the genetic causes of unexplained polyposis patients with monoallelic MUTYH PV. The analysis focused on 26 patients with suspected MAP, belonging to 23 families. Ten probands carried also one or more additional MUTYH variants of unknown significance. METHODS: Based on variant type and on the collected clinical and molecular data, these variants were reinterpreted by applying the ACMG/AMP rules. Moreover, supplementary analyses were carried out to investigate the presence of other variants and copy number variations in the coding and promoter regions of MUTYH, as well as other polyposis genes (APC, NTHL1, POLE, POLD1, MSH3, RNF43, and MCM9). RESULTS: We reclassified 4 out of 10 MUTYH variants as pathogenic or likely pathogenic, thus supporting the diagnosis of MAP in only four cases. Two other patients belonging to the same family showed a previously undetected deletion of the APC gene promoter. No PVs were found in the other investigated genes. However, 6 out of the 18 remaining families are still interesting MAP candidates, due to the co-presence of a class 3 MUTYH variant that could be reinterpreted in the next future. CONCLUSION: Several efforts are necessary to fully elucidate the genetic etiology of suspected MAP patients, especially those with the most severe polyposis/tumor phenotype. Clinical data, tumor molecular profile, family history, and polyposis inheritance mode may guide variant interpretation and address supplementary studies.

Toward a better definition of EPCAM deletions in Lynch Syndrome: Report of new variants in Italy and the associated molecular phenotype.

BACKGROUND: Inherited epimutations of Mismatch Repair (MMR) genes are responsible for Lynch Syndrome (LS) in a small, but well defined, subset of patients. Methylation of the MSH2 promoter consequent to the deletion of the upstream EPCAM gene is found in about 1%-3% of the LS patients and represents a classical secondary, constitutional and tissue-specific epimutation. Several different EPCAM deletions have been reported worldwide, for the most part representing private variants caused by an Alu-mediated recombination. METHODS: 712 patients with suspected LS were tested for MMR mutation in our Institute. EPCAM deletions were detected by multiplex ligation-dependent probe amplification (MLPA) and then defined by Long-Range polymerase chain reaction (PCR)/Sanger sequencing. A comprehensive molecular characterization of colorectal cancer (CRC) tissues was carried out by immunohistochemistry of MMR proteins, Microsatellite Instability (MSI) assay, methylation specific MLPA and transcript analyses. In addition, somatic deletions and/or variants were investigated by MLPA and next generation sequencing (NGS). RESULTS: An EPCAM deletion was found in five unrelated probands in Italy: variants c.556-490_*8438del and c.858+1193_*5826del are novel; c.859-1430_*2033del and c.859-670_*530del were previously reported. All probands were affected by CRC at young age; tumors showed MSI and abnormal MSH2/MSH6 proteins expression. MSH2 promoter methylation, as well as aberrant in-frame or out-of-frame EPCAM/MSH2 fusion transcripts, were detected in CRCs and normal mucosae. CONCLUSION: An EPCAM deletion was the causative variant in about 2% of our institutional series of 224 LS patients, consistent with previously estimated frequencies. Early age and multiple CRCs was the main clinical feature of this subset of patients.

A Specific Mutational Signature Associated with DNA 8-Oxoguanine Persistence in MUTYH-defective Colorectal Cancer.

8-Oxoguanine, a common mutagenic DNA lesion, generates G:C>T:A transversions via mispairing with adenine during DNA replication. When operating normally, the MUTYH DNA glycosylase prevents 8-oxoguanine-related mutagenesis by excising the incorporated adenine. Biallelic MUTYH mutations impair this enzymatic function and are associated with colorectal cancer (CRC) in MUTYH-Associated Polyposis (MAP) syndrome. Here, we perform whole-exome sequencing that reveals a modest mutator phenotype in MAP CRCs compared to sporadic CRC stem cell lines or bulk tumours. The excess G:C>T:A transversion mutations in MAP CRCs exhibits a novel mutational signature, termed Signature 36, with a strong sequence dependence. The MUTYH mutational signature reflecting persistent 8-oxoG:A mismatches occurs frequently in the APC, KRAS, PIK3CA, FAT4, TP53, FAT1, AMER1, KDM6A, SMAD4 and SMAD2 genes that are associated with CRC. The occurrence of Signature 36 in other types of human cancer indicates that DNA 8-oxoguanine-related mutations might contribute to the development of cancer in other organs.

Type and frequency of MUTYH variants in Italian patients with suspected MAP: a retrospective multicenter study.

To determine prevalence, spectrum and genotype-phenotype correlations of MUTYH variants in Italian patients with suspected MAP (MUTYH-associated polyposis), a retrospective analysis was conducted to identify patients who had undergone MUTYH genetic testing from September 2002 to February 2014. Results of genetic testing and patient clinical characteristics were collected (gender, number of polyps, age at polyp diagnosis, presence of colorectal cancer (CRC) and/or other cancers, family data). The presence of large rearrangements of the MUTYH gene was evaluated by Multiplex Ligation-dependent Probe Amplification analysis. In all, 299 patients with colorectal neoplasia were evaluated: 61.2% were males, the median age at polyps or cancer diagnosis was 50 years (16-80 years), 65.2% had <100 polyps and 51.8% had CRC. A total of 36 different MUTYH variants were identified: 13 (36.1%) were classified as pathogenetic, whereas 23 (63.9%) were variants of unknown significance (VUS). Two pathogenetic variants were observed in 78 patients (26.1%). A large homozygous deletion of exon 15 was found in one patient (<1.0%). MAP patients were younger than those with negative MUTYH testing at polyps diagnosis (P<0.0001) and at first cancer diagnosis (P=0.007). MAP patients carrying the p.Glu480del variant presented with a younger age at polyp diagnosis as compared to patients carrying p.Gly396Asp and p.Tyr179Cys variants. A high heterogeneity of MUTYH variants and a high rate of VUS were identified in a cohort of Italian patients with suspected MAP. Genotype-phenotype analysis suggests that the p.Glu480del variant is associated with a severe phenotype.

Concomitant mutation and epimutation of the MLH1 gene in a Lynch syndrome family.

Lynch syndrome (LS) is an inherited predisposition cancer syndrome, typically caused by germline mutations in the mismatch repair genes MLH1, MSH2, MSH6 and PMS2. In the last years, a role for epimutations of the same genes has also been reported. MLH1 promoter methylation is a well known mechanism of somatic inactivation in tumors, and more recently, several cases of constitutional methylation have been identified. In four subjects affected by multiple tumors and belonging to a suspected LS family, we detected a novel secondary MLH1 gene epimutation. The methylation of MLH1 promoter was always linked in cis with a 997 bp-deletion (c.-168_c.116+713del), that removed exon 1 and partially involved the promoter of the same gene. Differently from cases with constitutional primary MLH1 inactivation, this secondary methylation was allele-specific and CpGs of the residual promoter region were totally methylated, leading to complete allele silencing. In the colon tumor of the proband, MLH1 and PMS2 expression was completely lost as a consequence of a pathogenic somatic point mutation (MLH1 c.199G>A, p.Gly67Arg) that also abrogated local methylation by destroying a CpG site. The evidences obtained highlight how MLH1 mutations and epimutations can reciprocally influence each other and suggest that an altered structure of the MLH1 locus results in epigenetic alteration.

MUTYH c.933+3A>C, associated with a severely impaired gene expression, is the first Italian founder mutation in MUTYH-Associated Polyposis.

MUTYH variants are differently distributed in geographical areas of the world. In MUTYH-associated polyposis (MAP) patients from North-Eastern Italy, c.933+3A>C (IVS10+3A>C), a transversion causing an aberrant splicing process, accounts for nearly 1/5 of all mutations. The aim of this study was to verify whether its high frequency in North-Eastern Italy is due to a founder effect and to clarify its impact on MUTYH transcripts and protein. Haplotype analysis and age estimate performed on members of eleven Italian MAP families and cancer-free controls provided evidence that c.933+3A>C is a founder mutation originated about 83 generations ago. In addition, the Italian haplotype associated with the c.933+3A>C was also found in German families segregating the same mutation, indicating it had a common origin in Western Europe. Altogether c.933+3A>C and the two common Caucasian mutations p.Tyr179Cys and p.Gly396Asp represent about 60% of MUTYH alterations in MAP patients from North-Eastern Italy, suggesting the opportunity to perform targeted molecular screening for these variants in the diagnostic setting. Expression analyses performed on lymphoblastoid cell lines supported the notion that MUTYH c.933+3A>C alters splicing causing the synthesis of a non functional protein. However, some primary transcripts escape aberrant splicing, producing traces of full-length transcript and wild-type protein in a homozygote; this is in agreement with clinical findings that suggest a relatively mild phenotypic effect for this mutation. Overall, these data, that demonstrate a founder effect and further elucidate the splicing alterations caused by the MUTYH c.933+3A>C mutation, have important implications for genetic counseling and molecular diagnosis of MAP.

Capsule endoscopy is useful and safe for small-bowel surveillance in familial adenomatous polyposis.

BACKGROUND: Duodenal cancer and ampullary cancer are major causes of death after a prophylactic colectomy in patients with familial adenomatous polyposis (FAP). Forward-viewing endoscopy and side-viewing endoscopy are recommended in patients with FAP for surveillance of periampullary and duodenal polyposis. The study of polyps distal to the duodenum in FAP is limited. A capsule endoscopy (CE) allows visualization of the mucosa of the entire small bowel. OBJECTIVE: The objective was to detect whether CE has clinical value or any utility for the surveillance of small-bowel polyps in patients with FAP and to evaluate whether there are genotypic factors that predict which patients are at a lower risk of small-bowel polyps. SETTING: Two Italian tertiary-referral centers. PATIENTS: Twenty-three patients with FAP who presented for a CE. MAIN OUTCOME MEASUREMENTS: Patients with FAP were examined by CE to assess the location, size, and number of small-bowel polyps. Patient age at CE, sex, years of observation after surgery, type of surgery, duodenal adenomas, and colorectal cancer at surgery were analyzed. All patients were selected for mutation analysis, and the germline adenomatous polyposis coli (APC) gene mutation was detected. RESULTS: Eleven of 23 patients with FAP had duodenal polyps. During CE, jejunal-ileal polyps were detected in 7 of 23 FAPs, with a total number of 15 polyps in the ileum. The presence of duodenal adenomas was the only clinical feature predictive of small-bowel polyps. Identification of the ampulla of Vater was not achieved with CE; duodenal polyps were only seen in 4 of 11 patients identified endoscopically, with an underestimation of polyp numbers. APC mutations between codons 499 and 805 were associated with the absence of small-bowel polyps. CONCLUSIONS: CE is useful and safe for the surveillance of jejunal-ileal polyps in selected patients with FAP. CE is not useful in the surveillance of the duodenum where the majority of small-bowel cancers occur.

An investigation on the nature of the peptide at m/z 904, overexpressed in plasma of patients with colorectal cancer and familial adenomatous polyposis.

In an investigation devoted to the search for plasma markers for colorectal cancer (CRC), carried out by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, a series of overexpressed peptides were identified in the plasma of patients. Among them the peptide with molecular weight 903 Da was the most abundant one, with a mean +/- (SD) relative abundance of 37 +/- 17% and a frequency over 60%. Interestingly, also in plasma samples of ten subjects affected by familial adenomatous polyposis (FAP), the peptide with molecular weight 903 was overexpressed. In this investigation, MALDI/MS/MS experiments were carried out on the ion at m/z 904 detected in the MALDI mass spectra of CRC and FAP patients. The data analysis by SwissProt.2007.01.09 indicates that this peptide is due to the sequence RPPGFSPF, found in the kininogen-1 precursor, which is an alpha-2-thiol proteinase inhibitor. In the case of subjects affected by a particular FAP syndrome, the MALDI/MS/MS spectra were quite different from those obtained from CRC and FAP patients. In fact, two sequences have been evidenced: RPPGFSPF belonging to kininogen-1 precursor, and PRKSSSSR belonging to Forkhead box protein 01A.

Twelve years of endoscopic surveillance in a family carrying biallelic Y165C MYH defect: report of a case.

PURPOSE: We report the case of two siblings, clinically andendoscopically followed for 12 years, who displayed anattenuated adenomatous polyposis coli phenotype. METHODS: On workup for rectal bleeding with colonoscopy, we found multiple adenomas mainly right-sided in a 21-year-old female and the same colonic phenotype was observed in her 27-year-old brother. We made a clinical diagnosis of attenuated adenomatous polyposis coli and performed APC gene testing. Because they had refused the proposed ileorectal anastomosis surgical option, we planned a periodic, endoscopic follow-up. RESULTS: Gene testing did not confirm the clinical suspicion of attenuated adenomatous polyposis coli. Actually, we did not find anypathogenic mutation in APC gene and we recently identified a biallelic Y125C MYH defect. During the endoscopic follow-up, a progressive reduction of adenomas was seen. CONCLUSIONS: New insight colorectal cancer genetics have allowed definition of a new class of polyposis that applies to some patients with attenuated adenomatous polyposis coli phenotype as in the siblings we have described. To prevent colorectal cancer without recurring to surgery, colonoscopic polypectomy may be a suitable tool in controlling MYH polyposis.

Retinoic acid inhibits IL-6-dependent but not constitutive STAT3 activation in Epstein-Barr virus-immortalized B lymphocytes.

IL-6-mediated B-cell growth promotion is involved in the pathogenesis of EBV+ lymphoproliferative disorders of immunosuppressed patients. Since retinoic acid (RA) inhibits the proliferation of EBV-immortalized lymphoblastoid B-cell lines (LCLs), we have investigated the effects of RA on IL-6 signaling in these cells. RA down-regulated IL-6-receptor components with IL-6 agonist activity (membrane and soluble gp80) and increased the levels of soluble gp130, an IL-6 antagonist. These changes, however, were not related to the enhanced production of endogenous IL-6 induced by RA in LCLs. RA-induced modulation of IL-6 receptor components did not abolish IL-6-mediated phosphorylation of gp130, whereas JAK1 and STAT3 phosphorylation and activation induced by IL-6 were markedly inhibited. Overall, the effects of RA resulted in the induction of a complete resistance of LCLs to IL-6-mediated growth promotion. Conversely, RA did not inhibit the constitutive activation of JAK1, TYK2, STAT3 and ERK1/2, ruling out that the JAK/STAT and MAPK pathways may mediate the antiproliferative activity of RA. The finding that RA severely impairs IL-6-dependent signalings in LCLs and inhibits their growth despite the presence of constitutively active JAK/STAT and MAPK cascades provide additional support for a role of RA in the prevention and treatment of EBV-related lymphoproliferative disorders of immunosuppressed patients.

Simian virus 40 sequences in human lymphoblastoid B-cell lines.

Human Epstein-Barr virus-immortalized lymphoblastoid B-cell lines tested positive by PCR for simian virus 40 (SV40) DNA (22 of 42 cell lines, 52.3%). B lymphocytes or tissues from which B-cell lines derived were also SV40 positive. In situ hybridization showed that SV40 DNA was present in the nucleus of a small fraction (1/250) of cells. SV40 T-antigen mRNA was detected by reverse transcription-PCR. Lymphoblastoid B-cell lines (n = 4) infected with SV40 remained SV40 positive for 4 to 6 months. SV40-positive B-cell lines were more tumorigenic in SCID mice than were SV40-negative cell lines (4 of 5 [80%] SV40-positive cell lines versus 2 of 4 [50%] SV40-negative cell lines). These results suggest that SV40 may play a role in the early phases of human lymphomagenesis.

Different molecular mechanisms underlie genomic deletions in the MLH1 Gene.

In this study we examined a series of 52 patients belonging to hereditary nonpolyposis colorectal cancer (HNPCC) or HNPCC-related families, all who had previously tested negative for mismatch repair (MMR) gene point mutations. Southern blot mutational screening of MLH1 and MSH2 genes was carried out with the aim of detecting large genomic rearrangements and of identifying the molecular mechanisms underlying the inactivation of the MMR genes. Three patients had abnormal restriction patterns and were found to carry distinct MLH1 internal deletions. Long-range PCRs identified the loss of DNA tracts spanning exon 6 (about 2.4 kb in proband A-AV20 and 0.8 kb in proband A-PD5) and exon 3 (about 2.5 kb in proband R-RM2). In A-AV20 the breakpoints occurred into identical 33-bp regions in introns 5 and 6 and a mechanism of classical Alu-mediated homologous recombination was evident. Also, in patient A-PD5 the breakpoints were located in these introns, but without direct involvement of repetitive sequences. In patient R-RM2 the breakpoints were located within repetitive L1 elements with poor homology in intron 2 and 3 and the rearranged allele was characterized by a complex insertion deletion (delCCinsACATAGTA), giving rise to a palindromic CTTAACATAGTATGTTAAG sequence in proximity of the fusion site. This study confirms that genomic rearrangements are an important component of the spectrum of MMR mutations. Although Alu repeats are likely to be implicated in the majority of cases, different molecular mechanisms may also be responsible for the observed MLH1 intragenic deletions. In particular, HNPCC resulting from L1-mediated recombination has been identified as a novel mechanism for MMR inactivating mutation.