Elsebet Ostergaard
email: elsebet.ostergaard@dadlnet.dk

Mustafa Batbayli

Morten Duno

Kaj Vilhelmsen

Thomas Rosenberg

Mutations in PCDH21 cause autosomal recessive cone-rod dystrophy

Background: Cone-rod dystrophy is a retinal dystrophy with early loss of cone receptors and a parallel or subsequent loss of rod receptors. It may be syndromic, but most forms are nonsyndromic with autosomal dominant, autosomal recessive or X-linked recessive inheritance. Methods and results: We identified a small consanguineous family with six patients with cone-rod dystrophy from the Faroe Islands. Homozygosity mapping revealed a single homozygous locus of 4.2 Mb on chromosome 10q23.1-q23.2, encompassing 11 genes. All patients were homozygous for a 1 bp duplication in PCDH21, c.524dupA, which results in a frameshift and a premature stop codon (p.Q175QfsX47). Conclusion: To our knowledge, this is the first report of mutations in PCDH21 as a cause of human disease. PCDH21 is highly expressed in the retinal photoreceptor cells. It encodes protocadherin 21, which belongs to the cadherin superfamily of large cell surface proteins characterised by a variable number of extracellular cadherin domains. A PCDH21 knockout mouse model has previously shown loss of photoreceptor cells and abnormal cone and rod function, similar to the findings in the patients. Cone-rod dystrophy (CORD, [MIM 120970]) is a group of inherited disorders that belong to the generalized retinal dystrophies. The prevalence of CORD is 1/40.000. CORD is characterised by early loss of cone receptors accompanied by loss of rod receptors, in contrast to retinitis pigmentosa (RP), where the loss of cone photoreceptors occurs secondarily to rod degeneration. The loss of cone photoreceptor cells leads to visual loss, visual field loss, abnormalities of color vision, and variable degrees of photophobia and nystagmus. The loss of rod function leads to night blindness. The onset is usually in late childhood or early adulthood. Fundus examination shows focal macular retinal pigment epithelial (RPE) atrophy or a bull's eye maculopathy in the early stages, and in the later stages peripheral RPE atrophy, retinal hyperpigmentation, arteriolar attenuation, and optic disc pallor occurs. Absent or severely impaired cone function is found on electroretinography (ERG) and psychophysical testing.

 In addition, four loci are known, where the disease-causing gene has not been yet been identified.

 No major bottlenecks are thought to have occurred.[7] A founder effect is responsible for

a high incidence of some disorders in the Faroe Islands, e.g. cystic fibrosis [START_REF] West | The emergence of a nation[END_REF] and mitochondrial encephalomyopathy with elevated methylmalonic acid. [START_REF] Jorgensen | Linkage disequilibrium and demographic history of the isolated population of the Faroe Islands[END_REF]

METHODS

Human subjects

The consanguineous family consists of three males and three females affected with CORD (fig 1A).

Genealogical studies revealed a common ancestor six generations back, born in 1687, of the parents of patients II:4, II:6 and II:10. The Faroese population has been studied extensively for hereditary ophthalmological disorders, but to our knowledge, no other Faroese persons have a diagnosis of CORD.

The study complied with the ethical guidelines of the Declaration of Helsinki. It was approved by the Faroese Ethical Committee and carried out in collaboration with the governmental Genetics Resource Centre of the Faroe Islands. The family gave informed consent for the study.

The pedigree analysis provided strong evidence of autosomal recessive mode of inheritance and all affected persons were assumed homozygous for a mutant allele.

DNA and RNA extraction and cDNA synthesis

DNA was extracted from peripheral blood using standard procedures. RNA was extracted from cultured fibroblasts with the RNAeasy Mini kit (Qiagen, Inc., Valencia, CA) and reverse transcribed to cDNA with the SuperScript II Reverse Transcriptase kit (Invitrogen, Carlsbad, CA).

Microarray analysis

DNA from the six affected patients (II:4, II:6, II:10, IV:1, IV:2 and IV:3) was used for a genomewide search for homozygosity with the Affymetrix GeneChip 50K Xba array (Affymetrix Inc., Santa Clara, CA). In brief, 250 ng of DNA was digested with the restriction enzyme XbaI, mixed with Xba adapters and ligated with the T4 DNA ligase. The ligated DNA was PCR-amplified in four PCR reactions, pooled and purified. The purified PCR product was fragmented with DNase I and end-labelled with biotin. The samples were hybridised to an array for 18 hours in a hybridisation oven. The array was washed, stained and scanned with an Affymetrix GeneChip scanner 3000. Affymetrix software was used to analyse the data, which were exported to an Excel file.

Linkage analysis

Multipoint parametric linkage analysis was performed with the GeneHunter program on the EasyLINKAGE plus platform, version 5.08. [START_REF] Lindner | easyLINKAGE: a PERL script for easy and automated two-/multi-point linkage analyses[END_REF] An autosomal recessive mode of inheritance with complete penetrance was assumed, and the disease allele frequency was set at 0.001.

Mutation analysis

PCR was performed with the Promega kit and the following conditions: 0.2 mM dNTPs, 1 x buffer, 1.5 mM MgCl 2 , 0.5 mM of each primer, 10 ng template and 1.5U polymerase in a total volume of 50 µl. The PCR program was: 94° C for 2 min, 35 cycles of denaturing at 94° C for 30 sec, annealing at 60° C for 30 sec, and extension at 72° C for 30 sec, and a final extension step of 72° C for 7 min. Primer sequences are available on request.

Sequencing was performed with ABI Big Dye Terminator v. 1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, USA). The sequencing reactions were vacuum-purified with the Montage Seq96 Sequencing Reaction Cleanup Kit (Millipore SA, SaintQuentin, France) and analyzed on an ABI 3130xl Gene Analyzer (Applied Biosystems).

The data were analysed with Sequencing Analysis Software Version 5.2 from Applied Biosystems, and mutation screening was performed using Mutation Surveyor v.3.1 Software (SoftGenetics).

RESULTS

Clinical findings

The three oldest affected siblings (patients II:4, II:6 and II:10) reported decreased vision from around age 17 years. In addition to CORD, two of the patients (patients IV:1 and IV:3) had oculocutaneous albinism, which caused nystagmus and reduced visual acuity from early infancy.

The genetic background for their albinism is unknown, but no mutations were found by sequencing TYR and OCA2. Patient IV:2 denied visual difficulty in daylight when examined at the age of 15.

He had, however, always had difficulties with nighttime orientation. Patient II:10 complained of impaired vision at night and of delayed adaptation to sudden changes in light intensity.

Visual acuity varied between 0.67 and 0.02 (Table 1). In patient II:4, large inter-ocular differences in visual acuity were observed due to a large scar in the left macula, which was likely caused by a fetal toxoplasmosis infection. The inter-ocular difference in visual acuity in patient II:10 was explained by a left-sided squint in childhood.

Retinal examination showed both central and peripheral pigment abnormalities (fig 2). The foveal changes consisted of irregular pigmentation and atrophy of the RPE with a horizontal oval-shaped or ill-defined "moth-eaten" appearance. In patient IV:2, the foveal regions had an extensive sheen.

In the two patients with oculocutaneous albinism (patients IV:1 and IV:3), the changes in the foveal regions were less obvious due to the scarcity of pigment. The peripheral pigment disturbances included diffuse RPE atrophy and black hyperpigmentations of polymorphic shape, some of which were filiform and sheeted the vessels. The amount of pigmentation varied considerably among patients of the same age, but showed some correlation to the extent of visual field constriction. In the oldest patients the optic discs looked moderately atrophic and the retinal vessels were universally constricted.

Visual field measurements with a large object showed considerable variation; patient II:4 had only narrow peripheral remnants left in the lower visual fields and a paracentral preserved island of 10°, whereas patients II:6 and II:10 had normal or slightly constricted outer field limits. In patient II:6, a central scotoma measuring 5° was found. The smallest test objects revealed concentric constriction in patients IV:1 and IV: The ERG of patient II:10 showed severely reduced amplitudes and prolonged photopic implicit times (Table 1). Patient IV:1 had normal amplitudes at the age of 2 years but prolonged cone implicit times. Eight years later the ERG showed absent response to low-intensity stimuli and a 50% reduction in the response to high-intensity flash. In patient IV:2, the ERG response was absent at age 17 years.

Patients II:6 and II:10 reported normal smell sense.

Mapping of a gene for CORD to chromosome 10

A frequent cause of CORD is mutations in ABCA4, and we therefore analysed the SNP data for linkage to ABCA4. Assuming autosomal recessive inheritance and a homozygous or compound heterozygous mutation, we did not find evidence of linkage to ABCA4 (Supplementary table 1). Genomewide analysis of SNP data for homozygosity revealed a single homozygous region on chromosome 10q23.1-q23.2 (Supplementary table 2); a significant LOD score of 3.1 was found by multipoint linkage analysis (fig 1B). The linkage interval had a size of 4.2 Mb and was flanked by the heterozygous markers rs6584455 and rs868042 (fig 1C).

The region encompasses 11 genes, of which three were candidate genes: RGR, which had previously been shown to cause autosomal recessive retinitis pigmentosa in humans, LRIT1, which is highly expressed in retina, and PCDH21, where a knockout mouse model had shown loss of cone and rod photoreceptor cells. [START_REF] Morimura | Mutations in RGR, encoding a light-sensitive opsin homologue, in patients with retinitis pigmentosa[END_REF][START_REF] Gomi | Molecular cloning of a novel membrane glycoprotein, pal, specifically expressed in photoreceptor cells of the retina and containing leucine-rich repeat[END_REF][START_REF] Rattner | A photoreceptor-specific cadherin is essential for the structural integrity of the outer segment and for photoreceptor survival[END_REF]

Screening of candidate genes

No mutations were found in RGR or LRIT1. Sequencing of PCDH21 revealed a 1 bp insertion, c.524dupA (fig 1D). The six patients were homozygous for the mutation and 12 family members were heterozygous carriers (fig 1A). Individuals II:1 and II:3 did not carry the mutation; no DNA was available from the remaining family members.

The 1 bp duplication is expected to cause a frameshift resulting in a premature stop codon (p.Q175QfsX47), which was confirmed by analysis of cDNA from fibroblasts (fig 1E).

Screening of 159 anonymous Faroese controls for the mutation revealed three heterozygous carriers, corresponding to a carrier frequency of 1.9%. None of the controls were homozygous for the mutation.

DISCUSSION

To our knowledge, this is the first report of mutations in PCDH21 as a cause of human disease. In a previous study, two heterozygous missense mutations were found in three patients with autosomal recessive RP, but a second mutation was not found, and hence the pathogenicity of the mutations is unclear. [START_REF] Bolz | Protocadherin-21 (PCDH21), a candidate gene for human retinal dystrophies[END_REF] The PCDH21 mutation was identified in a small consanguineous family from the Faroe Islands and a carrier frequency of 1:53 (1.9%) was found. A high carrier frequency is found in other disorders in the Faroe Islands that are caused by founder mutations, e.g. cystic fibrosis (1:24) and mitochondrial encephalomyopathy with elevated methylmalonic acid (1:33). [START_REF] West | The emergence of a nation[END_REF][START_REF] Jorgensen | Linkage disequilibrium and demographic history of the isolated population of the Faroe Islands[END_REF] PCDH21 was identified by Rattner et al. by subtractive hybridization and high throughput screening in a search for genes specifically expressed in the retina. [START_REF] Rattner | A photoreceptor-specific cadherin is essential for the structural integrity of the outer segment and for photoreceptor survival[END_REF] At the same time, Nakajima et al. identified PCDH21 in a search for genes that encode proteins with cadherin domains. [START_REF] Nakajima | Identification of three novel non-classical cadherin genes through comprehensive analysis of large cDNAs[END_REF] PCDH21 is conserved among different vertebrate species, but has not been found in invertebrates. It is a member of the protocadherin family, the largest subgroup within the cadherin superfamily of large cell surface proteins characterised by a variable number of extracellular cadherin domains. [START_REF] Morishita | Structure of the cadherin-related neuronal receptor/protocadherin-alpha first extracellular cadherin domain reveals diversity across cadherin families[END_REF] The cadherins are involved in calcium-dependent cell-cell adhesion and are important in nervous system function.

Cadherin/protocadherin genes have previously been shown to be involved in disorders of the retina: mutations in the protocadherin gene PCDH15 are responsible for Usher syndrome type 1F, and mutations in the cadherin genes CDH23 and CDH3 were shown to cause Usher syndrome type 1D and hypotrichosis with juvenile macular dystrophy, respectively. [START_REF] Ahmed | PCDH15 is expressed in the neurosensory epithelium of the eye and ear and mutant alleles are responsible for both USH1F and DFNB23[END_REF][START_REF] Bolz | Mutation of CDH23, encoding a new member of the cadherin gene family, causes Usher syndrome type 1D[END_REF][START_REF] Sprecher | Hypotrichosis with juvenile macular dystrophy is caused by a mutation in CDH3, encoding P-cadherin[END_REF] PCDH21 has six predicted cadherin repeats and an intracellular domain, which is not homologous to that of other cadherins. PCDH21 is highly expressed the retina, more specifically in the junction between the inner and outer segments (OS) of rod and cone photoreceptors; the expression is also high in olfactory bulbs, with a lower expression in the septum and olfactory cortex. [START_REF] Rattner | A photoreceptor-specific cadherin is essential for the structural integrity of the outer segment and for photoreceptor survival[END_REF][START_REF] Nakajima | Identification of three novel non-classical cadherin genes through comprehensive analysis of large cDNAs[END_REF] PCDH21 expression is very low or absent in heart, kidney, liver, lung, spleen, testis and skeletal muscle. The photoreceptor OS is a derivative of a non-motile cilium that contains phototransduction proteins organized in a stack of over 1,000 disks. The disks are continuously renewed from the base, and the older disks are shedded from the tip. The proteins are synthesized in the inner segment and transported to the OS via the connecting cilium.

PCDH21 undergoes a specific proteolytic cleavage, which releases ectodomain as a soluble fragment. In rds knockout mice, where the OS assembly is disturbed, the proteolytic cleavage is inhibited and full-length PCDH21 accumulates. [START_REF] Rattner | Proteolytic shedding of the extracellular domain of photoreceptor cadherin. Implications for outer segment assembly[END_REF] Mutations in PROM1 have been identified in patients with autosomal recessive RP, and PCDH21 protein has been shown to interact with PROM1 by co-immunoprecipitation studies. [START_REF] Yang | Mutant prominin 1 found in patients with macular degeneration disrupts photoreceptor disk morphogenesis in mice[END_REF] PROM1 is a transmembrane protein associated with plasma membrane protrusions, and it co-localizes with PCDH21 to the base of the photoreceptor OS. In transgenic mice expressing human PROM1 with a heterozygous missense mutation, PROM1 and PCDH21 protein was found throughout the photoreceptor layers, instead of the normal localization at the base of the photoreceptor OS. In Pcdh21 -/-mice, PROM1 mislocalized throughout the OS. These findings suggest that the interaction between PCDH21 and PROM1 is necessary for both proteins to function normally.

Taken together, these findings suggest that PCDH21 is involved in OS structure and/or assembly, but not in phototransduction in rods and cones. This hypothesis is supported by the localization of PCDH21 to the base of the OS at the earliest stages of differentiation of rods, before other OS proteins, e.g. rhodopsin, RP1 and ROM1, are found. [START_REF] Rattner | Proteolytic shedding of the extracellular domain of photoreceptor cadherin. Implications for outer segment assembly[END_REF] A PCDH21 knockout mouse model showed normal viability and fertility without anatomical or behavioural abnormalities. [START_REF] Rattner | A photoreceptor-specific cadherin is essential for the structural integrity of the outer segment and for photoreceptor survival[END_REF] At age 1 month, the retinas appeared normal, but this was followed by a progressive loss of photoreceptors, and a 50% reduction in the number of photoreceptor cells was observed by age 6 months, which likely is the result of apoptosis. Analysis of photoreceptor electrical signalling showed 2-to 3-fold diminished saturating a-wave amplitude, which reflects rod photoreceptor function, in Pcdh21 -/-mice compared to Pcdh21 +/+ and Pcdh21 +/-mice. The saturating amplitude of the cone-driven b-wave response was diminished about 2-fold in Pcdh21 -/-mice.

Electron microscopy showed that in Pcdh21 -/-mice, the OS were shorter with swirls of membranes with imperfectly stacked disks. The inner segments and RPE appeared unaffected. The knockout mouse thus had abnormal cone and rod function, but the dysfunction of rods and cones was not as severe as expected from the degree of disorganization of the OS. The ERG findings and the loss of photoreceptor cells in patients with PCDH21 mutations were similar to those found in the knockout mouse model. Despite the high PCDH21 expression in olfactory bulbs, the patients showed no signs of decreased olfactory function.

The frameshift mutation found in PCHD21, one of three sequenced genes of 11 candidate genes in the linkage interval, the segregation of the mutation with the disease in the family, the high and almost exclusive expression of the gene in the retina, and the similarity of the phenotype of the patients with that of a knockout mouse heavily imply that autosomal recessive cone-rod dystrophy in this family is caused by PCDH21 mutations. Since several of the previously reported genes involved in CORD have been shown to be involved in e.g. Stargardt disease, Leber congenital amaurosis and RP, PCDH21 should be considered a candidate gene for other retinal dystrophies.

2 .

 2 Color vision was severely impaired in the two patients tested (patients II:6 and II:10 at ages 36 and 38 years, respectively). Dark adaptation was tested in two patients (patients II:10 and IV:2 at ages 38 and 15 years, respectively). It showed a monophasic and protracted course, with only a slight elevation of the final threshold of ½-1 log unit in patient II:10 (fig 3).

( 5 -

 5 95%), implicit time median 29.0 msec, range 27.0-34.0 msec (5-95%). Rod low-intensity flash response: b-wave amplitude median 210 μV, range 124-302 μV (5-95%), implicit time median 103.0 msec, range 81.0-123.0 msec (5-95%). Cone-rod high-intensity flash response: a-b wave amplitude median 427 μV, range 285-674 μV (5-95%), implicit time median 48.0 msec, range 41.0-55.0 msec (5-95%).

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. Fundus pictures of the right eye. A. Diffuse atrophy of the retinal pigment epithelium

Figure 3 .

 3 Figure 3. Dark adaptation in patients with CORD due to PCDH21 mutations. The dark adaptation

Table 1 .

 1 Clinical and electrophysiological characteristics of patients with CORD caused by

	PCDH21 mutations.
	Patient Age at	Gender Visual	Refraction	ERG (right	ERG (right	ERG (right
		examination		acuity	(right/left)	eye) cone	eye) rod	eye) cone-rod
		(years)		(right/left)		response a	response a	response a
						(amplitude	b-wave	a-b-wave
						(μV)/	(amplitude	(amplitude
						implicit	(μV)/	(μV)/
						time	implicit time	implicit time
						(msec))	(msec))	(msec))
	II:4	29	M	0.67/	-3.00/-3.00 No response No response No response
				0.02
	II:6	36	F	0.10/0.10 -2.25-1.50 x	10/42	132/80	140/62
					25°/
					-2.50-1.00 x
					0°
	II:10 37	M	0.60/	0.00/	16/40	No response 89/59
				0.10	0.00
	IV:1	2	F	0.08/	+5.50-3.00	44/39	206/112	280/66
				0.08	x 5°/
					+4.50-2.50
					x 170°
	IV:2	17	M	0.16/	0.00/	No response No response No response
				0.67	0.00
	IV:3	½	F	0.20/	+4.50-1.50	55/32	No response 165/56
				0.20	x 0°/
					+4.50-1.50
					x 0°

a ERG normal values. Cone scotopic flicker response: Amplitude median 94 μV, range 53-145 μV

Acknowledgements:

We thank the family for their participation.

Funding: This work was supported by grants from The Danish Association of the Blind and The Danish Medical Research Council.

Electronic database information:

GenBank, http://www.ncbi.nlm.nih.gov/Genbank/ (for PCDH21 [accession number NM_033100]).

Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim (for CORD).

Competing interests: None declared

Patient consent: Obtained

License statement

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in JMG and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence (http://group.bmj.com/products/journals/instructions-for-authors/licence-forms).


Franck Bourdeaut

Aurélie Hérault

David Gentien

Gaëlle Pierron

Stelly Ballet

Stéphanie Reynaud

Régine Paris

Gudrun Schleiermacher

Clarisse Baumann

Pascale Philippe-Chomette

Marion Gauthier-Villars

Michel Peuchmaur

François Radvanyi

Olivier Delattre
email: olivier.delattre@curie.fr

Mosaicism for oncogenic G12D KRAS mutation associated with epidermal nevus, polycystic kidneys and rhabdomyosarcoma

Keywords: mosaicism, KRAS, rhabdomyosarcoma, epidermal nevus

Epidermal nevus (EN) is a congenital disorder characterized by hyperpigmented epidermal thickening following a Blaschko's line. It is due to somatic mutations in either FGFR3 or PIK3CA in half of cases, and remains of unknown genetic origin in the other half.

EN is also observed as part of complex developmental disorders or in association with bladder carcinomas, also related to FGFR3 and PIK3CA mutations. Mosaic mutations of these genes have been occasionally evidenced in syndromic EN. In this report, we describe the cooccurrence of EN, rhabdomyosarcoma, polycystic kidneys and growth retardation in an infant. An oncogenic G12D KRAS mutation was detected in both the epidermal component of the EN and in the rhabodmyosarcoma but neither in the dermal component of the EN lesion nor in unaffected tissues including normal skin or blood. This report identifies for the first time that a KRAS mutation in epiderma causes EN. The observation of the same G12D KRAS mutation in two distinct regions of the body strongly suggests a somatic mosaicism. Finally, this report highlights the potentially underestimated importance of mosaic oncogenes mutations in childhood cancers.

Epidermal naevus (EN) is a congenital benign acanthotic skin tumour that presents at birth as a localized epidermal thickening with hyperpigmentation. It usually follows a Blaschko's line and is always diagnosed within the first days to months of life. Histologic examination reveals acanthosis, hyperkeratosis and papillomatosis. Somatic mutations of the FGFR3 gene are the causative lesion of several benign skin tumours first shown in seborrheic keratosis [START_REF] Logie | Activating mutations of the tyrosine kinase receptor FGFR3 are associated with benign skin tumors in mice and humans[END_REF] and subsequently in EN [START_REF] Hafner | Mosaicism of activating FGFR3 mutations in human skin causes epidermal nevi[END_REF]. Interestingly, FGFR3 mutations identified in EN are mostly R248C substitutions, which were previously reported as fully oncogenic variants in malignant tumors ([3], review in [4]). Activating PIK3CA mutations are also detected in one third of patients, either alone or in association with FGFR3 alterations [5]. Unlike FGFR3, the main PIK3CA variants observed in EN (E545G) differ from those reported in malignant lesions (E545K, E542K or H1047R) and may present a weaker kinase activity (review in [4]).

Finally, no mutation is identified in more than half of the cases, indicating that other causative lesions remain to be identified.

Epidermal nevus can occasionally be associated with complex developmental abnormalities in so called epidermal nevus syndromes [6]. It is also observed in the Proteus syndrome which is related to deleterious PTEN mutations that enhance the PIK3CA activity [7,8]. Germline mutations in PTPN11, KRAS and BRAF resulting in Noonan (NS) and Cardio-facio-cutaneous (CFC) syndromes also predispose to epidermal hyperplasia and acanthosis [START_REF] Niihori | Germline KRAS and BRAF mutations in cardio-faciocutaneous syndrome[END_REF][START_REF] Schubbert | Germline KRAS mutations cause Noonan syndrome[END_REF]. Similarly, epidermal hyperplasia, acanthosis and epidermal nevi are also reported in Costello syndrome (CS) [START_REF] Nguyen | Cutaneous manifestations of Costello syndrome[END_REF], a congenital developmental disorder due to germline HRAS mutations and characterized by cardiac malformation, short stature, failure to thrive, feeding difficulties, facial dysmorphy, and predisposition to cancers such as early rhabdomyosarcomas, neuroblastomas and transitional bladder carcinomas [START_REF] Nguyen | Cutaneous manifestations of Costello syndrome[END_REF][START_REF] Aoki | Germline mutations in HRAS proto-oncogene cause Costello syndrome[END_REF]. Since CS, CFC and NS are all due to a constitutive activation of the RAS-RAF cascade, this signaling, PAGE together with FGFR3 and PIK3CA, most likely plays a major role in epithelium growth control.

We now report on an infant showing a large hyperkeratotic lesion present at birth and following a Blaschko's line, fully consistent with the diagnosis of EN (Figure 1A). The child was born with normal weight, stature and head circumference (2.890g, 49cm and 33cm respectively) after an unremarkable pregnancy. She had no dysmorphy. The biopsy of the epidermal lesion confirmed the diagnosis by showing hyperkeratosis, acanthosis and papillomatosis (Figure 1B). At 6 months of age, the girl developed an utero-vaginal rhabdomyosarcoma with both embryonal and alveolar phenotypes (Figure 1E). Surprisingly, the abdominal CT-san performed at time of the tumor diagnosis revealed bilateral micropolystic kidneys, a feature that was not found in the mother (no data from the father) (Figure 1D). Moreover, the child presented failure to thrive (short stature -2DS, low weight < -2DS) linked to uncommon feeding difficulties that sustained after the end of chemotherapy.

No organic cause of this growth defect was found, in particular the renal function was normal and no sign of intestinal malabsorption was detected. Skeleton X-ray was normal, the echocardiography revealed no malformation. No developmental delay was noticed The unusual association of several clinical abnormalities prompted us to explore further the genetics of the epidermal nevus and the rhabdomyosarcoma. Neither PIK3CA nor FGFR3 mutations were found in the DNA extracted from the nevus biopsy (Sanger method and SNaPshot methods, respectively). Considering that the tumor was of epithelial origin, we microdissected the hyperproliferative epithelium from the dermal tissue by using laser capture-microdissection (Leica system, Figure 2A andC). In order to search for genetic lesion possibly causative for EN, DNA from the epidermal part was amplified by whole genomic amplification (phi29 DNA polymerase, New England Biolabs); array-CGH was then performed, using the oligonucleotide Nimbelgen array-CGH platform. As shown in Figure 1C, no copy number alteration was evidenced.

Despite the presence of an alveolar component, no FKHR translocation was evidenced in the cDNA from the rhabdomyosarcoma. To more thoroughly characterize this atypical mixed tumour, genomic DNA was extracted from the primary biopsy and analyzed by array-CGH. This analysis showed a gain of whole chromosome 8, an abnormality commonly observed in embryonnal rhabdomyosarcomas, and other less common alterations (Figure 1F).

Given the coexistence of four rare symptoms, i.e. rhabdomyosarcoma, epidermal nevus, micropolystic kidneys, and failure to thrive with feeding difficulties, we postulated that the child may bare a single genetic lesion likely to account for the whole clinical phenotype.

Knowing that i) the mutations observed in epidermal nevi lead to a constitutive activation of the RAS-RAF cascade, ii) mutations in the RAS pathway are occasionally detected in rhabdomyosarcomas, and iii) germline mutations in the RAS pathway predispose to both epidermal hyperproliferation and rhabdomyosarcomas, we explored this pathway by gene sequencing. Interestingly enough, whereas no mutation was found in NRAS nor HRAS, an oncogenic G12D KRAS mutation was evidenced in both the rhabdomyosarcoma and the epidermal component of the nevus (Figure 2B and2D). In contrast, no mutation was found in the dermal component of the skin lesion (Figure 2D), nor in a normal skin area; wild-type sequence was also observed in blood lymphocytes, cheeck swab and bone trephine biopsies.

No tissue was available to explore the status of KRAS in the polycystic kidney. To sensitize the detection of the G12D mutation, an allele specific PCR was performed according to previously published methods [START_REF] Case | Mutation of genes affecting the RAS pathway is common in childhood acute lymphoblastic leukemia[END_REF]. No variant was detected in the blood, bone biopsy and normal skin. This report indicates for the first time that oncogenic KRAS mutation causes EN.

Mutations in the RAS pathways are found in approximately 15% of RMS [START_REF] Chen | Mutations of the PTPN11 and RAS genes in rhabdomyosarcoma and pediatric hematological malignancies[END_REF][START_REF] Martinelli | RAS signaling dysregulation in human embryonal Rhabdomyosarcoma[END_REF][START_REF] Stratton | Detection of point mutations in N-ras and K-ras genes of human embryonal rhabdomyosarcomas using oligonucleotide probes and the polymerase chain reaction[END_REF]; hence, the KRAS mutation in the malignant tumour could be more expected. However, most of the mutations involve the NRAS codon 61 and no mutation in KRAS has been observed in the two most recent series of 31 and 30 primary rhabdomyosarcomas [START_REF] Chen | Mutations of the PTPN11 and RAS genes in rhabdomyosarcoma and pediatric hematological malignancies[END_REF][START_REF] Martinelli | RAS signaling dysregulation in human embryonal Rhabdomyosarcoma[END_REF]. Only one mutation in KRAS codon 12 has been reported in the whole literature [START_REF] Stratton | Detection of point mutations in N-ras and K-ras genes of human embryonal rhabdomyosarcomas using oligonucleotide probes and the polymerase chain reaction[END_REF]. Although KRAS mutation has not been involved in polycystic kidney in human, it is noteworthy that, in the mouse, selective hyperexpression of Ras in developing renal tubules results in the development of multiple renal cysts [17]. This experimental observation provides strong support in favor of a causative role of the KRAS mutation in our patient's renal malformation.

Although we can not formally rule out that the two identical KRAS alterations occur by chance, it has to be noted that KRAS mutations are rare in rhabdomyosarcoma, and that not a single G12D KRAS mutation in rhabdomyosarcoma is reported in the COSMIC cancer mutation database. Moreover, the whole clinical phenotype is consistent with a broad hyperactivation of the Ras pathway. Altogether, these data rather suggest a mosaicism than an isolated and independent occurrence of the two mutations. This mutation is present in both mesodermally and ectodermally derived tissues, suggesting that it may have occur relatively early during development. Consistently, previous reports support the hypothesis of oncogenic mosaic mutation being responsible for EN. First, FGFR3 and PIK3CA mutations observed in the abnormal Blaschko's line are systematically absent from the normal skin counterpart; this has for long suggested that EN results from somatic mutations occurring in a metameric pattern during development and leading to a mosaicism [START_REF] Hafner | Mosaicism of activating FGFR3 mutations in human skin causes epidermal nevi[END_REF]5]. Hernandez et al reported several patients baring multiple EN with the same FGFR3 R248C mutation, but without the severe achondroplastic phenotype that is usually associated with R248C germline mutations [18]. Of even greater interest, an undoubtful mosaic FGFR3 mutation has been reported in a patient showing multiple EN, seizures and mental retardation [6]. The higher risk of cancer conferred by mosaic mutations in FGFR3 or PIK3CA is all the more plausible that several cases of young patients harboring both EN and low-grade bladder cancers have been reported (case report and review in [19]). To our knowledge, rhabdomyosarcoma has been associated with EN in only two patients [7,20]. However, as mentioned above, the constitutive activation of RAS pathway, characteristic of CS, predisposes to both rhabdomyosarcomas and hyperproliferative skin disorders [START_REF] Aoki | Germline mutations in HRAS proto-oncogene cause Costello syndrome[END_REF]21]. Noticeably, the patient of our present report shows a clinical phenotype that can be partially related to CS, since growth defect, feeding difficulties and rhabdomyosarcoma are frequent features of CS. On a molecular point of view, the present case could also be related to a mosaic form of NS or CFC, a proportion of which are characterized by germline KRAS mutations and occasionally associated with epidermal naevus. However, the germline mutations reported in CFC and NS almost never involve codons 12, 13 or 61 (review in [22,23]) since only one CFC patient with a KRAS G12S mutation has been reported [24]. Analyses of the functional consequences of KRAS variants responsible for NS and CFC demonstrate their obvious property to hyper-activate the RAS signaling, that is nevertheless less pronounced than the one mediated by the G12D variant [START_REF] Niihori | Germline KRAS and BRAF mutations in cardio-faciocutaneous syndrome[END_REF][START_REF] Schubbert | Germline KRAS mutations cause Noonan syndrome[END_REF]. Of note, ubiquitous expression of G12D K-ras mutant in mice leads to early embryonic lethality [25]. Altogether, KRAS activating germline mutations responsible for NS and CFC might remain viable because KRAS activity is lower than that of G12D variant. The present report suggests that widespread KRAS G12D mutation remains compatible with life provided nevertheless that it is restricted to a specific mosaic pattern.

Segmental Neurofibromatosis type 1 is a well-known cancer-prone developmental disorder due to a mosaic NF1 mutation that leads to a RAS hyperactivation in a specific pattern of tissues. Likewise, mosaicism in HRAS has been described in Costello patients [26].

Our present report describes a comparable genetic abnormality in the RAS pathway that PAGE results in a new developmental disorder with cancer predisposition. This observation of segmental G12D KRAS mutation highlights the potentially underestimated importance of oncogenes mosaic mutations in childhood cancers.

PAGE

Legend to figures

Figure 1 :

 1 Figure 1: Phenotype and copy number analysis of the two tumours:

Figure 2 :

 2 Figure 2: G12D KRAS mutation in the epidermal component of the lesion.

  Stabley DL, Nicholson L , et al. Somatic mosaicism for an HRAS mutation causes Costello syndrome. Am J Med Genet A 2006;140(20):2163-9.

	21 The Corresponding Author has the right to grant on behalf of all authors and Gripp KW. Tumor predisposition in Costello syndrome. Am J Med Genet C Semin Med Genet
	does	2005;137C(1):72-7. grant on behalf	of	all	authors,	an	exclusive	licence	(or	non	exclusive	for
	22 government Aoki Y, Niihori T, Narumi Y, et al. The RAS/MAPK syndromes: novel roles of the RAS pathway employees) on a worldwide basis to the BMJ Publishing Group Ltd to
	permit	in human genetic disorders. Hum Mutat 2008;29(8):992-1006. this article (if accepted) to be published in JMG	and	any	other	BMJPGL
	23 products Gelb BD, Tartaglia M. Noonan syndrome and related disorders: dysregulated RAS-mitogen and sublicences such use and exploit all subsidiary rights, as set out
	activated protein kinase signal transduction. Hum Mol Genet 2006;15 Spec No 2:R220-6. in our licence.
	24	Nava C, Hanna N, Michot C, et al. Cardio-facio-cutaneous and Noonan syndromes due to
		mutations in the RAS/MAPK signalling pathway: genotype-phenotype relationships and
	overlap with Costello syndrome. J Med Genet 2007;44(12):763-71. Competing Interest: None declared
	25	Tuveson DA, Shaw AT, Willis NA, et al. Endogenous oncogenic K-ras(G12D) stimulates
		proliferation	and	widespread	neoplastic	and	developmental	defects.	Cancer	Cell
		2004;5(4):375-87.
	19 20 26	Fibroblast growth factor receptor 3 mutations in low grade bladder tumors. J Invest Dermatol Garcia de Jalon A, Azua-Romeo J, Trivez MA, et al. Epidermal naevus syndrome (Solomon's epidermal nevi and associated 2007;127(7):1664-6. syndrome) associated with bladder cancer in a 20-year-old female. Scand J Urol Nephrol 2004;38(1):85-7. Rongioletti F, Rebora A. Epidermal nevus with transitional cell carcinomas of the urinary tract. J Am Acad Dermatol 1991;25(5 Pt 1):856-8. PAGE 14 Gripp KW, PAGE 15

Acknowledgement: we thank Dr Daniel Orbach, Dr Françoise Boman, Pr Jean-François Mercier, Pr Jean-Pierre Hugo for their kind help in the care of the child and for providing the biological samples. We thank Dr Helene Cave for her kind advice in reviewing the manuscript. This work was supported by grants from the INCa (plateforme de génétique moléculaire des cancers) and the Ligue Nationale Contre le Cancer (Equipes labellisées: FB, OD and AH, FR).

.