In this paper, we report the spectrum of PTPN11 (hotspot exons) mutations in a cohort of 31 patients affected by Noonan syndrome, discuss their pathogenicity, and compare our mutation rate with various populations throughout the world. In a second part, we assess the clinical data of this cohort, in order to figure out any possible association with a particular epigenetic (non-genetic) risk factor.
Molecular and functional prediction finding
We have sequenced seven exons (2, 3, 4, 7, 8, 12, 13) that were proved earlier to host 99.9% of PTPN11 mutations [3], which means that about 1% of mutations might be overlooked in this study. Three of detected mutations (D61G, Y63C, and A72S) are clustered in the most mutated exon (exon 3). This exon encodes a part of N-SH2 domain, which is involved in interaction with upstream proteins, as well as with phosphotyrosine-phosphatase domain (PTP) to ensure SHP2 self-inhibition. N308S is localized in PTP, which is the SHP2 catalytic domain that ensures the dephosphorylation of phosphor-tyrosine residues of targeted proteins, besides its contribution in SHP2 self-inhibition [24].
Multiple alignment of SHP2 sequence with various species shows that the affected amino acids are highly conserved (Fig. 4). Furthermore, according to in silico prediction tools, D61G, Y63C, A72S, and N308S are deleterious or disease causing. It was shown in different studies that these mutations segregate with the phenotype in the affected families, and was not observed in healthy populations.
This pathogenicity is basically related to the position of the affected residues. In fact, mutations located within, or near to, N-SH2-PTP interaction area seem to disrupt the SHP2 inactive conformation, leading to the exposure of PTP catalytic site, and thus a continuously activated protein [24, 33].
Such gain-of-function mutations result in permanent signal transduction through molecular signalization pathway RAS/MAPK (rat sarcoma viral oncogene homolog/mitogen-activated protein kinase), even in the absence of extra-cell ligand upstream of this pathway.
Since RAS/MAPK is a ubiquitous pathway, involved in proliferation and differentiation of numerous tissues [34, 35], mutation disrupting its function could have a drastic impact on various organs, as it is seen in Noonan syndrome features.
In addition to the identified missense mutations, we detected the synonymous variant H85H (c.255C>T) in patient P28 who carries A72S mutation as well. Human Splicing Finder (HSF) shows that H85H may have a potential altering effect on splicing function through impairing splicing regulation consensus motifs. Such effect could entail a loss of a whole exon during the splicing process, resulting in a non-functional truncated protein. Therefore, it is important to conduct functional studies in that particular case to determine whether the observed phenotype is due to a gain-of-function mutation (A72S) or to a loss-of-function mutation (H85H).
Besides these exonic variants, we have detected three novel duplications located in the intronic region, c.525+129_525+132dup, c.525+125_525+132dup, and c.525+121_525+132dup. These duplications concern ATTT motif (Fig. 2) and seem to be mainly due to the presence of a long series of ATTT tandem repeats at this region. According to HSF program, these duplications, in addition to the fourth intronic variant c.854-21C>T, seem to have no pathogenic effect on splicing.
Statistical finding
According to these findings, the PTPN11 mutation rate in our study is around 16.13% (5/31). In order to check out the putative involvement of additional environmental factors, we proceeded to the comparison of our rate with those of different populations from all over the world (Table 4).
Our rate seems to be statistically close to rates of North-African and most of European populations. A significant difference was seen between our rate and all American and most of Asian studies.
The lowest rates are seen in the African countries, 4.7–31.6%. The ranges of rates in European and Asian populations are higher, respectively 23–59.6% and 27.1–55.6%, while the highest levels are reported in American studies, with rates set between 42 and 50.7%.
In a second step, in order to determine whether these differences are significant or not, we compared, on the one hand, the rates of countries belonging to the same continent with each other, and on the other hand, we compared combined rates of different continents with each other.
The performed comparisons show predominance of statistically close rates between populations from the same continent, and significant differences between most populations belonging to different continents, as well as between continental combined rates.
These variations in PTPN11 mutation rates throughout geographical regions suggest the involvement of, besides genetic factors, further ethnical and/or environmental factors, which widely vary across populations, and may include, among others, the following:
Eating habits, such as healthy dietary habits in Mediterranean and Asian regions
Sociocultural and/or religious convictions related to restriction of tobacco and alcohol consumption (i.e., Muslim communities) and favored consanguineous marriages (i.e., tribalist communities)
Industrialization of countries resulting in more industrialized food, exposure to polluted air, and unhealthy lifestyle in urban zones of developed countries
Clinical findings
In order to best characterize these environmental factors in our NS cohort, we proceeded to the statistical assessment of available data related notably to consanguinity and parental age at proband birth (Table 1), in comparison either with previous studies or with a Moroccan cohort from general population that we interrogated for this purpose.
The average age of included patients at diagnosis was 6.5 years with a median of 5 years. Sex ratio of our cohort is around 1.2, meaning a higher incidence among males. This seems to be consistent with previous studies [14, 36].
Congenital heart disease (CHD) is present in 50 to 90% of NS cases [37,38,39]. In our study, 84% of subjects have CHD, with a predominance of pulmonary valvular stenosis, observed in 71.4% of subjects, followed by atrial septal defect, then cardiomyopathy which is consistent with previous studies, though some of them reported more, or as many, cardiomyopathies as ASD among Noonan syndrome patients [4, 40, 41].
Consanguinity was noticed in 36% of patients. This rate seems to be quite high compared to that reported in the Moroccan general population, which is set around 15.25% [42]. The significant difference (p = 0.01) puts the consanguinity among the prominent risk factors of NS occurrence.
The averages of paternal and maternal ages at proband pregnancy, in our study, are 40 and 31 years, respectively. These values were compared with the corresponding averages of a group of 29 mothers and fathers chosen randomly from general population, which are 26 and 33 years, respectively. Comparisons show significant differences in both cases, with p values of 0.03 and 0.01, respectively. Moreover, in our NS-mutated population, paternal ages were interestingly further higher. Paternal age ranges indeed between 30 and 49 with an average of 38 years and a median of 39 years, which seems to be particularly higher than that found in the study of Tartaglia et al., in which they found that the average paternal age of NS patient births (35.6 years) is significantly higher than the general population analyzed in their study [36]. These findings confirm, on the one hand, the association of advanced paternal age to higher risk of Noonan syndrome occurrence among offspring and suggest, on the other hand, the possible involvement of advanced maternal age as well.
It is worth to mention that the main limitation of the present study is the small size of the studied population. Moreover, due to unavailability of parent DNAs, we could not assess the inheritance pattern of identified variants.
