ABSTRACT
MMR genes mutations and HNPCC syndrome: in vitro assessment of
the missense variants pathogenicity
Hereditary Nonpolyposis Colorectal Cancer Syndrome (HNPCC Syndrome,
or Lynch Syndrome), is an autosomal dominant hereditary cancer
syndrome, which accounts for 5% of all colorectal cancers. HNPCC is
associated with an increased (90% for men, 70% for women) lifetime risk
of endometrial, ovarian and other extra colonic cancers. HNPCC is
associated with a defective MisMatch Repair (MMR) as a consequence of
germline mutations in one of the corresponding MMR genes, mostly MLH1
(50%) or MSH2 (39%). MMR is a multi-enzymatic system which contributes
to genomic stability maintenance, by correcting mismatched base pairs or
slippages in repeated sequences generated during DNA replication.
Approximately 30% of MLH1 mutations and 20% of MSH2 mutations are
nontruncating missense variants, which lead to a single amino acid
substitution. These alterations of uncertain genetic significance may
change the shape of the protein and affect its ability to interact with other
MMR components, leading to cancer predisposition, as a consequence of
the loss of MMR functionality.
The aim of this study was to assess the pathogenicity of MLH1 and MSH2
missense mutations. Ascertainment the causal relationship with the
pathology could represent a useful tool in clinical practice and in genetic
counselling. As clinical criteria are frequently insufficient to address
pathogenic significance to these variants, several functional assays have
been developed.
Direct sequencing of the genomic DNA of 130 patients with suspected
HNPCC Syndrome was performed. Eight missense mutations in MMR
genes were found: 5 alterations of MLH1 (P496R, L559R, K618A, Y646C,
P648S), found in 6 patients of unrelated families, and 3 mutations of
MSH2 (G162R, D167H, R359S), found in 6 patients, 3 of which belonging
to the same family. No one of these mutations was listed in the InSiGHT
mutation database.
As The lack of the mutated protein in vivo and a high MSI are the typical
hallmarks
of
HNPCC,
immunoistochemical
staining
(IHC)
and
microsatellite instability (MSI) analysis were performed.
To address a pathogenic significance to these mutations, functional
studies dealing with expression level, interaction and localization analysis
were developed and performed.
The expression levels of the MLH1 and MSH2 mutated proteins were
investigated by transfecting an human MMR(-) expression system
(HCT116 and LoVo cells respectively), with vectors expressing the mutated
form of the MLH1 or MSH2 gene, reproduced by site-directed mutagenesis.
The interaction assay was developed to investigate the MLH1 or MSH2
missense variants abilty to disrupt the corresponding protein interaction
with its MMR biochemical partner, PMS2 or MSH6 respectively. The
inability to form functional heterodimers leads to the loss of MMR
functionality, suggesting the correlation between the variant and the
pathology. The localization assay analysed the effect of MSH2 missense
mutations in the correct subcellular localization of the corresponding
repair protein: defects in nucleocytoplasmic import are considered as
indicative of a MMR-deficient phenotype.
Clinical, genetic, in silico and biochemical data correlation allowed to
assess a probably pathogenic role to P648S and L559R MLH1 variants and
to G162R e R359S MSH2 variants, and to exclude the pathogenicity of
P496R MLH1 one. Pathogenicity of K618A and Y646C MLH1 mutations
was questionable, as their correlation with HNPCC features was low and
results of functional analysis were ambiguous.
These results suggests that the MLH1-PMS2 interaction assay and the
MSH2 localization analysis we developed, together with clinical, genetic
and expression analyses, could represent a useful tool to evaluate a
disease causing role of MMR missense variants.