A new class of genetic disorders, the “RASopathies”, has recently emerged. To date, this nosologic group comprises NS, LS, Hereditary gingival fibromatosis type 1, Capillary Malformation-AV malformation syndrome, NF1, NFLS, Costello syndrome, Cardio-facio-cutaneous syndrome (CFC), and Autoimmune Lymphoproliferative syndrome (ALPS) . Collectively, these conditions represent relatively common monogenic disorders. Although each one exhibits typical phenotypic features, they share many clinical signs, including craniofacial anomalies; congenital heart defects; short stature; varying degrees of neurocognitive impairment; cutaneous and musculoskeletal abnormalities and predisposition to malignancies. Thus, differential diagnosis can represent a clinical dilemma, particularly among NF1, NS, LS, and NFLS.
Several case reports of patients with borderline phenotypes that illustrate this clinical overlap have been published. For example, Wu et al.  reported a de novo NF1 mutation in a woman with a prior diagnosis of LS. Moreover, Carcavilla et al.  reported three children who filled NF1 clinical criteria but were diagnosed with LS and carried the PTPN11 mutation p.(Thr468Met), the same mutation detected in our patients. Carcavilla et al.  suggested that a distinguishing feature for LS might be the diffuse pattern of lentigines, even if they seem to appear later in life than CALMs; this is in accord with our personal observations. The patients described in this previous report had cardiomyopathy but lacked other features typical of LS, such as deafness or genital abnormalities.
Our patients similarly lacked these types of clinical signs. We noted a strong dysmorphic feature overlap between our young patient and the one described by Nystrom et al. . The child described by Nystrom et al. , who had a clinical diagnosis of NS because of pulmonic stenosis and NS facial dysmorphisms, was discovered to carry the BRAF mutation p.(Lys499Glu). The authors proposed a gene-based classification of RASopathies and suggested that NS and CFC are allelic disorders of BRAF. A BRAF analysis was performed for our proband, and no mutation was found. Taking into account the dysmorphic overlap between our proband (clinically diagnosed with LS and carrying a PTNP11 mutation) and Nystrom’s patient (diagnosed with NS due to BRAF mutation), we enforce the opinion that a straightforward genotype–phenotype relationship is not always present among RASopathies.
In the last 10 years, we did experience with other similar cases. A 3-year-old boy was addressed to us because of CALMs (n° 5) in suspicion of NF1. He showed mild Noonan like dymorphisms, pulmonary stenosis and was a sporadic case. We performed PTPN11 analysis as first genetic test that led us to NS diagnosis. The other patient was a 18 months-old female with few CALMs and lentigines who initially received diagnosis of NF1. Intriguingly, her parents were healthy. During the following two years she received diagnosis of HCM and developmental delay. Sparse and slow growing hair and Noonan dysmorphisms were also present. The PTPN11 analysis detected a mutation compatible with LS. All these observations point to the PTPN11 gene, instead of NF1, as the first gene to be ruled out if the differential diagnosis of NFNS/NFLS/LS. On the contrary, a male with CALMs, joint laxity, frontal bossing, macrocephaly, pulmonary stenosis and no lentigines at the age of 5, suspected to be affected by NS, received diagnosis of NF1 after the molecular analysis (firstly performed because of the number of CALMs = 6).
We also report diagnosis of NFLS in 4 patients (3 familiar cases, and one sporadic), and their affected relatives, who previously received diagnosis of NF1. Due to the lacking of NF1 typical stigmata, NF1 analysis was performed but any mutation was detected. Nevertheless the clinical diagnosis of NF1 was confirmed. After a long follow-up (4–10 years), and after the detection of SPRED1 mutations as the genetic cause of NFLS, the diagnosis was changed.
The recent description of NFLS produced new questions, particularly about the limits of the diagnostic criteria for NF1 and the clinical homogeneity of this condition. The current clinical diagnostic criteria for RASopathies seem to be inadequate, especially when the patient is observed during childhood, when some age-dependent manifestations are not yet present. Patients who are young and who appear to lack a familial history of RASopathy, need to be clinically reviewed and other specific consultations (e.g., ophthalmological, dermatological, cardiological and audiometric) should be performed in order to reach a correct diagnosis. Unfortunately, these additional evaluations may be associated with burdensome health care costs. Muram et al.  reported a cost saving approach by SPRED1 analysis in patients with a probable NF1 diagnosis. The genetic testing was cost saving between the ages of 9 and 15 years in individuals with multiple CALMs with or without freckling compared to the no-testing approach with routine follow-up. Furthermore, Lepri et al.  reported a next generation analysis (integrated by Sanger sequencing of the remaining not-covered regions) resulting 6 time less expensive than protocols entirely based on Sanger sequencing in 10 patients affected by RASopathies.
Our experience with the proband and his father, taken together with the known broad intrafamilial variability of the phenotype in RASopathies, is a reminder of how clinical examinations of parents and other first-degree relatives (although referred to as healthy) can be useful in designing suitable diagnostic approaches to children with suspected NF1/NS/LS/NFLS. In our case report, the clinical evaluation of the father would have helped to orientate the stratified molecular diagnoses, priorizing PTPN11 analysis. The clinical examination of both parents should be considered in the genetic counselling of couples subjected to the in vitro fertilization.
Finally, to our knowledge, this is the third report of the occurrence of Arnold Chiari malformation [24, 25] and the second one of syringomyelia with LS . Instead NS is already known to have a high incidence of spinal cord malformations  and there are some evidences that this is also true for NF1 . This confirms that LS and NS belong to a clinical spectrum together with NFNS .