Mosaic RASopathies: Comprehensive Genetic and Clinicopathological Analysis for Diagnosis and Management

1. Introduction

Molecular genetic studies of epidermal nevus syndromes (ENS) clearly defined them as mosaic disorders caused by postzygotic mutation in various genes, primarily those involved in RAS/MAPK signaling pathway [1,2,3]. Epidermal nevi (EN) are cutaneous hamartomas, which include keratinocytic or organoid nevi that may involve melanocytes or other skin appendages (epidermis, hair follicles, sweat and sebaceous glands). In addition to skin lesions presenting as various types of nevi, these syndromes are characterized by a wide range of extracutaneous abnormalities affecting virtually all organ systems. Currently, however, the nevus subtype (sebaceous, spilus, melanocytic, etc.) remains the main diagnostic feature used to classify the disease [4].

At the same time, there is substantial genetic and clinical overlap within this group of mosaic disorders. Congenital craniofacial linear nevus sebaceous (LNS) is a hallmark of Schimmelpenning-Feuerstein-Mims syndrome (SFMS), but also, a necessary component of phakomatosis pigmentokeratotica (PPK), when is occurs in association with a nevus spilus (speckled lentiginous nevus) [5,6]. The combination of EN and hypophosphatemic rickets (HR) is classified as a cutaneous skeletal hypophosphatemia syndrome (CSHS) [7]. Genetic overlap between these clinical subtypes has been identified, and the same mosaic mutations in RAS/MAPK pathway genes, primarily HRAS and KRAS, have been found in SFMS, PPK, CSHS, keratinocytic epidermal nevus (KEN), congenital melanocytic nevus, and others [2,5,8,9,10,11,12]. The same KRAS variants are present in oculoectodermal syndrome (OES) and encephalocraniocutaneous lipomatosis (ECCL); the latter is also characterized by nevus sebaceous, typically in obligatory combination with nevus psiloliparus, while congenital aplasia cutis, alopecia, and skin pigmentation are also common features. Mutations in other genes, such as BRAF, PIK3CA, FGFR1-3, PTEN, may also be detected in nevi and can be accompanied by syndromic manifestations [3].

Last studies proved that involved genes play a key role in the character of extracutaneous disorders, prognosis of the disease and possible treatment options [13,14]. There was strong evidence for the significance of genetic classification, encompassing ENS under the general term “mosaic RASopathies” [8,15,16,17,18]. To date, the fundamental mechanisms underlying the development of ENS, as well as the reasons for the variability of clinical manifestations, remain largely unknown. We previously conducted a systematic review of genetically confirmed clinical cases of SFMS and identified correlations between the presence of KRAS or HRAS mutations and specific phenotypic manifestations [13].

In this study, we present a series of 18 clinical cases with cutaneous manifestations and overlapping phenotypes characteristic of mosaic RASopathies to demonstrate the importance of genetic testing for accurate diagnosis and for predicting the extracutaneous phenotypic manifestations most typically associated with pathogenic variants in various genes of the RAS/MAPK signaling pathway. We also analyzed an expanded sample of patients diagnosed with SFMS and PPK, using data from previously described clinical cases with a genetically confirmed diagnosis, in order to more clearly delineate the genotype–phenotype relationship. These data provide a better understanding of the molecular basis of mosaic diseases and may also have implications for patient management.

2. Materials and Methods

The study included 18 patients (10 males, 8 females) aged from 1 month to 21 years with clinical features of ENS. Next-generation sequencing (NGS) was performed on DNA isolated from blood leukocytes, nevus tissue, normal skin, and tumor using the QIAmp DNA Mini Kit (Qiagen, Hilden, Germany). Libraries were prepared with the KAPA HyperPrep Kit (Roche, Basel, Switzerland) according to the manufacturer's protocol, hybridized to the coding regions of 198 genes (Data S1), then pooled and sequenced on a NextSeq 2000 (Illumina, San Diego, CA, USA) (paired-end sequencing, 300 cycles, 250–300× coverage depth). Exome sequencing of blood samples was performed for a number of patients. The identified pathogenic variants were verified by Sanger sequencing.

Sequencing data were processed using an automated algorithm that included alignment of reads to the reference human genome sequence (hg38), post-processing of alignment, variant detection, and quality filtering. The annotation and interpretation of detected variants were performed as described previously [19]. Statistical analysis was performed using GraphPad Instat software (GraphPad Software, La Jolla, CA, USA) (Fisher's exact test). Differences were considered significant at p < 0.05. The false discovery rate (FDR) was used to adjust for multiple comparisons.

3. Results

This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, and the experimental conclusions that can be drawn. In total, 18 patients were included in the study. Mutations in the genes KRAS (n=6), HRAS (n=8), BRAF (n=1), PIK3CA (n=1), and FGFR2 (n=1) were present only in affected tissues (nevus, tumor) and were absent from the peripheral blood DNA and skin without lesions. The diagnosis was assigned taking into account nevus type, extracutaneous abnormalities, and genetic data. The clinical and genetic characteristics of the patients are presented in Table S1 (Supplementary). A more detailed description of these clinical cases is provided below.

3.1. Schimmelpenning-Feuerstein-Mims Syndrome

Patient ID1, a 9-year-old boy, had congenital craniofacial LNS and EN on the trunk (Figure1 A-D). Subsequently, numerous Spitz nevi on the face developed, some of them were surgically excised (Figure 1, E-G). The patient has a delay in physical development, and from 12 months onwards, skeletal deformities began (Figure 1, C,D), and radiographs showed dysplastic foci of mixed lytic and sclerotic bone, as well as classic signs of rickets. Treatment of HR (phosphate and alfacalcidol) was ineffective, so at 4 years, pathogenetic therapy with burosumab (monoclonal antibody against fibroblast growth factor 23) was started [20]. After 15 months, the pain was relieved, independent walking was restored, and biochemical values (alkaline phosphatase (ALP), serum phosphorus and calcium) were normalized. Exome sequencing of blood DNA did not reveal any mutations. Genetic testing of the nevus identified a pathogenic variant HRAS c.182A>G (p.Gln61Arg) with a variant allele frequency (VAF) of 62%.

Patient ID2, a 8-month-old girl had craniofacial LNS (Figure 1, H-K), a pigmented nevus in the right axillary region (Figure 1J), right palpebral ptosis and a patent arterial duct since birth. MRI showed moderate expansion of the subarachnoid spaces. At 3 months, paroxysmal atrial tachycardia with atrial flutter and aplasia cutis on the vertex were diagnosed. Genetic testing of the LNS revealed a mutation HRAS c.182A>G (p.Gln61Arg) (VAF 28-36%). The patient is under follow-up due to the possible risk of developing disorders in the future.

Patient ID3, a 4-year-old boy, had an LNS on the head, face, trunk, and extremities (Figure 2 A-D). For the first 3 months, he was in intensive care unit due to CNS depression, pseudobulbar syndrome, and aspiration pneumonia. MRI revealed encephalopathy, and at 4 months, the seizures were recorded. At 6 months, he underwent a left orchiopexy – a soft tissue tumor of paratesticular region was found and subsequently removed. The tumor was identified as alveolar rhabdomyosarcoma (RMS) T1N1M1, IRS – IV, without FOXO1 gene rearrangements (the sign of favorable prognosis) and with metastases to the soft tissues of the left femur and right hand (Figure 2, E-G). After 9 courses of adjuvant polychemotherapy (PCT), the patient has been in remission for over two years. At 20 months, central precocious puberty (CPP) was diagnosed, and triptorelin acetate was prescribed. Also, he had a fracture of the left femur, and high level of ALP, hypophosphatemia and phosphaturia were defined, indicating HR. Phosphate and alfacalcidol were prescribed, followed by burosumab starting at 3 years and 9 months. After 6 months of the treatment, the laboratory values became normal, but without clinical improvement. A pathogenic variant HRAS c.37G>C (p.Gly13Arg) was revealed with a VAF from 17% to 22% in the nevus and 84% in the tumor.

Patient ID4, a 1-year-old boy was born with LNS on the scalp, face, and trunk (Figure2, H-N). Other findings were patent foramen ovale, cardiomegaly, right cryptorchism and an enlarged left testis. At 1 month, a tumor in the scrotum with intensive vascularization (spindle cell RMS, T1N0M0, IRS – IA) was revealed and left orchiectomy was performed followed by PCT. At 4.5 months, epilepsy was diagnosed, and anticonvulsive treatment was started. A mutation HRAS c.181C>A (p.Gln61Lys) was identified with a VAF from 18% to 37% in the nevus, and 76% in the tumor.

Patient ID5, a 14-years-old girl, at birth had LNS on the scalp and face, ptosis, deformation of the right palpebral region (Figure3, A-D) and congenital heart defect. At 1 month, clipping of the ductus arteriosus was performed and an orbital lipoma was removed, while during the next 2 years, multiple papillomatous lesions of the skin and palate were excised. Due to suspected Wilms' tumor at 2 years, a right nephrectomy was performed, but histology confirmed nephroblastomatosis. At 2.5 years, gliosis in the right occipital-parietal region and arachnoid cyst were detected on MRI, and focal epilepsy was diagnosed and unsuccessfully treated. At 3 years, the patient had a stroke with right-sided hemiparesis and another cerebrovascular accident after half a year. Hyperhomocysteinemia (homocysteine 12 mkmol/l) was defined and treated by dipyridamole. At the same age, CPP (thelarche, adrenarche) was observed, several benign tumors appeared (extramedullary neoplasm of the 2–3 cervical vertebrae, syringocystadenoma papilliferum in LNS), and plexiform neurofibroma which was operated on the age of 7 years (Figure 3, E-G). Now, the sexual development has normalized, but the patient has severe neurological and intellectual impairments. Exome sequencing of blood DNA did not reveal any changes. A pathogenic variant KRAS c.35G>A (p.Gly12Asp) was identified in the nevus (VAF 35%).

Patient ID6, a 5-year-old girl, was born at 36 weeks of pregnancy with low weight (1880g) and body length (42cm). At birth, LNS was presented on the scalp, face and neck (Figure 3, H-Q). At 4 months, she had seizures, and MRI revealed bilateral lipomatous lesions in the trigeminal nerves, ventriculomegaly, dysplasia of the right temporal-parietal-occipital region, white matter atrophy and hydrocephaly. Finally, focal epilepsy was diagnosed, and the patient took antiepileptic therapy without noticeable effect. At 6 months, echocardiography revealed rhabdomyoma in the right ventricle; then a central giant cell granuloma in the upper and lower jaws on the right was diagnosed, but the operation was postponed until somatic status improves. The patient has severe cognitive impairments. A mutation KRAS c.35G>A (p.Gly12Asp) was revealed in the nevus (VAF 23–28%).

Patient ID7, a 6-month-old boy had just congenital LNS on the face (Figure 3, R,S). Genetic examination of the nevus revealed a mutation KRAS c.35G>A (p.Gly12Asp), that was not observed in other tissues. Now, he is under dynamic monitoring due to suspected SFMS.

Patient ID8, a 12-year-old boy had congenital LNS on the right-sided face (Figure 3, T). In local hospital an attempt was made to partially remove the nevus surgically. Since birth he had epibulbar dermoids in the right eye which were removed at 5 months. He also had mixed non-permanent monocular non-accommodative strabismus and partial vascularized corneal opacity in the right eye (Figure 3, U). At 1.5 months he was diagnosed with epilepsy, and at 4 years, a resection of the temporal lobe was performed followed by histological examination that revealed gangliocytoma (WHO grade 1). Since then, he has not suffered from seizures; however, significant developmental delay was observed (he started walking at 1 year and 7 months, and speaking at 5 years). Genetic testing of nevus tissue revealed a mutation KRAS c.35G>A (p.Gly12Asp), that was not observed in blood DNA or skin without lesions.

3.2. Phacomatosis Pigmentokeratotica

Patient ID9, a 9-year-old girl was born with LNS involving the scalp and face and a nevus spilus on the trunk and limbs (Figure 4, A-C). At 4 months, she had seizures and was prescribed tetracosactide with positive effect. At 8 months, a hypervascular tumor (presumably rhabdomyosarcoma after biopsy) with invasion of the bladder and vagina was identified. She received 9 courses of neoadjuvant PCT, and then cystectomy, hysterectomy and vaginal resection were performed, and the tumor was defined as pelvic ectomesenchymoma, T2bN1M0, IRS-IIIA (Figure 4, D-K). A pathogenic variant HRAS c.37G>C (p.Gly13Arg) was detected (VAF 25–36% in the nevus and 41% in the tumor).

Patient ID10, a 19-year-old boy, had extensive congenital LNS, while later, a wide-spreaded nevus spilus and café-au-lait macules appeared on the body (Figure 5, A-F). At 8 years, he underwent surgery using a transpedicular system due to severe scoliosis [21]. Also, he has corneal opacity in the left eye, delayed speech development, and intellectual disability. A mutation HRAS c.37G>C (p.Gly13Arg) was revealed in the nevus (VAF 37%).

Patient ID11, a 14-year-old girl, had LNS since birth, and later, nevus spilus developed in the lumbar region (Figure 5, G, H). At 2 years, the first fracture occurred, and HR was diagnosed. The patient received phosphate and alfacalcidol for 8 years, but had multiple fractures and shortening of the left leg by 8 cm. From 11 years, she became limited in mobility, and currently receives burosumab. A pathogenic variant HRAS c.182A>G (p.Gln61Arg) was identified in the nevus tissue (VAF 33%). Now she is under follow-up without other abnormalities.

Patient ID12, a 17-year-old boy has had LNS on the scalp and nevus spilus on the right side of the face, as well as on the midline of neck, back, and chest since birth (Figure 5, I-N). Ophthalmological examination diagnosed a hemangioma of the right eye. Brain MRI revealed a dystopia of the cerebellum into the foramen magnum and vascular anomalies (hypoplasia of the proximal part of the left anterior cerebral artery, anterior trifurcation of the right internal carotid artery). Also, he was diagnosed with a bone cyst of the upper part of the right humerus at the age of 14 years. Pathology in other systems of organs was not seen. Genetic examination of both nevi types revealed a mutation HRAS c.182A>G (p.Gln61Arg), that was not observed in other tissues or blood.

Patient ID13, a 21-year-old girl, was born with LNS on the scalp, ear and neck (Figure 5, O-Q). Later, facial angiofibromas appeared, and nevus spilus was detected on the face and trunk. From 2 months of age, she began to suffer from pharmacoresistant epilepsy. MRI revealed focal cortical dysplasia in the right frontal-temporal lobe, and at 5 years, a lobectomy was done. At 14 years, she underwent embolization of an arteriovenous malformation at the level of the 4-8 thoracic vertebrae, and after 3 years, angiomyolipomas and a cyst of the kidney were identified. Also, retinal hamartomas and mild pulmonary valve stenosis were found. The patient is poorly socialized and receives antipsychotic therapy (risperidone) due to self-aggression. Exome sequencing of blood DNA did not reveal any changes, but target sequencing of the nevus identified a mutation KRAS c.35G>A (p.Gly12Asp) with a VAF of 32%.

Patient ID14, an 18-year-old boy, at birth had LNS on the face and back, and a nevus spilus on the chest (Figure 5, R, S). At the age of 1 year, his left low extremity became swollen and at 4 years, a venous malformation was detected by MR-angiography and surgically corrected. Brain MRI revealed hypoplasia in the left anterior cerebral artery and aplasia of the left posterior connective artery. Other abnormalities were not identified. A mutation KRAS c.34G>A (p.Gly12Ser) was identified in the nevus (VAF 30%). Because clinical features of both Parks-Weber syndrome (venous malformation of the leg and hypoplasia of the cerebral arteries) and PPK (congenital LNS and nevus spilus) were present simultaneously, he was diagnosed with a combination of the two syndromes.

Patient ID15, a 1-year-old boy, was born with congenital LNS on the scalp and neck (Figure 5, T). At the age of 1 month, a nevus spilus appeared in the lumbar region. Further, the patient was diagnosed with focal epilepsy, hypoplasia of the corpus callosum, a retrocerebellar cyst, nystagmus, strobism, and sensorineural hearing loss. Also, he suffered from severe recurrent infections and died from pneumonia at the age of 2 years. Primary immunodeficiency or other hereditary diseases were excluded after exome sequencing of blood DNA. A likely pathogenic variant BRAF c.1406G>C (p.Gly469Ala, rs121913355) was found only in the nevus (VAF 26%). The differential diagnosis for this patient between mosaic form of cardio-facial-cutaneous (CFC) syndrome and PPK was discussed [22].

3.3. Other Clinical Cases

Patient ID16, a 10-year-old girl (Figure 6, A, B), since birth had an EN on the scalp, face and neck [23]. At the age of 2 months, focal epilepsy was diagnosed, and MRI revealed hemimegalencephaly. At 10 years of age, she was found to have left-sided hemiparesis, delayed psychomotor development, strabismus of the left eye and nystagmus. A mutation PIK3CA c.1633G>A (p.Glu545Lys) was identified in the nevus (VAF 29%), indicating diagnosis of PIK3CA-Related Overgrowth Spectrum (PROS).

Patient ID17, a 2-year-old boy (Figure 6, C, D), was born with a hairless, well-defined linear plaque on the scalp, but without other abnormalities. Surgical excision of the skin lesion with subsequent plastic surgery of the right auricle was performed, and histology verified cerebriform nevus. Genetic testing of the nevus revealed a mutation FGFR2 c.1144T>C (p.Cys382Arg) with a VAF of 37%. He was diagnosed with rare subtype of cutis verticis gyrate and upon follow-up, there were no other pathologies.

Patient ID18, a 14-year-old girl (Figure 6, E) was born with an EN in the right scalp. No other organ involvement was detected during follow-up. At 14 years of age, surgical excision of the nevus was performed, and genetic testing did not reveal any mutations in the studied genes.

4. Discussion

The differential diagnosis of mosaic RASopathies remains a significant challenge for clinicians due to the rarity of these conditions and the similarity of their phenotypes. Typically, the first sign is characteristic skin lesions in the form of extensive epidermal nevi, which appear primarily in the craniofacial region. It is crucial for both parents and clinicians to understand the spectrum of possible clinical manifestations during the child's development and to discuss the individual patient’s prognosis. The primary factor believed to influence on disease severity is the timing of the postzygotic mutation during embryogenesis. It mainly defines the extent of tissue and organ involvement—whether ectodermal, mesodermal, or endodermal, or a combination—and, consequently, the spectrum and expressiveness of syndromic clinical manifestations [2,3,24].

We also examined the variability in clinical manifestations and disease course in relation to the specific gene mutated, drawing on both our own data and the published literature. In our study, eight patients were diagnosed with SFMS and seven with PPK. In both groups, mutations in the HRAS or KRAS genes were predominant. We combined these two clinical groups and compared our results with those from previously published studies described SFMS and PPK cases [7,9,11,14,15,17,18,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94] to analyze the spectrum of gene variants, the variability of cutaneous and extracutaneous anomalies, and genotype-phenotype correlations (Table S1).

In our study, the most common substitution in HRAS was Q61R (50%), followed by G13R (38%), whereas in the literature, the most frequent was HRAS G13R (57%). The G12D substitution predominated among all KRAS mutations, both in our study (83%) and in cases from the literature (64%) (Figure 7, A). All our patients with HRAS mutations had craniofacial LNS, and four of them (50%) also had nevus spilus. Other cutaneous anomalies included EN, melanocytic nevi, and café-au-lait spots. According to the literature, LNS was present in 67% of 45 HRAS-mutated cases, often in combination with nevus spilus (40%), EN, and KEN [13,15,22]. All six of our patients with KRAS mutations also had congenital craniofacial LNS, and two of them had nevus spilus. Other skin lesions included café-au-lait spots and angiofibromas. In 35 KRAS-mutated cases from the literature, LNS was noted in 68%, and only one patient also had nevus spilus, a finding indicative of PPK.

Despite the apparent overlap of HRAS- and KRAS-mutated phenotypes, the spectrum of extracutaneous anomalies differed significantly between the two groups (Table S2, Figure 7, B). The most notable difference was the association of KRAS mutations with ophthalmic disorders (70% in KRAS-mutated vs. 19% in HRAS-mutated, OR = 10.4, p* = 0.001); coloboma, choristoma, and epibulbar/bulbar dermoid were detected only in patients with KRAS mutations, both in our study and in the literature (Table S2). Neurological disorders (including epilepsy and various brain malformations) were also more common in KRAS-mutated than in HRAS-mutated patients (78% vs. 38%, OR = 5.9, p = 0.001), as were cardiovascular disorders and lymphatic malformations (48% vs. 19%, OR = 3.7, p* = 0.013; and 22% vs. 4%, OR = 7.2, p* = 0.014, respectively). In our cases and in the literature, there is a strict association between HRAS mutations and HR, which was diagnosed exclusively in HRAS-mutated patients, occurring in 30–40% of cases (p* = 0.0004) [7,14,26,27,48,56,67,75]. Other skeletal abnormalities (fibrous dysplasia, scoliosis, limb asymmetry, cystic bone lesions, etc.) were found in HRAS- and KRAS-mutated patients, and the difference was not significant.

In total sample, benign neoplasms occurred with equal frequency in both groups (33% in HRAS-mutated patients and 35% in KRAS-mutated patients), whereas malignancies were more frequent in the HRAS-mutated group (36% vs. 12%, OR = 4.0, p* = 0.002). Among the malignancies, embryonal/alveolar RMS was more common in childhood [18,26,44,56,65,68], while skin cancer [53,56,57,63,64,93], urothelial carcinoma [59,90], and astrocytoma [32] have been described in adults. In our study, two HRAS-mutated patients (ID3 and ID4) developed embryonal RMS, and one patient (ID9) developed ectomesenchymoma; all tumors occurred in the groin area before the age of 3 years, and the pathogenic mutation had the highest VAF in the tumor tissue (84%, 76%, and 41%, respectively). KRAS mutations have previously been described in Wilms' tumor [45], embryonal RMS [17,71,91], and yolk sac tumor [62], all occurring under the age of 1 year. Besides nephroblastoma, renal anomalies (cysts, hamartomas, angiolipomas, etc.) were a very characteristic feature of KRAS-associated mosaicism (ID5) [17,28,30,37,91], as was CPP (ID3, ID4, and ID5) [46,69,77]. Hearing loss (ID13) [17,34,37,57,60,61,78,92] has been reported with equal frequency in both groups (Table S2). We have previously suggested that the expression of genetic information and the formation of specific phenotypic patterns in mosaic syndromes may depend not only on the timing of the mutation, but also on the expression levels of different RAS family genes in various organs and tissues during embryonic development [13]. In this study, we observed very similar genotype–phenotype correlations when using a significantly larger sample. Further research in this area may help elucidate this question.

Differential diagnosis of ENS associated with postzygotic mutations in RAS/MAPK pathway is complicated, and DNA testing plays a key role in establishing the diagnosis. Germline BRAF mutations can lead to CFC [95], while postzygotic mutations have been described in PPK [12,76,78]. Patient ID15 had severe psychomotor delay, minor cardiac abnormalities, immunodeficiency, and facial dysmorphism, which are characteristic of CFC. On the other hand, a diagnosis of PPK could also be considered based on the mosaic BRAF variant, congenital LNS, and nevus spilus. Patients with PROS may also have EN, besides asymmetric tissue growth [96]. In patient ID16 with EN, lower limb asymmetry, and hemimegaloencephaly, SFMS was suspected, but the presence of a mosaic PIK3CA variant allowed the correct diagnosis of PROS to be made. Germline variants in the FGFR2 gene may be associated with severe skeletal disorders [97], while mosaic mutations can lead to phacomatosis resembling SFMS [98]. The somatic mutation FGFR2 c.1144T>C (VAF 23–40%) was found in 5/8 patients with cerebriform nevus of the sebaceous glands [99], and also in patient ID17 in our study. It is also important to exclude germline mutations in patients with mosaic RASopathies. Previously, we described a patient with a congenital facial epidermal nevus (Figure 6, F) who was found to have a pathogenic PTEN variant first in the nevus, and this was subsequently confirmed by blood DNA analysis [100]. Thus, the final diagnosis was type 2 segmental Cowden disease, which is associated with linear Cowden nevus, and a lifelong surveillance was prescribed due to the high cancer risk.

In the nevus tissue of patient ID18, genetic analysis did not reveal any mutations in any of the genes associated with ENS. Furthermore, the patient had only skin manifestations, which were successfully corrected by surgery, and during further follow-up (more than 5 years) no symptoms characteristic of ENS were noted. Therefore, the case was considered as isolated skin malformation.

Based on this study, we propose the following algorithm for accurately determining somatic mosaicism in ENS. Given the varying VAF in affected tissues, biopsies should be taken from at least four different areas of the nevus (and different types of nevi, if feasible), and genetic testing of peripheral blood should be performed to exclude germline mutations. This approach can help differentiate mosaic disorders from hereditary syndromes with incomplete phenotypic manifestations. A mutation in a specific gene can certainly influence the range of clinical manifestations; however, it is important to recognize that these correlations are probabilistic in nature. Therefore, all patients with characteristic skin lesions (epidermal nevus) and suspected mosaic forms of the disease should be referred for assessment by a multidisciplinary team of specialists [15].

This study has some limitations. Due to the rarity of these mosaic diseases, we had a limited number of cases for the statistical analysis. The comparison of small patient groups led to wide confidence intervals, indicating some inconsistency in the results. Another aspect is that extracutaneous anomalies can develop over a long period, while some of patients from our study and literature cases were only 0–3 years old. Therefore, certain syndromic manifestations may not yet have become fully apparent. It is also worth noting that in the cases reported in the literature, not all patients underwent a comprehensive examination of all organ systems, which could also have influenced the final statistical calculations. Further investigations involving more genetically confirmed cases will make it possible to identify clearer correlations and the molecular basis underlying the diverse phenotypic patterns.

5. Conclusions

Genetic testing plays a crucial role in accurately diagnosing mosaic forms of the disease. The spectrum and severity of clinical manifestations of mosaic RASopathies may depend not only on the timing of the postzygotic mutation, but also on the specific gene involved. For example, it can be assumed that patients with KRAS-mediated RASopathy will have an increased risk of ophthalmological disorders (especially coloboma or choristoma), as well as neurological, renal, and vascular abnormalities, while HRAS-mediated mosaic disorder is likely to be associated with hypophosphatemic rickets and malignancies in both children and adults. The specific mosaic genotype may be taken into consideration for prognosis, patient management, and the development of targeted therapies.