Diagnosis of Noonan syndrome and related disorders using target next generation sequencing

Noonan syndrome (NS, OMIM 163950) is an autosomal dominant developmental disorder [1] with a prevalence ranging between 1:1.000 and 1:2.500 live births [2]. This disorder is characterized by wide phenotype variability and shares some clinical features, as facial dysmorphisms, congenital heart defect (CHD), postnatal growth retardation, ectodermal and skeletal defects, and variable cognitive deficits [1, 2], with other rare conditions, including LEOPARD syndrome (LS, OMIM 151100) [3], cardiofaciocutaneous syndrome (CFCS, OMIM 115150) [4], Noonan-like syndrome with loose anagen hair (NS/LAH, OMIM 607721) [5], and Costello syndrome (CS, OMIM 218040) [6]. This group of related disorders is caused by germline mutations in distinct genes, encoding for components of the RAS-MAPK signalling pathway. Based on common pathogenetic mechanisms and clinical overlap, these diseases have been grouped into a single family, the so-called neuro-cardio-facial-cutaneous syndromes (NCFCS), recently coined RASopathies [7, 8]. NS is associated with PTPN11, SOS1, KRAS, NRAS, RAF1, BRAF, SHOC2, MEK1 and CBL gene mutations [9‐19], LS with PTPN11, RAF1 and BRAF gene mutations [13, 17, 20, 21], NS/LAH with SHOC2 gene mutations [22], CFCS with KRAS, BRAF, MEK1 and MEK2 gene mutations [23, 24], CS with HRAS gene mutations [25].

So far, the molecular characterization can be reached in approximately the 75-90% of affected individuals. Some distinct phenotypes are emerged in association with definite gene mutations.

Nowadays, due to high genetic heterogeneity of these disorders, which affect genes that all together span about 30 kb of genomic DNA, the standard diagnostic testing protocol requires a multi-step approach, using Sanger sequencing. The selection of the genes to investigate on a first diagnostic level depends on the frequency of their association with this disorder and their relationship with a distinct phenotype. For this reason, accurate clinical evaluation and close interaction between clinical and molecular geneticists are mandatory for selecting the genes to be first studied. By using this approach, the causative mutations can be identified in most of the cases. Some mutations cannot be identified during the first screening level since some phenotypes may be related to mutations in different causative genes or some clinical features associated with NS related disorders may not be evident at younger ages, or some extremely rare mutations are not routinely screened at first analysis. To detect these mutations, an additional screening level is required with a second panel of genes, which again should be guided by clinical geneticist. In these latter cases the molecular diagnosis requires a longer time before identifying the pathogenic mutation. Moreover, standard Sanger sequencing for multiple genes is also an expensive technique. Based on these notions, genetically heterogeneous disorders demand innovative diagnostic protocols, in order to be able to identify disease-causing mutations in a rapid and routinely way.

Here we report our personal experience on the use of targeted Next Generation Sequencing (NGS) for diagnosis of RASopathies. Our study suggests that this protocol can be easily used as a standard diagnostic tool to identify disease-causing mutations, with a straightforward workflow from genomic DNA up to genomic variants identification.

The term RASopathy applies to a group of genetic disorders characterized by similar phenotypes, caused by mutations in the RAS MAPK pathway. These phenotypes are characterized by a high degree of genetic heterogeneity, since individual diseases can arise from mutations in different genes. In addition, since different RASopathies share similar clinical features, their molecular characterization is complex, time consuming and expensive.

In order to improve the molecular testing of RASopathies, in this study we investigated a protocol based on a targeted NGS using MiSeq Illumina platform enabling the analysis of all known causative genes in up to 96 patients in a single sequencing run. In particular, we analyzed 80 patients and identified 38 mutations in 6 of 11 RAS-pathway genes, including PTPN11 (22/38 = 58%), SOS1 (9/38 = 23%), BRAF (2/38 = 5%), MEK2 (2/38 = 5%), RAF1 (2/38 = 5%), CBL (1/38 = 3%). The relative frequency of mutations in the tested genes was in agreement with published results [30, 36, 37]. As shown in Table 3, in many patients the causal mutation was identified in the gene considered the most suitable candidate, based on frequency of mutations and the phenotypic characteristics. As expected, most NS patients had a PTPN11 mutation, while the second most frequently mutated gene was SOS1, followed by RAF1. Three patients with LEOPARD syndrome had one of the PTPN11 recurrent mutation previously associated with this phenotype (T468M or R498W) [20, 33]. Among patients heterozygous for a pathogenic BRAF mutation, one was clinically diagnosed as being affected by CFCS, while in the other case clinical evaluation was unable to conclude whether he was affected by NS or CFCS. The patients’ age at diagnosis is obviously important: this latter subject was 2 year-old at time of clinical evaluation, when he displayed only some features of CFCS [38]. Two other patients with CFCS had a mutation in MEK2 gene, which is less commonly mutated in this disorder. In addition, one NS patient had a mutation in CBL, a gene rarely associated with this disorder. Since all genes have been analyzed in one run, the present protocol allowed to reach the simultaneous identification of mutations affecting both the most frequent and rare genes, with a significantly reduction of time needed to reach the molecular characterization of the patient. This point is important for early diagnosis and classification of the different RASopathies, allowing a more appropriate management and counseling.

A wide spectrum of PTPN11 mutations has been associated with NS, including some rare mutations: I221V, Q256R, F285S, V428L (Table 4). All these variants are associated with the distinct facial gestalt of NS, which was markedly expressed in one patient (case no.10) with F285S and mildly expressed in the others. Familial transmission from an affected individual has been found in one instance, and suspected in another case (no.8), where the affected proband’s brother was referred to have membranous subaortic stenosis and cryptorchidism. Mental retardation or cognitive deficit was not associated with these mutations, with the exception of F285S mutation. Interestingly, the patient heterozygous for this latter mutation had a congenital pancreatic cyst, an unusual malformation in the RASopathies. In one patient (case no.16) we identified two unpublished mutations affecting two consecutive PTPN11 aminoacids, D395Y and Y396H. Both mutations were inherited from the affected father. Variability of clinical expression in this family was recognizable, since facial anomalies of NS were associated with developmental delay in the son only. The father had congenital total alopecia as distinctive feature. It is evident that the phenotype related to these mutations is quite atypical, showing common Noonan-like facial anomalies associated with variable additional neural and ectodermal features.

The other 43 patients enrolled in this study were negative for the investigated RAS genes. The proportion of negative patients was higher compared to previous reports, likely because clinical inclusion criteria were less stringent compared to other studies [39‐41], being based on NS facial anomalies and almost only one additional feature. Interestingly, the mother of patient n°18 was included in this study being the mutated parent of an affected child, although the minimal clinical criteria for diagnosis of NS were not present.

All the variations have been confirmed by Sanger sequencing as well as the 4 negative control patients. These data indicated a 100% detection rate of mutations involved in RASopathies and, most important, all these results have been obtained with MiSeq on board analysis, being the bioinformatics examination performed only adopting the user friendly Miseq Reporter software.

The absence of any false negative and false positive results and the possibility to have an easy and accurate data analysis make this approach a good diagnostic tool. Furthermore, the library preparation workflow is easy and the TSCA kit performance is stable. In fact, all different experiments for the RAS pathway genes in each run provided the same results in terms of coverage and quality (Figure 4).

Two major points to be considered in the diagnostic protocols include the time needed to complete the entire workflow and the costs that this approach requires. Targeted NGS analysis of the complete coding sequences of the 11 genes in the RAS pathway for 96 patients takes about two months, including ten days for library preparation and data analysis and about 45 days to characterize uncovered regions using Sanger sequencing. Conversely, the use of Sanger sequencing to analyze the full coding sequence of the 11 genes would take about 16-18 months for the same number of patients. We also calculated that the cost of NGS analysis applied to the 92% of the regions of interest, plus Sanger sequencing of for the remaining regions, would cost 6 time less than the cost of a protocol entirely based on Sanger sequencing (Figure 5). However, time and cost could be further reduced, by designing a new panel which includes also RIT1, a gene recently associated to NS [42], resulting in a 100% coverage of the cumulative region. This result likely makes the Sanger sequencing irrelevant for the analysis, further reducing the time and the cost of the entire process.

This study demonstrates that NGS can be successfully applied to the molecular testing of RASophaties with a remarkable gain of time and less cost, while maintaining the high quality of the results. Consistent with available records, our data confirm that the genetic mechanism underlying the RASopathies is due to germline mutations in different genes encoding for components of the RAS-MAPK signalling mutations, with PTPN11, followed by SOS1, being the most frequently mutated genes in our cohort. Moreover, the use of NGS protocol has allowed, with a high standard in terms of coverage and quality, an early detection of rare mutations in other RAS-MAPK genes, avoiding the use of standard Sanger sequencing approach and the related enlarged cost and time consuming issues. Taken all together, these data highlight the usefulness of a molecular characterization that lead to an early diagnosis especially for patients with mild, nonspecific or atypical features and might direct to a more appropriate genetic counselling and clinical management.