Biallelic inactivation of the NF1 tumour suppressor gene in juvenile myelomonocytic leukaemia: Genetic evidence of driver function and implications for diagnostic workup.

Juvenile myelomonocytic leukaemia (JMML) is characterized by gene variants that deregulate the RAS signalling pathway. Children with neurofibromatosis type 1 (NF-1) carry a defective NF1 allele in the germline and are predisposed to JMML, which presumably requires somatic inactivation of the NF1 wild-type allele. Here we examined the two-hit concept in leukaemic cells of 25 patients with JMML and NF-1. Ten patients with JMML/NF-1 exhibited a NF1 loss-of-function variant in combination with uniparental disomy of the 17q arm. Five had NF1 microdeletions combined with a pathogenic NF1 variant and nine carried two compound-heterozygous NF1 variants. We also examined 16 patients without clinical signs of NF-1 and no variation in the JMML-associated driver genes PTPN11, KRAS, NRAS or CBL (JMML-5neg) and identified eight patients with NF1 variants. Three patients had microdeletions combined with hemizygous NF1 variants, three had compound-heterozygous NF1 variants and two had heterozygous NF1 variants. In addition, we found a high incidence of secondary ASXL1 and/or SETBP1 variants in both groups. We conclude that the clinical diagnosis of JMML/NF-1 reliably indicates a NF1-driven JMML subtype, and that careful NF1 analysis should be included in the genetic workup of JMML even in the absence of clinical evidence of NF-1.

Achromatopsia-Visual Cortex Stability and Plasticity in the Absence of Functional Cones.

Purpose: Achromatopsia is a rare inherited disorder rendering retinal cone photoreceptors nonfunctional. As a consequence, the sizable foveal representation in the visual cortex is congenitally deprived of visual input, which prompts a fundamental question: is the cortical representation of the central visual field in patients with achromatopsia remapped to take up processing of paracentral inputs? Such remapping might interfere with gene therapeutic treatments aimed at restoring cone function. Methods: We conducted a multicenter study to explore the nature and plasticity of vision in the absence of functional cones in a cohort of 17 individuals affected by autosomal recessive achromatopsia and confirmed biallelic disease-causing CNGA3 or CNGB3 mutations. Specifically, we tested the hypothesis of foveal remapping in human achromatopsia. For this purpose, we applied two independent functional magnetic resonance imaging (fMRI)-based mapping approaches, i.e. conventional phase-encoded eccentricity and population receptive field mapping, to separate data sets. Results: Both fMRI approaches produced the same result in the group comparison of achromatopsia versus healthy controls: sizable remapping of the representation of the central visual field in the primary visual cortex was not apparent. Conclusions: Remapping of the cortical representation of the central visual field is not a general feature in achromatopsia. It is concluded that plasticity of the human primary visual cortex is less pronounced than previously assumed. A pretherapeutic imaging workup is proposed to optimize interventions.

Unusual phenotypes in patients with a pathogenic germline variant in DICER1.

Pathogenic germline DICER1 variants are associated with pleuropulmonary blastoma, multinodular goiter, embryonal rhabdomyosarcoma and other tumour types, while mosaic missense DICER1 variants in the RNase IIIb domain are linked to cause GLOW (global developmental delay, lung cysts, overgrowth, and Wilms' tumor) syndrome. Here, we report four families with germline DICER1 pathogenic variants in which one member in each family had a more complex phenotype, including skeletal findings, facial dysmorphism and developmental abnormalities. The developmental features occur with a variable expressivity and incomplete penetrance as also described for the neoplastic and dysplastic lesions associated with DICER1 variants. Whole exome sequencing (WES) was performed on all four cases and revealed no further pathogenic or likely pathogenic dominant, homozygous or compound heterozygous variants in three of them. Notably, a frameshift variant in ARID1B was detected in one patient explaining part of her phenotype. This series of patients shows that pathogenic DICER1 variants may be associated with a broader phenotypic spectrum than initially assumed, including predisposition to different tumours, skeletal findings, dysmorphism and developmental abnormalities, but genetic work up in syndromic patients should be comprehensive in order not to miss additional underlying /modifying causes.

Integration of genomic analysis and transcript expression of ABCC8 and KCNJ11 in focal form of congenital hyperinsulinism.

Background: The focal form of CHI is caused by an autosomal recessive pathogenic variant affecting the paternal homologue of genes ABCC8 or KCNJ11 and a second somatic event specifically occurring in the affected islet of Langerhans. The approach of this study was to integrate the genetic changes occurring in pancreatic focal lesions of CHI at the genomic and transcriptional level. Research Design and Methods: Patients receiving therapeutic surgery and with proven ABCC8 or KCNJ11 pathogenic variants were selected and analyzed for loss of heterozygosity (LOH), changes in copy number and uniparental disomy (UPD) on the short am of chromosome 11 by molecular microarray analysis and methylation-specific MLPA. Gene expression was analyzed by RT-PCR and Massive Analysis of cDNA Ends (MACE). Results: Both genes, ABCC8 and KCNJ11, are located in proximity to the Beckwith-Wiedemann (BWS) imprinting control region on chromosome 11p15. Somatic paternal uniparental isodisomy (UPD) at chromosome 11p was identified as second genetic event in focal lesions resulting in LOH and monoallelic expression of the mutated ABCC8/KCNJ11 alleles. Of five patients with samples available for microarray analysis, the breakpoints of UPD on chromosome 11p were different. Samples of two patients were analyzed further for changes in gene expression. Profound downregulation of growth suppressing genes CDKN1 and H19 was detected in focal lesions whereas growth promoting gene ASCL2 and pancreatic transcription factors of the endocrine cell lineage were upregulated. Conclusions: Paternal UPD on the short arm of chromosome 11 appears to be the major second genetic event specifically within focal lesions of CHI but no common breakpoint for UDP can be delineated. We show for the first time upregulation of growth promoting ASCL2 (achaete-scute homolog 2) suggestive of a driving factor in postnatal focal expansion in addition to downregulation of growth suppressing genes CDKN1C and H19.

A Case Report: First Long-Term Treatment With Burosumab in a Patient With Cutaneous-Skeletal Hypophosphatemia Syndrome.

Epidermal nevus syndromes encompass a highly heterogeneous group of systemic disorders, characterized by epidermal nevi, and a spectrum of neuromuscular, ocular, and bone abnormalities. Cutaneous-skeletal hypophosphatemia syndrome (CSHS) constitutes a specific sub-entity in which elevated levels of fibroblast growth factor-23 cause hypophosphatemic rickets that are, to date, not amenable to causal therapy. Here, we report the first long-term follow-up of causal treatment with burosumab in a 3-year-old female patient with CSHS. 4 weeks after initiation of burosumab treatment, serum phosphate normalized to age-appropriate levels. Furthermore, long-term follow-up of 42 months revealed significant improvement of linear growth and gross physical functions, including respiratory insufficiency. Radiographic rickets severity as well as subjective bone pain were strongly reduced, and no side effects were observed over the course of treatment. In summary, we, here, report about a successful treatment of hypophosphatemic rickets in CSHS with burosumab over the time course of 42 months. In our patient, burosumab showed convincing efficacy and safety profile, without any loss of effect or increase of dose.

Recurrent Mandibular Giant Cell Lesion in Neurofibromatosis Type 1: Second Hit Mutation on the NF1 Gene in the Osseous Lesion.

BACKGROUND/AIM: In the autosomal dominant hereditary disease neurofibromatosis type 1 (NF1), lesions of the jaw develop in isolated cases, which are diagnosed as central giant cell granuloma (CGCG). This study aimed to clarify the genetic basis of a bone lesion in a syndromic patient. CASE REPORT: The NF1 patient had developed a CGCG that recurred after local excision. Blood and tumor tissue were studied for NF1 mutations using advanced molecular genetic methods. Examinations of blood and tumor tissue provided evidence of the constitutive mutation in both samples. A further mutation was detected in the tumor, which was interpreted as a somatic mutation. The detection of somatic mutation in the tissue was successful both on native and routinely fixed material. CONCLUSION: The study supports current assessments of CGCG as a benign neoplasm. In NF1 patients, the phenotype seems to imply bi-allelic loss of the NF1 gene. The detection of both mutations in routinely fixed tissue allows studies of archived tissue samples with this diagnosis.

Correlation of PET-MRI, Pathology, LOH, and Surgical Success in a Case of CHI With Atypical Large Pancreatic Focus.

Congenital hyperinsulinism (CHI) is a rare cause of severe hypoglycemia in newborns. In focal CHI, usually one activity peak in fluorine-18-L-dihydroxyphenylalanine (18F-DOPA) positron emission tomography-magnetic resonance imaging (PET-MRI) indicates one focal lesion and its resection results in cure of the child. We present the case of a 5-month-old girl with CHI. Mutational screening of genes involved in CHI revealed a heterozygous pathogenic variant in the ABCC8 gene, which was not detectable in the parents. 18F-DOPA PET-MRI revealed 2 distinct activity peaks nearby in the pancreatic body and neck. Surgical resection of the tissue areas representing both activity peaks resulted in long-lasting normoglycemia that was proven by a fasting test. Molecular analysis of tissue samples from various sites provided evidence that a single second genetic hit in a pancreatic precursor cell was responsible for the atypical extended pancreatic lesion. There was a close correlation in the resected areas of PET-MRI activity with focal histopathology and frequency of the mutant allele (loss of heterozygosity) in the tissue. Focal lesions can be very heterogenous. The resection of the most affected areas as indicated by imaging, histopathology, and genetics could result in complete cure.

Cutis marmorata telangiectatica congenita being caused by postzygotic GNA11 mutations.

Cutis marmorata telangiectatica congenita (CMTC) is characterized by coarse-meshed capillary malformations arranged in asymmetrically distributed patches. The disorder may be associated with hyper- or hypoplastic limbs, syndactyly, cleft palate, and glaucoma. Because the disease usually occurs sporadically, the concept of a lethal mutation surviving by mosaicism was proposed about 30 years ago. Here we describe three children with CMTC due to a postzygotic GNA11 mutation c547C > T (p.Arg183Cys), documented in saliva (patient 1) or lesional cutaneous tissue (patients 2 and 3). All three individuals had widespread and asymmetric CMTC which was present from birth and became fainter during the first years of life. Variably associated anomalies included glaucoma, choroidal capillary malformation, and body asymmetry. In previous case reports, postzygotic GNA11 mutations were documented in two cases of phacomatosis cesiomarmorata, being characterized by CMTC coexisting with segmental dermal melanocytosis. Moreover, postzygotic GNA11 mutations were noted in two CMTC patients described under the incorrect diagnosis of "nevus vascularis mixtus". Hence, the present cases convincingly support the concept that CMTC can be caused by mosaic GNA11 mutations and thus belongs to the GNA11-Related Capillary Nevus (GNARCAN) spectrum. In two other bona fide cases of CMTC, however, we were unable to find a mutation in GNA11, which may be explained either by our inability to detect a very low percentage of mutant cells or by genetic heterogeneity of the phenotype.

Oral HRAS Mutation in Orofacial Nevus Sebaceous Syndrome (Schimmelpenning-Feuerstein-Mims-Syndrome): A Case Report With a Literature Survey.

BACKGROUND/AIM: The aim of this study was to present the long-term course of a patient with nevus sebaceous syndrome (NSS). Recent genetic studies place the syndrome in the emerging group of so-called RASopathies. The focus of the report is on surgical treatment and morphological and genetic findings of the face and oral cavity. CASE REPORT: A female patient was treated for congenital alterations of facial skin and oral mucosa. The oral lesions were removed repeatedly. Eruption of teeth on the lesion sites was made easier by the measures taken. However, after repeated ablation of the affected gingiva, the periodontal papillomatous epithelium re-differentiated into the same reddish, conspicuous, hyperplastic epithelium. The teeth in the affected region showed noticeable changes in position, surface, and shape. A HRAS mutation was detected only in the regions of altered oral epithelia and not in adjacent soft tissues. CONCLUSION: Reports on NSS rarely address oral manifestations. The recorded alterations of oral soft and hard tissues in NSS indicate a topographical relationship between the development of oral mucosa and teeth as well as the long-lasting impact of a sporadic mutation on organ development at this site.

Structural changes to primary visual cortex in the congenital absence of cone input in achromatopsia.

Autosomal recessive Achromatopsia (ACHM) is a rare inherited disorder associated with dysfunctional cone photoreceptors resulting in a congenital absence of cone input to visual cortex. This might lead to distinct changes in cortical architecture with a negative impact on the success of gene augmentation therapies. To investigate the status of the visual cortex in these patients, we performed a multi-centre study focusing on the cortical structure of regions that normally receive predominantly cone input. Using high-resolution T1-weighted MRI scans and surface-based morphometry, we compared cortical thickness, surface area and grey matter volume in foveal, parafoveal and paracentral representations of primary visual cortex in 15 individuals with ACHM and 42 normally sighted, healthy controls (HC). In ACHM, surface area was reduced in all tested representations, while thickening of the cortex was found highly localized to the most central representation. These results were comparable to more widespread changes in brain structure reported in congenitally blind individuals, suggesting similar developmental processes, i.e., irrespective of the underlying cause and extent of vision loss. The cortical differences we report here could limit the success of treatment of ACHM in adulthood. Interventions earlier in life when cortical structure is not different from normal would likely offer better visual outcomes for those with ACHM.

Structural Differences Across Multiple Visual Cortical Regions in the Absence of Cone Function in Congenital Achromatopsia.

Most individuals with congenital achromatopsia (ACHM) carry mutations that affect the retinal phototransduction pathway of cone photoreceptors, fundamental to both high acuity vision and colour perception. As the central fovea is occupied solely by cones, achromats have an absence of retinal input to the visual cortex and a small central area of blindness. Additionally, those with complete ACHM have no colour perception, and colour processing regions of the ventral cortex also lack typical chromatic signals from the cones. This study examined the cortical morphology (grey matter volume, cortical thickness, and cortical surface area) of multiple visual cortical regions in ACHM (n = 15) compared to normally sighted controls (n = 42) to determine the cortical changes that are associated with the retinal characteristics of ACHM. Surface-based morphometry was applied to T1-weighted MRI in atlas-defined early, ventral and dorsal visual regions of interest. Reduced grey matter volume in V1, V2, V3, and V4 was found in ACHM compared to controls, driven by a reduction in cortical surface area as there was no significant reduction in cortical thickness. Cortical surface area (but not thickness) was reduced in a wide range of areas (V1, V2, V3, TO1, V4, and LO1). Reduction in early visual areas with large foveal representations (V1, V2, and V3) suggests that the lack of foveal input to the visual cortex was a major driving factor in morphological changes in ACHM. However, the significant reduction in ventral area V4 coupled with the lack of difference in dorsal areas V3a and V3b suggest that deprivation of chromatic signals to visual cortex in ACHM may also contribute to changes in cortical morphology. This research shows that the congenital lack of cone input to the visual cortex can lead to widespread structural changes across multiple visual areas.

Functional assessment of two variants of unknown significance in TEK by endothelium-specific expression in zebrafish embryos.

Vascular malformations are most often caused by somatic mutations of the PI3K/mTOR and the RAS signaling pathways, which can be identified in the affected tissue. Venous malformations (VMs) commonly harbor PIK3CA and TEK mutations, whereas arteriovenous malformations (AVMs) are usually caused by BRAF, RAS or MAP2K1 mutations. Correct identification of the underlying mutation is of increasing importance, since targeted treatments are becoming more and more relevant, especially in patients with extensive vascular malformations. However, variants of unknown significance (VUSs) are often identified and their pathogenicity and response to targeted therapy cannot be precisely predicted. Here, we show that zebrafish embryos can be used to rapidly assess the pathogenicity of novel VUSs in TEK, encoding for the receptor TIE2, present on endothelial cells of VMs. Endothelium-specific overexpression of TEK mutations leads to robust induction of VMs, whereas MAP2K1 mutations cause AVMs in our zebrafish model. TEK mutations are often found as double mutations in cis; using our model, we show that double mutations have an additive effect in inducing VMs compared with the respective single variants. The clinically established mTOR-inhibitor sirolimus (rapamycin) efficiently abrogates the development of VMs in this zebrafish model. In summary, endothelium-specific overexpression of patient-derived TEK variants in the zebrafish model allows assessment of their pathogenic significance as well as testing of candidate drugs in a personalized and mutation-specific approach.

Mosaic RASopathy due to KRAS variant G12D with segmental overgrowth and associated peripheral vascular malformations.

Oncogenic RAS variants lead to constitutive overactivation and increased signal transduction into downstream pathways. They are found as somatic driver events in various types of human cancer. In a somatic mosaic status, the same RAS variants have been associated with a wide spectrum of focal or segmental tissue dysplasia and overgrowth including various types of congenital nevi, vascular malformations, and other changes (mosaic RASopathies). We present a 3-year-old male patient with segmental overgrowth of the subcutaneous fatty tissue of the right lower extremity with colocalized arteriovenous and capillary malformations and dysplastic draining veins in combination with talipes equinovarus of the right foot. In tissue biopsies of the affected extremity, we identified a mosaic KRAS variant, c.35G>A (p.Gly12Asp), while this variant was absent in the DNA extracted from a biopsy of the normal extremity. This report provides further evidence for the wide clinical and phenotypic variability associated with mosaic KRAS variants. The described pattern confirms that the combination of segmental overgrowth and vascular anomalies in the form of arteriovenous and capillary malformations is a possible manifestation of a mosaic RASopathy. The accurate genetic diagnosis is crucial for molecular-targeted therapy, which might be a future therapeutic target for mosaic RASopathies.

Neurofibromatosis Type 1 With Cherubism-like Phenotype, Multiple Osteolytic Bone Lesions of Lower Extremities, and Alagille-syndrome: Case Report With Literature Survey.

BACKGROUND/AIM: Neurofibromatosis type 1 (NF) is an autosomal dominant hereditary disease. The cardinal clinical findings include characteristic skeletal alterations. Difficulties in diagnosis and therapy can arise if an individual has further illnesses. CASE REPORT: This is a case report of a 16-year-old patient affected by NF1. She also suffered from Alagille syndrome and the consequences of fetal alcohol exposure. The patient's facial phenotype showed findings that could be assigned to one or more of the known diseases. The patient was referred for treating a cherubism-like recurrent central giant cell granuloma (CGCG) of the jaw. The patient developed bilateral, multilocular non-ossifying fibromas (NOF) of the long bones of the lower extremity. Treatment of the skeletal lesions consisted of local curettage. While NOF regressed after surgery, the CGCG of the jaw remained largely unchanged. Extensive genetic tests confirmed a previously unknown germline mutation in the JAG1 gene, the germline mutation of the NF1 gene, and the somatic mutation in the NF1 gene in the diffuse plexiform neurofibroma, but not in the CGCG. CONCLUSION: Assigning facial findings to a defined syndrome is ambiguous in many cases and especially difficult in patients who have multiple diseases that can affect the facial phenotype. Surgical therapy should be adapted to the individual findings.

KRAS Mutation in an Implant-associated Peripheral Giant Cell Granuloma of the Jaw: Implications of Genetic Analysis of the Lesion for Treatment Concept and Surveillance.

The aim of this case report was to detail diagnosis and therapy in a case of implant-associated peripheral giant cell granuloma (IA-PGCG) of the jaw. Case Report: The 41-year-old female attended the outpatient clinic for treatment of recurrent mandibular IA-PGCG. The lesion was excised and the defect was closed with a connective tissue graft of the palate. Healing of oral defects was uneventful, and no local recurrence has occurred during a follow-up of 7 months. Genetic examination of the lesion identified a somatic mutation in KRAS. Conclusion: The lesions are assessed as reactive-inflammatory changes in the mucous membrane of the oral cavity. The cause of the lesion is unknown. KRAS mutations are commonly found in various cancer tissues, but also in germline and mosaic RASopathies. Recently, KRAS mutations have been identified in several IA-PGCG. The clinical course of a frequently locally recurring lesion gives rise to the assumption that lesions of this type show characteristics known in benign neoplasms.

Possible New Strategies for the Treatment of Congenital Hyperinsulinism.

Objective: Congenital hyperinsulinism (CHI) is a rare disease characterized by persistent hypoglycemia as a result of inappropriate insulin secretion, which can lead to irreversible neurological defects in infants. Poor efficacy and strong adverse effects of the current medications impede successful treatment. The aim of the study was to investigate new approaches to silence ß-cells and thus attenuate insulin secretion. Research Design and Methods: In the scope of our research, we tested substances more selective and more potent than the gold standard diazoxide that also interact with neuroendocrine ATP-sensitive K+ (KATP) channels. Additionally, KATP channel-independent targets as Ca2+-activated K+ channels of intermediate conductance (KCa3.1) and L-type Ca2+ channels were investigated. Experiments were performed using human islet cell clusters isolated from tissue of CHI patients (histologically classified as pathological) and islet cell clusters obtained from C57BL/6N (WT) or SUR1 knockout (SUR1-/-) mice. The cytosolic Ca2+ concentration ([Ca2+]c) was used as a parameter for the pathway regulated by electrical activity and was determined by fura-2 fluorescence. The mitochondrial membrane potential (ΔΨ) was determined by rhodamine 123 fluorescence and single channel currents were measured by the patch-clamp technique. Results: The selective KATP channel opener NN414 (5 µM) diminished [Ca2+]c in isolated human CHI islet cell clusters and WT mouse islet cell clusters stimulated with 10 mM glucose. In islet cell clusters lacking functional KATP channels (SUR1-/-) the drug was without effect. VU0071063 (30 µM), another KATP channel opener considered to be selective, lowered [Ca2+]c in human CHI islet cell clusters. The compound was also effective in islet cell clusters from SUR1-/- mice, showing that [Ca2+]c is influenced by additional effects besides KATP channels. Contrasting to NN414, the drug depolarized ΔΨ in murine islet cell clusters pointing to severe interference with mitochondrial metabolism. An opener of KCa3.1 channels, DCEBIO (100 µM), significantly decreased [Ca2+]c in SUR1-/- and human CHI islet cell clusters. To target L-type Ca2+ channels we tested two already approved drugs, dextromethorphan (DXM) and simvastatin. DXM (100 µM) efficiently diminished [Ca2+]c in stimulated human CHI islet cell clusters as well as in stimulated SUR1-/- islet cell clusters. Similar effects on [Ca2+]c were observed in experiments with simvastatin (7.2 µM). Conclusions: NN414 seems to provide a good alternative to the currently used KATP channel opener diazoxide. Targeting KCa3.1 channels by channel openers or L-type Ca2+ channels by DXM or simvastatin might be valuable approaches for treatment of CHI caused by mutations of KATP channels not sensitive to KATP channel openers.

Mosaic Neurofibromatosis Type 1 With Multiple Cutaneous Diffuse and Plexiform Neurofibromas of the Lower Leg.

BACKGROUND: Neurofibromatosis type 1 (NF1) is an autosomal dominant hereditary disease with complete penetrance and a very variable phenotype. Recent research has shown that postzygotic NF1 gene mutations occur to a far greater extent than previously thought. The phenotype of affected individuals reflects the time of somatic mutation and the phenotype is correspondingly diverse. This report describes histological and genetic findings in a case of mosaic NF1, the clinical control of which documents almost stationary skin findings over a period of 9 years. CASE REPORT: The 55-year-old female first presented for advice on a strip of nodular skin tumours of the calf skin. She had no hallmarks of NF1. It was only 9 years later that she had the skin tumours removed, all of which were partially diffuse and partially plexiform neurofibroma. The genetic examination showed an atypical large deletion of the NF1 gene in the skin tumours, but not in overlying skin or blood. CONCLUSION: Segmental NF1 is a distinct type of mosaic/somatic NF1 mutation. The phenotype of diffuse and plexiform skin neurofibromas can resemble cutaneous neurofibroma. Surgical therapy for segmental neurofibromatosis does not differ from the concepts for treating nerve sheath tumours in NF1 patients with a germline NF1 mutation.

Expansion of the phenotypic spectrum and description of molecular findings in a cohort of patients with oculocutaneous mosaic RASopathies.

BACKGROUND: Postzygotic KRAS, HRAS, NRAS, and FGFR1 mutations result in a group of mosaic RASopathies characterized by related developmental anomalies in eye, skin, heart, and brain. These oculocutaneous disorders include oculoectodermal syndrome (OES) encephalo-cranio-cutaneous lipomatosis (ECCL), and Schimmelpenning-Feuerstein-Mims syndrome (SFMS). Here, we report the results of the clinical and molecular characterization of a novel cohort of patients with oculocutaneous mosaic RASopathies. METHODS: Two OES, two ECCL, and two SFMS patients were ascertained in the study. In addition, two subjects with unilateral isolated epibulbar dermoids were also enrolled. Molecular analysis included PCR amplification and Sanger sequencing of KRAS, HRAS, NRAS, and FGFR1 genes in DNA obtained from biopsies (skin/epibulbar dermoids), buccal mucosa, and blood leukocytes. Massive parallel sequencing was employed in two cases with low-level mosaicism. RESULTS: In DNA from biopsies, mosaicism for pathogenic variants, including KRAS p.Ala146Thr in two OES subjects, FGFR1 p.Asn546Lys and KRAS p.Ala146Val in ECCL patients, and KRAS p.Gly12Asp in both SFMS patients, was demonstrated. No mutations were shown in DNA from conjunctival lesions in two subjects with isolated epibubar dermoids. CONCLUSION: Our study allowed the expansion of the clinical spectrum of mosaic RASopathies and supports that mosaicism for recurrent mutations in KRAS and FGFR1 is a commonly involved mechanism in these rare oculocutaneous anomalies.