Mutazioni germinali di geni correlati alla Sindrome di Lynch

La Sindrome di Lynch (anche chiamata Hereditary Non Polyposis Colorectal Cancer, HNPCC) è il termine usato per indicare la predisposizione genetica a sviluppare il tumore del colon-retto non poliposico e, in misura minore, in altri distretti. La sindrome di Lynch è trasmessa con modalità autosomica dominante a penetranza incompleta, e rende conto del 5%-10% di tutte le neoplasie del grosso intestino. A livello molecolare, la sindrome di Lynch è correlata a mutazioni germinali di uno dei geni che caratterizzano il MMR (MisMatch Repair), un complesso multienzimatico preposto alla riparazione degli errori di replicazione del DNA e che svolge un ruolo fondamentale nel mantenimento della stabilità dell’informazione genetica. I geni coinvolti nel sistema del MMR, presumibilmente implicati nella sindrome di Lynch finora identificati, sono: MLH1 (3p21), MLH3 (14q24.3), PMS2 (7p22), MSH2 (2p22), MSH3 (5q11) e MSH6 (2p16); di questi MLH1 e MSH2 rappresentano quelli più frequentemente mutati con un’ incidenza pari al 50 e 40 %. L’identificazione dei soggetti candidati allo studio si è basata sulle caratteristiche dell’anamnesi familiare e sulle linee guida di Bethesda, riviste nel 2003. Lo studio della sequenza nucleotidica di tutti gli esoni dei geni MLH1, MSH2 e MSH6 è stato effettuato tramite sequenziamento diretto, usando un sequenziatore automatico a capillare e MLPA (Multiplex Ligation Probe Amplification). Fino ad oggi, sono entrati in studio 313 pazienti, di cui 181 donne e 132 uomini, con un’età mediana di 48 anni (range 16-85). Sono state identificate 11 diverse mutazioni di MLH1 in 19 pazienti differenti, di cui 7 missenso, 1 silente e 3 mutazioni nel sito di splicing. Nel gene MSH2, sono state individuate 16 diverse mutazioni germinali in 24 pazienti: 8 di tipo missenso, 3 mutazioni frameshift, 2 mutazioni non-senso, 1 nel sito di splicing e 2 riarrangiamenti genici, quest’ultimi rilevati tramite MLPA. A carico del gene MSH6 sono state trovate 4 diverse mutazioni germinali in 5 pazienti: 2 mutazioni missenso, 1 frameshift e 1 mutazione non-senso. L’analisi genetica è stata inoltre estesa a 69 soggetti sani, appartenenti a famiglie in cui è stata riscontrata una mutazione nei geni MLH1, MSH2 o MSH6, per identificare i portatori sani della mutazione ed avviarli a programmi di screening. Ventisette dei 64 soggetti analizzati sono risultati portatori del difetto molecolare individuato nella famiglia, mentre 37 soggetti sono risultati negativi per la mutazione caratteristica della famiglia di appartenenz

Prognostic impact of mismatch repair genes germline defects in colorectal cancer patients: are all mutations equal?

Background: Lynch syndrome (LS) is the most common hereditary colorectal cancer (CRC) syndrome, caused by germline mutations in MisMatch Repair (MMR) genes, particularly in MLH1, MSH2 and MSH6. Patients with LS seem to have a more favourable prognosis than those with sporadic CRC, although the prognostic impact of different mutation types is unknown. Aim of our study is to compare survival outcomes of different types of MMR mutations in patients with LS-related CRC. Methods: 302 CRC patients were prospectively selected on the basis of Amsterdam or Revised Bethesda criteria to undergo genetic testing: direct sequencing of DNA and MLPA were used to examine the entire MLH1, MSH2 and MSH6 coding sequence. Patients were classified as mutation-positive or negative according to the genetic testing result. Results: A deleterious MMR mutation was found in 38/302 patients. Median overall survival (OS) was significantly higher in mutation-positive vs mutation-negative patients (102.6 vs 77.7 months, HR:0.63, 95%CI:0.46-0.89, p = 0.0083). Different types of mutation were significantly related with OS: missense or splicing-site mutations were associated with better OS compared with rearrangement, frameshift or non-sense mutations (132.5 vs 82.5 months, HR:0.46, 95%CI:0.16-0.82, p = 0.0153). Conclusions: Our study confirms improved OS for LS-patients compared with mutation-negative CRC patients. In addition, not all mutations could be considered equal: the better prognosis in CRC patients with MMR pathogenic missense or splicing site mutation could be due to different functional activity of the encoded MMR protein. This matter should be investigated by use of functional assays in the future

A germline missense mutation in exon 3 of the MSH2 gene in a Lynch syndrome family: correlation with phenotype and localization assay

Lynch syndrome is caused by germline mutations in any of the MisMatch Repair (MMR) genes. About 37% of MSH2 variants are missense variants causing single amino-acid substitutions. Whether missense variants affect the normal function of MMR proteins is crucial both to provide affected families a more accurate risk assessment and to offer predictive testing to family members. Here we report one family, fulfilling both Amsterdam I and II criteria and Bethesda guidelines, referred to our center for genetic counselling. The proband and some of her relatives have been investigated for microsatellite instability (MSI), immunohistochemical MMR protein staining, direct sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA). Also Subcellular Localization Assay and Splice site predictions analyses were used. A germline missense variant of uncertain significance (exon 3, p.Val161Asp) was found in MSH2 gene in proband and in some relatives. The variant was associated with lack of expression of MSH2 protein (DMMR) and MSI-High status in tumour tissues. The localization assay of the MSH2 protein showed an abnormal subcellular localization pattern of the corresponding protein. Finally, splice-site prediction analysis ruled out a potential role of new splice sites as the cause behind the lack of expression of MSH2 protein and we suppose a potential correlation with other forms of post-transcriptional regulation (circular RNAs). The variant here reported shows a high correlation with phenotype and is located in an evolutionary conserved domain. The localization assay also suggest a potential pathogenic role, thus supporting further research on this matter

Lynch syndrome-associated lung cancer: pitfalls of an immunotherapy-based treatment strategy in an unusual tumor type

: Lynch syndrome is a hereditary cancer predisposition syndrome caused by germline alterations in mismatch repair (MMR) genes leading to increased risk of colon cancer as well as other cancer types. Non-small cell lung cancer (NSCLC) is not among typical Lynch syndrome-associated tumors: pembrolizumab, an immune checkpoint inhibitor, is actually approved for the treatment of NSCLC patients and represents a promising treatment option for patients with advanced metastatic MMR-deficient cancer, regardless of tumor origin. This case report describes the clinical presentation and management of a 74-year-old female with a history of rectal adenocarcinoma and ovarian cancer, who has a documented frameshift pathogenic variant in the exon 8 of MSH6 gene and an intronic variant in the BRCA2 gene (classified as a variant of uncertain significance), affected by NSCLC with brain metastases. Despite these premises, the patient was treated with pembrolizumab and she did not benefit from this kind of treatment