Lynch syndrome is a heterogeneous disorder in respects to its molecular basis, as well as its phenotypic expression. Therefore, the selection of the putative mutation carriers as well as the detection of the causative germline alterations is a challenging task. A variety of point mutations, such as substitutions, small insertions/deletions and splice site alterations, as well as large genomic rearrangements have been reported in the international InSiGHT (LOVD) database [10
]. Particularly, genomic deletions account for approximately 10% of MLH1
mutations, while genomic duplications have been observed in approximately 1% of the Lynch syndrome cases [11
]. Furthermore, the fact that there are multiple susceptible genes that predispose to Lynch syndrome, immunohistochemistry and/or MSI should be used, where possible, as a pre-screening method to successfully identify the high risk families. In our series of experiments, tumor samples from the patients studied were not available and therefore, direct sequencing analysis of the MMR genes was performed.
Nine pathogenic point mutations and one genomic rearrangement have been detected in twelve out of seventeen (70.5%) AMS+
families studied. A 29.5% of AMS+
families that included at least two colorectal or other Lynch syndrome-associated cancer cases remained unresolved. Additionally, no deleterious mutations were detected in patients with young age of onset for CRC, i.e. < 40 years, but with no family history. Our results coincide with observations of previous studies which have demonstrated that a proportion as high as 50% of AMS+
families harbour no pathogenic mutations in either of the major MMR genes, namely MLH1
]. Nevertheless, the detection rate among AMS+
families was much higher than in families selected upon Bethesda guidelines, indicating the already reported higher specificity of the modified Amsterdam criteria in clinical practice.
Since the particular study includes a relatively small sample size, which can be a limitation when trying to provide a complete mutational spectrum, all available information on Greek putative Lynch syndrome families have been combined [15
]. Thirty-three families have been screened for the presence of germline mutations in the MLH1
genes, while thirteen of them have been screened for the presence of germline mutations in the MSH6
gene. Sixteen mutations have been detected in eighteen of these families (Figure ). To the best of our knowledge, eight of the pathogenic mutations recorded have never been described before.
Figure 4 MLH1 and MSH2 mutational spectrum in two Greek patients' cohorts. This configuration reviews all the pathogenic germline mutations detected in two Greek patients' cohorts. The black circles represent the families harboring deleterious mutations which (more ...)
Genomic rearrangements in the MLH1
genes represent 6% of the mutations identified in Greece, a percentage that falls within the range reported previously [9
]. More specifically, in a German patients' cohort genomic deletions account for 10.6% of colorectal cancer families, while in the Dutch population genomic aberrations are encountered in 6.5% of AMS+
]. If only AMS+
families are taken into account, this proportion rises up to 8.3%, which is in accordance with the observation made by Martínez-Bouzas et al
]. Nonsense mutations and splice site changes seem to prevail among point alterations identified in the Greek patients' cohort. A quite interesting finding of this study is the high rate of novel mutations, which is calculated to be 53.3%, highlighting the distinct heterogeneous nature of the Greek mutational spectrum of the MLH1
Despite the aforementioned heterogeneity, two of the mutations described, the MLH1
, c.790+1G > A transition and the MLH1
, c.116+5G > C transversion were detected in two distinct families during this study. The MLH1
, c.790+1G > A has been reported several times in different populations and further analysis is required in order to investigate the possible founder effect of this mutation within the Greek or Cypriot population. The extensive clustering of cancer cases within members of family 41 is of particular interest, since thirteen family members spread across three successive generations were diagnosed with five different cancer types. Interestingly, two of the family members were diagnosed with breast and thyroid cancer, respectively. These two types of cancer are not typical phenotypic features of the Lynch syndrome. Both patients were genotyped and found not to harbour the particular pathogenic mutation, indicating that they were probably sporadic cancer cases. On the contrary, other reports have been able to identify breast cancer in MMR mutation carriers, enhancing the theory that breast cancer can be a rare phenotypic feature of Lynch syndrome [27
On the other hand, the MLH1
, c.116+5G > C has been reported only once before [16
]. There is a possibility that the two families (F656, F1376) harbouring the MLH1
, c.116+5G > C mutation may be related, but it was not feasible to track down all the family relatives, since most of them live abroad. Further investigations are required in order to elucidate whether the particular alteration has a founder effect within the Greek population.
The aforementioned data underscore the heterogeneity of the MLH1 and MSH2 mutational spectrum in the Greek population, since each Greek family harbours a distinct deleterious germline mutation, which can be of any type. Furthermore, the first germline pathogenic mutation in MSH6 gene in a Greek family is reported here. Therefore, a combination of techniques that will be able to detect both small size alterations within the three genes and large deletions/insertions is required for routine genetic testing of putative Lynch syndrome patients, which is in accordance with other studies.
Moreover, we have achieved to determine the breakpoints of the MLH1
, c.454-?_545+?del and subsequently to develop diagnostic primers for its rapid detection. This deletion was described initially by Viel and her colleagues [32
] and has been reported several times since then. The possible founder effect of the specific mutation within the Greek population has been excluded when 951 unselected colorectal cancer cases were screened and no carriers of the specific mutation have been identified.
It is of great significance to obtain systematically data about the exact nature and frequency of the pathogenic mutations encountered in a particular patients' cohort, in order to customize the genetic testing to the population' s needs, which will eventually result in the reduction of the high cost of genetic testing along with the optimization of the detection rate.
Our results indicate the distinct, heterogeneous nature of Lynch syndrome's associated mutations in a Greek colorectal cancer patients' cohort, as well as the significant contribution of Amsterdam criteria in the identification of putative Lynch syndrome families. Consequently, the compilation of an accurate and detailed family history by the physician is a critical step for the diagnosis of Lynch syndrome.