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

Abstract 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.

In order to expand the knowledge on the clinical and genetic features of mosaic RASopathies, we describe here the phenotypic and molecular characteristics of a cohort of Mexican patients with a clinical diagnosis of these disorders. Additionally, two patients with unilateral isolated epibulbar dermoid were also genotyped for the search of somatic mutations in Ras/MAPK signaling pathway genes. Our study allowed the expansion of the clinical spectrum of mosaic RASopathies and supports that somatic mosaicism for recurrent mutations in KRAS and FGFR1 is a commonly involved mechanism in these rare oculocutaneous developmental anomalies.

| Patients
The study was approved by local Institutional Review Board and Ethics committees and was conducted according to the principles expressed in the Declaration of Helsinki. Parents of patients signed a written informed consent for clinical and molecular studies and for tissue biopsies.
Six unrelated patients with suspected oculocutaneous somatic mosaicism were included in the study. All of them had a complete ophthalmologic and dermatologic examination. When possible, imaging studies such as cranial computed tomography and magnetic resonance imaging, ocular ecography, echocardiogram, and abdominal ultrasonography were performed. In addition, two patients with a diagnosis of isolated unilateral epibulbar dermoids were enrolled in the study.
For patient #1 and #2, small biopsies were obtained from aplasia cutis lesions on the head and from hyperpigmented skin regions on the neck. A biopsy of a nevus psiloliparus was obtained in patient #3 while biopsies from sebaceous nevi from patients #4 and #5 were obtained. Patients #6-8 underwent biopsies from epibulbar dermoids. In all cases, tissue samples were used for DNA isolation. In addition, peripheral leukocytes and buccal mucosa cells were employed | 3 of 12 CHACON-CAMACHO et Al. for DNA isolation in all six patients (#1-6) with syndromic entities.

| Molecular analysis
Tubes containing skin biopsies were placed in a TissueLyser Adapter Set for disruption and homogenization of tissues, following the manufacturer's recommendations (TissueLyser II system, Qiagen, Hilden, Germany). Genomic DNA from biopsy tissues, peripheral blood leukocytes, and buccal mucosa cells was isolated using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), and Gentra Puregene Buccal Cell Kit (Qiagen, Hilden, Germany), following the manufacturer´s instructions. The coding regions of KRAS, HRAS, NRAS, and FGFR1 genes and their adjacent intronic sequences were amplified by PCR using pairs of primers corresponding to Ensembl reference sequences (KRAS, ENST00000311936.7; HRAS, ENST00000451590.5; NRAS, ENST00000369535.4; FGFR1, ENST00000447712.6). Direct automated sequencing of exons of KRAS, HRAS, NRAS, and FGFR1 was performed with the BigDye Terminator 3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA). All samples were analyzed either on an ABI 3130 or 3500xl Genetic Analyzer (Applied Biosystems) and sequences were compared against the respective reference sequences. The mosaicism level of the identified variants was estimated by comparing the area under the curve of electropherograms for wildtype and mutant peaks using the Mutation Surveyor software, as previously reported (Minton, Flanagan, & Ellard, 2011).
In patients #3 and #6, massive parallel sequencing was applied using a TruSeq Custom Amplicon Low Input Library Prep Kit (Illumina) as recommended by the supplier for DNA enrichment. Massive sequencing was performed on a nextgeneration sequencing platform (MiSeq System, Illumina). The target regions included hotspots for mosaic mutations in the genes KRAS, HRAS, NRAS, FGFR1-3, among others. Target amplicons showed a read depth between 1,000 and 5,000 reads. Sequence data were analyzed by Variant Studio Data Analysis Software 3.0 (Illumina). Application of this procedure achieved a detection limit as low as 1% for mutation hot spots.

| Patient #1
A 4-year-old girl was referred due to a congenital anomaly in her left eye and was clinically diagnosed with OES. She was the third child of unrelated, healthy parents and her family history was uneventful. Her mother did not receive prenatal care but denied exposure to teratogenic agents. The proband was born at 41 weeks of gestation via spontaneous vaginal delivery and her birth length was 46 cm (3-10th centile), weight 2,700 g (10-25th centile), and Apgar scores of 7/8. Psychomotor development was normal. At birth, a left eye conjunctival mass and scalp lesions (Figure 1-a1-3) were noted. At present examination, she exhibited left parietal alopecia and two areas of aplasia cutis near to vertex capitis ( Figure 1-a1,2). Inspection of the left eye revealed a subconjunctival temporal tissue mass extending to the corneal limbus and temporal cornea, with no apparent interference with the visual axis (Figure 1-a3). On funduscopic examination, optic nerve excavations of 50% and 80% in right and left eyes, respectively, were observed. In addition, bilateral epicanthal folds, downslating palpebral fissures, low-set ears, broad nasal tip and bridge, downturned corners of the mouth, and left facial hemihyperplasia were also seen. Skin examination disclosed hyperpigmented lesions on the left side to the body, spreading-out over the left hemimandible ( Figure  1-a4) and zygomatic region, left side of the neck, and downward following the middle line involving the suprasternal notch, upper back, and left arm. Left polythelia (Figure 1-a5) and hypertrichosis were also evident. Echocardiogram evidenced atrial septal defect (ASD) and persistent ductus arteriosus (PDA) ( Table 1). Cranial computed tomography did not demonstrate brain anomalies. No neurological symptoms were present.

| Patient #2
A 12-year-old male was clinically diagnosed with OES. He is the fifth child of healthy, nonconsanguineous parents and was referred due to bilateral epibulbar dermoids. All of his living siblings were healthy and his father had a history of scapular lipectomy for cutaneous lipomatosis. At birth he had a weight of 4,475 g (>97th centile), length of 51 cm (25-50th centile), and Apgar scores of 8/9. Examination at the age of 11 months disclosed bilateral epibulbar dermoids, microcornea, endotropia, and nystagmus. The visual axes were involved by dermoid tissue extension to both corneas. Cardiovascular examination and electrocardiogram revealed hypertrophic cardiomyopathy at 4 years of age. At present examination, several somatic anomalies were evident including frontal bossing, aplasia cutis verticis of 1 × 1 cm (Figure 1-b1

| Patient #3
This 6-month-old boy exhibited a round and soft mass of 4 × 2 cms located at the right zygomatic and conjunctival areas and was clinically diagnosed with ECCL. The propositus is the second child of nonconsanguineous, healthy parents. His mother received regular prenatal care and was not exposed to teratogenic agents. He was born by spontaneous vaginal delivery and his birth length was 43 cm (<3rd centile), and weight 2,045 g (<3rd centile). He exhibited alopecia in the anterior midline, parietal, and temporal areas as well as a smooth hairless fatty nevus (nevus psiloliparus) at the right zygomatic and temporo-parietal regions ( Figure  1-c1,3). Two small nodular skin tags outside of the outer canthus and a left conjuctival lesion suggestive of choristoma were also identified. Ocular ultrasound demonstrated right optic nerve coloboma (Figure 1-c2). No other anomalies were found. No central nervous system imagenologic studies were carried out. At present, no neurological anomalies were present in this patient.

| Patient #4
This is a 2-year-old male referred due to cutaneous sebaceous lesions, epilepsy, and left eye leucoma. He was clinically diagnosed with SFMS. He is the second child of consanguineous (first cousin), healthy parents and was born at term by cesarean section with a birth length of 51 cm (25-50th centile), weight 4,100 g (75th centile), and Apgar score of 6/7. Tonic-clonic convulsions developed at 20 days of age. His examination demonstrated absence of hair in frontal midline and left parietal areas. Two large oval orange-yellowish nevus sebaceous lesions were observed in the forehead and left parietal region ( Figure  1-d1,2). Other patches were noted on the left cheek and left region of the upper lip (Figure 1-d3). A linear sebaceous lesion was seen over the nose tip whereas a temporal bulbar dermoid was observed in the left eye. Wide nasal bridge and anteverted nares were also noted. Ocular ecography showed left optic nerve coloboma while auditory evoked potentials identified bilateral hearing loss. A cranial computed tomography demonstrated cortical atrophy, interhemispheric asymmetry, left polymicrogyria, and ventriculomegaly.

| Patient #5
A 12-years-old female who was evaluated due to a sebaceous nevus on the head received a clinical diagnosis of SFMS. She was the third child of a nonconsanguineous and healthy couple. Her mother received adequate prenatal care and had no exposure to teratogenic agents. Her birth measurements were not provided but were reported as normal.
A biopsy of the nevus sebaceous lesion was performed at one year of age but no results were provided to the family. At present examination, she exhibited a linear nevus sebaceous at the left parotid, temporal, and parietal regions (4 × 12 cm) (Figure 1-e1), which extended to a round aplasia cutis lesion (1.5 × 1.5 cm) in the left parietal area (Figure 1-e1). An epibulbar dermoid was present in the left eye (Figure 1-e2). Skin hyperpigmentation was observed in the left frontal region (8 × 3 cm) (Figure 1-e2), around the mouth (Figure 1-e3), in both upper and lower gingival mucosa (Figure 1-e4,5), and in the tip of the tongue (Figure 1-e6). Additional café-au-lait spots were present through her body while multiple hyperpigmentated nevi were noted around the mouth (Figure 1-e3,4). Cranial computed tomography evidenced a left temporal arachnoid cyst (Figure 1-e7), hemimegalencephaly (Figure 1-e9), and left eye retinal coloboma (Figure 1-e8). Despite her brain anomalies, she was free of neurological symptoms at present examination.

| Patient #6
This is a 10 months-old male who was seen due to alopecia and epibulbar dermoid in the right eye. He was the second child of consanguineous and healthy parents. His mother received regular prenatal care and was not exposed to teratogenic agents. He was born at term by cesarean section and his birth length was 50 cm (50th centile), weight 3,500 g (50th centile) and Apgar score of 6/7. His examination revealed brachycephaly, focal alopecia in the right parietal area, and a right upper lid small nodular skin tag. An epibulbar dermoid expanding to right cornea was present. In addition, focal skin hypoplasia in the right zygomatic area, which extended to the temporal and preauricular regions was observed. Linear hyperpigmentation on the right lateral neck and on the cervical back were also identified. Right cryptorchidism was present. MR disclosed arachnoid cyst in right temporal region as well as right cortical atrophy. No neurological manifestations were present.

| Patients #7 and #8
These subjects were a boy aged 2 years and a girl aged 14 years, both exhibiting unilateral isolated epibulbar dermoids. No systemic features or neurological symptoms were identified in them and their family history was negative for congenital anomalies. Biopsy of dermoid tissue was performed during ocular surgery in both patients.

| Molecular results
No pathogenic variants in the KRAS, NRAS, HRAS, and FGFR1 genes were identified by Sanger sequencing in DNA isolated from blood leukocytes or buccal cells from any subject. However, different levels of mosaicism for specific mutations were demonstrated in DNA obtained from biopsies of skin lesions or epibulbar dermoids (Table 1)  2012; Peacock et al., 2015). OES is a very rare disease characterized for a consistent combination of scalp lesions and epibulbar dermoids (Toriello, Lacassie, Droste, & Higgins, 1993). Aplasia cutis congenita is the most frequent skin finding and other body structures are commonly involved, including central nervous system (arachnoid cyst, dilatation of ventricules) (Gardner & Viljoen, 1994;Martin, Lockspieler, & Slavotinek, 2007), bones (bone cyst) (Toriello et al., 1999), urogenital tract (bladder exstrophy, epispadias, bladder rhabdomyosarcoma) (Lees, Taylor, Atherton, & Reardon, 2000), cardiovascular system (aortic coarctation, Moyamoya disease) (Horev, Lees, Anteby, Gomori, & Ben-Neriah, 2011;Toriello et al., 1993). In our series, patients #1 and #2 received a clinical diagnosis of OES as they exhibited aplasia cutis and epibulbar dermoids, cardinal features of the disease. Of note, patient #1 presented with some features rarely reported in OES as facial asymmetry (Martin et al., 2007), glaucoma (Federici et al., 2004), lower limb lymphedema (Horev et al., 2011), ASD (Toriello et al., 1993), and PDA. Our data support that these anomalies can be considered as part of the disorder rather than coincidental findings in previous patients. Interestingly, ASD and PDA were both present in our patient #1, a previously undescribed coexistence. Similarly, anomalies as left hemihypertrophy and supernumerary nipples observed in patient #1, and short neck, microcornea, nystagmus, and hypertrophic cardiomyopathy, occurring in patient #2, have not been previously observed in OES (Table  1), expanding thus the disease's clinical spectrum. The genetic etiology of OES was recently identified through comparative whole-genome sequencing in DNA from a variety of tissues of unrelated affected individuals (Boppudi et al., 2016;Peacock et al., 2015). Using this approach, KRAS somatic mutations were demonstrated, including c.38G>A (p.Gly13Asp) and c.57G>C (p.Leu19Phe) (Peacock et al., 2015). More recently, three additional patients with OES were shown to carry the KRAS codon 146 variants c.436G>A (p.Ala146Thr) and c.437C>T (p.Ala146Val) (Boppudi et al., 2016). In the present work, we molecularly examined two OES patients (#1 and #2) and identified the two previously reported KRAS codon 146 mutations. Such pathogenic variants were firstly reported as cancer-associated somatic mutations, particularly in colorectal cancer (CRC), but have been also found in other malignancies such as multiple myeloma, pancreatic ductal adenocarcinoma, and non-small cell lung cancer (Feig & Cooper, 1988 ECCL is a distinct uncommon oculo-neuro-cutaneous disease with a prevalence of <1/1,000,000 (https://www.orpha. net/consor/cgi-bin/OC_Exp.php?Lng=EN&Expert=2396) which was first described by Haberlans and Perou in a subject with severe mental retardation, epilepsy, unilateral choristoma, and cutaneous lesions, and central nervous system abnormalities (Haberland & Perou, 1970). ECCL exhibits an extensive and variable clinical spectrum, but cardinal major and minor criteria have been already defined (Hunter, 2006;Moog, 2009). In our patient #3, an ECCL diagnosis was supported by the involvement of two systems classified with major criteria (eye choristoma and nevus psiloliparus) and at least one minor skin criteria (skin tags); in the patient #6, the diagnosis was supported by the involvement of three systems, with major criteria in ≥2 (choristoma, small skin tag, skin hypoplasia, arachnoid cyst, and cortical atrophy). Nevus psiloliparus and choristomas are observed in up to 80% of ECCL patients, whereas skin tags are another common sign that can be identified in up to 70% of cases (Moog, 2009). Optic nerve coloboma, identified in the right eye of our ECCL patient (#6), has been previously reported in at least two subjects with the disease (Chandravanhi, 2014;Hunter, 2006). Somatic FGFR1 and KRAS mutations were recently demonstrated in subjects with ECCL (Bennett et al., 2016;Boppudi et al., 2016). FGFRs belong to the RTKs (receptor tyrosine kinases) family of proteins which regulate a range of functions including cell growth differentiation/proliferation, tissue patterning, and organogenesis (Dorey & Amaya, 2010). The c.1638C>A (p.Asn546Lys) FGFR1 mutation has been recurrently identified in ECCL cases (Bennett et al., 2016) while one patient with clinical criteria for both OES and ECCL was found to carry the c.436G>A (p.Ala146Thr) KRAS mutation (Boppudi et al., 2016). Thus, the identification of the FGFR1 p.Asn546Lys variant in our ECCL patient #3 confirms this missense change as the major cause of ECCL. On the other hand, the c.437A>G (p.Ala146Val) KRAS variant detected in our ECCL patient #6 is a new mutation for ECCL, although other point mutations at the same codon has been previously associated with the disease (Boppudi et al., 2016). In 1895, Jadassohn first described a patient with a congenital cutaneous lesion of linear distribution, which was initially classified as naevi sebacei, and subsequently as nevus sebaceous of Jadassohn (Jadassohn, 1895;Robinson, 1932). In 1957, Schimmelpenning recognized the association of such cutaneous lesion with brain, eye, and skeletal anomalies (Schimmelpenning, 1957), and shortly after Feuerstein and Mims reported two cases with the clinical triad of linear nevus sebaceous with epilepsy and mental retardation (Feuerstein & Mims, 1962). The estimated incidence of nevus sebaceous of Jadassohn is around 1-3 per 1,000 live birth, but its incidence is unknown when it is associated with systemic abnormalities as in the SFMS (Warrenburg, Gulik, Renier, Lammens, & Doelman, 1998). Up to 60% of SFMS patients have brain abnormalities, particularly hemimegalencephly, ventriculomegaly, and cortical atrophy (Lantis, Leyden, Thew, & Heaton, 1968;Lin & Yan, 2010). Consistently, in our SFMS patients #4 and #5 brain defects were demonstrated, including uncommonly reported anomalies such as polymicrogyria (Clancy et al., 1985) or arachnoid cyst (Kamate Dumale & Hattiholi, 2009). Contrarily, intellectual disability, a finding in up to 70% of SFMS cases (Warrenburg et al., 1998), was absent in our two SFMS probands. Eye abnormalities can be recognized in ~60% of patients with SMFS and both of our SFMS patients exhibited eye choristoma, which is the most commonly identified ocular anomaly; optic nerve coloboma, a defect observed only in 7% of patients with this syndrome (Arevalo, Lasave, Arevalo, & Shields, 2013;Paulidis, Cantalupo, Boria, Cossu, & Pisani, 2012;Shields, Shields, Eagle, Arevalo, & DePotter, 1997), was also observed in our patients. Bilateral hearing loss, identified in SFMS patient #4, has been previously described unilaterally in a single subject (Gowdar, Nyamagoudar, & Chezhian, 2015). Our SFMS patients exhibited the linear cutaneous lesions on the scalp and face, which are the most common localization (Eisen & Michael2009). Interestingly, our SFMS patient #5 had a pattern of mucocutaneous hyperpigmentation reminiscent of that observed in Peutz-Jeghers syndrome patients (Beggs et al., 2010).
In conclusion, we have studied a cohort on Mexican patients with oculocutaneous mosaic RASopathies. Our results support the inclusion of molecular analysis of KRAS and FGFR1 genes as part of major criteria of OES, ECCL, and SFMS. The knowledge of the molecular cause has important implications for prognosis and therapy in patients suffering from such mosaic developmental syndromes.