Diversity, Parental Germline Origin, and
Phenotypic Spectrum of De Novo HRAS Missense
Changes in Costello Syndrome
Giuseppe Zampino, Francesca Pantaleoni, Claudio Carta, Gilda Cobellis, Isabella Vasta, Cinzia Neri, Edgar A. Pogna, Emma De Feo, Angelica Delogu, Anna Sarkozy, Francesca Atzeri, Angelo Selicorni, Katherine A. Rauen, Cheryl S. Cytrynbaum, Rosanna Weksberg, Bruno Dallapiccola, Andrea Ballabio, Bruce D. Gelb, Giovanni Neri, and Marco Tartaglia
Communicated by Nancy B. Spinner
Activating mutations in v-Ha-ras Harvey rat sarcoma viral oncogene homolog (HRAS) have recently been identified as the molecular cause underlying Costello syndrome (CS). To further investigate the phenotypic spectrum associated with germline HRAS mutations and characterize their molecular diversity, subjects with a diagnosis of CS (N=9), Noonan syndrome (NS; N=36), cardiofaciocutaneous syndrome (CFCS; N = 4), or with a phenotype suggestive of these conditions but without a definitive diagnosis (N=12) were screened for the entire coding sequence of the gene. A de novo heterozygous HRAS change was detected in all the subjects diagnosed with CS, while no lesion was observed with any of the other phenotypes. While eight cases shared the recurrent c.34G ? A change, a novel c.436G ? A transition was observed in one individual. The latter affected residue, p.Ala146, which contributes to guanosine triphosphate (GTP)/guanosine diphosphate (GDP) binding, defining a novel class of activating HRAS lesions that perturb development. Clinical characterization indicated that p.Gly12Ser was associated with a homogeneous phenotype. By analyzing the genomic region flanking the HRAS mutations, we traced the parental origin of lesions in nine informative families and demonstrated that de novo mutations were inherited from the father in all cases.We noted an advanced age at conception in unaffected fathers transmitting the mutation. Hum Mutat 28(3), 265�272, 2007. Published 2006 Wiley-Liss, Inc.y
KEY WORDS: HRAS; Costello syndrome; mutation analysis; parental origin of de novo mutations
INTRODUCTION
Costello syndrome is the eponymous name for the disorder originally described in 1971, and further delineated by the same author in 1977, as a condition characterized by prenatal overgrowth followed by postnatal feeding difficulties and severe failure to thrive, distinctive ��coarse�� facial features, mental retardation, short stature, cardiac defects (most commonly hypertrophic cardiomyopathy, septal defects, valve thickening and/or dysplasia, and arrhythmias), and musculoskeletal and skin abnormalities. Children and young adults with CS are predisposed to malignancies. Indeed, solid tumors, including rhabdomyosarcoma and less frequently neuroblastoma and bladder carcinoma, have been estimated to occur in approximately 15% of affected individuals throughout childhood, while benign cutaneous papillomata in the perinasal or/and perianal region(s) represent a distinctive and common feature associated with the disorder.
By using a candidate gene approach, Aoki et al demonstrated that germline heterozygous missense mutations in the v-Ha-ras Harvey rat sarcoma viral oncogene homolog (HRAS) protooncogene (MIM] 190020) cause CS. According to the available data, activating HRAS mutations have been identified in approximately 85% of subjects with a clinical diagnosis of CS. Due to the severely reduced fitness associated with the disorder, CS almost invariably arises sporadically from a de novo mutation. As observed for other autosomal dominant disorders, advanced paternal age and increased paternal�maternal age difference has been documented in CS suggesting that paternal germline mutations are the origin of disease.
The diagnosis of CS can be difficult and uncertain in the neonatal and infancy periods due to phenotypic overlap with cardiofaciocutaneous syndrome (CFCS; MIM] 115150) and Noonan syndrome (NS; MIM] 163950). CFCS and NS are genetically heterogeneous dominant traits, the former caused by mutations in the KRAS (MIM] 190070), BRAF (MIM] 164757), MEK1 (MIM] 176872), or MEK2 (MIM] 601263) genes in the majority of cases and the latter resulting from mutations in the PTPN11 gene (MIM] 176876) in approximately 50% of affected individuals and KRAS mutations in a small percentage of subjects exhibiting a severe phenotype. All of these disease-causing genes encode for proteins functioning as signaling transducers with positive modulatory roles in RAS signaling.
For this report, we screened cohorts including subjects with CS, NS, CFCS, or clinical features suggestive of these disorders but without a definitive diagnosis for the entire coding sequence of HRAS to investigate further the diversity of germline HRAS mutations, evaluate possible contribution of mutations in HRAS to the pathogenesis of disorders clinically related to CS, and provide a detailed portrait of the phenotype associated with HRAS lesions. By analyzing genomic regions flanking the de novo HRAS mutations causing CS, we also determined the parental origin of mutations in nine informative families.
MATERIALSANDMETHODS
Clinical Evaluation
Three cohorts including subjects with CS, NS, and CFCS were included in the study. Within groups, clinical features satisfied diagnostic criteria for each disorder (see below). A fourth clinically heterogeneous group (N512) included subjects who exhibited a phenotype suggestive of CS or NS but lacking sufficient signs for a definitive diagnosis. In some of these subjects features overlapped those of CFCS. Subjects were examined by clinicians experienced with these disorders (G.Z., A.S., R.W., and G.N.). CS, NS, and CFCS were diagnosed according to Hennekam [2003], van der Burgt et al. [1994], and Kavamura et al. [2002], respectively. Among the subjects grouped in the CS cohort, Cases CS-01 to CS-08 were evaluated in the Birth Defects Unit of the Pediatric Department of the Catholic University, Rome, and were systematically characterized by a detailed historical and clinical schedule, including evaluation of:
1) growth parameters, nutritional history through a diet diary, and growth hormone secretion with a provocative arginine test;
2) neurological history and examination, including electroencephalography (EEG), neuroradiologic imaging (MRI) and a complete neuropsychological profile by applying the age-appropriate development scale;
3) cardiologic status (echocardiogram, electrocardiogram [ECG], and 24-hr Holter recording);
4) sleep habits investigated with a sleep diary and nighttime polysomnographic recordings;
5) orthopedic and physiatric evaluation, including radiological investigation; and
6) ophthalmologic and otorhinolaryngologic evaluations.
Cases CS-01 and CS-04 had previously been described by Zampino et al. [1993]. Subject CS-32 was evaluated in the Ambulatorio Genetica Clinica of the Clinica Pediatrica de Marchi, Milan. Mutations in the coding sequences of the PTPN11 and KRAS genes had been excluded in all subjects included in the study. Moreover, no BRAF (exons 6, 11, 12, 14, and 15), MEK1 (exons 2 and 3), and MEK2 (exon 2) mutation was identified among the four subjects diagnosed with CFCS (G.Z. and M.T., unpublished results).
To investigate the parental germline origin of de novo HRAS mutations (see below) eight additional subjects with CS and a de novo c.34G4A substitution (Cases CS-10 to CS-12, CS16, CS19, CS29, CS-30, and CS-31) were also included in the study. Their clinical data were not available for this report. Cases CS-10 and CS-11, and Case CS-12 were seen in the Division of Clinical and Metabolic Genetics of the Hospital for Sick Children, Toronto, and Ambulatorio Genetica Clinica, Clinica Pediatrica de Marchi, Milan, respectively. Cases CS-30 and CS-31 were evaluated in the Birth Defects Unit of the Pediatric Department of the Catholic University, Rome, and Mendel Institute, Rome, while Cases CS16, CS19, and CS29 had previously been reported by Estep et al. [2006]. Informed consent was obtained for all subjects included in the study.
MolecularAnalyses
Genomic DNAs were isolated from peripheral blood leukocytes. For a few cases, DNA was also obtained from buccal brushings, hair bulbs and/or cultured fibroblasts. DNA extraction was performed according to standard methods. The entire HRAS coding region (exons 2, 3, 4, 5, and 6; GenBank accession numbers NM_005343.2, NM_176795.2, and NT_035113.6) was screened for mutations. PCR reactions were carried out in a 25-ml reaction volume containing 50�80 ng genomic DNA, 1U AmpliTaq Gold (Applied Biosystems, Foster City, CA; www.appliedbiosystems. com), 20 pmol of each primer (MWG-Biotech, Ebersberg, Germany; www.mwg-biotech.com/), 1.5mM MgCl2, 75mM each dNTP, 5% dimethylsulfoxide (DMSO), and 1 PCR Buffer II (Applied Biosystems, Foster City, CA), using a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA). Cycling parameters were as follows: 941C, 8 min (first denaturing step); 941C, 45 sec; 60�621C, 30 sec; 721C, 45 sec; 33 cycles; 721C, 10 min (last extension step). Primer pairs were designed to amplify exons, exon/intron boundaries, and short intron flanking stretches, and are listed in Supplementary Table S1 (available online at http://www.interscience.wiley.com/jpages/1059-7794/suppmat). Mutation analysis of the amplimers was carried out by denaturing high performance liquid chromatography (DHPLC) using the Wave 2100 System (Transgenomic, Omaha, NE; www.transgenomic. com/) at column temperatures recommended by the Navigator version 1.5.4.23 software (Transgenomic, Omaha, NE). Amplimers having abnormal denaturing profiles were purified (Microcon PCR, Millipore, Billerica, MA; www.millipore.com) and sequenced bidirectionally using the ABI BigDye terminator Sequencing Kit v.1.1 (Applied Biosystems, Foster City, CA) and an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). Mutation numbering was in reference to the A of the ATG translation initiation codon in the reference cDNA sequence.
Molecular graphics was performed using the University of California, San Francisco (UCSF) Chimera software (www.cgl. ucsf.edu/chimera) [Pettersen et al., 2004].
Primer pairs utilized to amplify the genomic portions flanking the disease causative mutations are listed in Supplementary Table S1, together with annealing temperatures and genomic location and size of PCR products. Occurrence of variation within these fragments was evaluated by DHPLC and direct sequencing. Position of polymorphic sites was in reference to nucleotide 474671 of the reference genomic sequence (NT_035113.6).
Genotyping of parental DNAs was performed by direct sequencing, while determination of haplotypes in affected individuals was attained by amplification and cloning (TA Cloning Kit, Invitrogen, Carlsbad, CA; www.invitrogen.com/) of the genomic fragments encompassing the exonic mutation and the flanking polymorphic site. Parental sex assignment was confirmed in all informative families by amelogenin gene (AMELX and AMELY) amplification, as previously described. Paternity was verified in all families by short tandem repeat (STR) genotyping, using the AmpDESTER Profiler Plus (Applied Biosystem, Foster City, CA).
RESULTS
HRAS Mutation Analysis
DHPLC screening of the entire HRAS coding sequence on peripheral blood leukocyte genomic DNA specimens allowed the identification of a heterozygous missense mutation in all the subjects diagnosed with CS. While the recurrent c.34G?A change (p.Gly12Ser) was observed in eight subjects, a novel c.436G?A transition, predicting the substitution of an alanine residue at codon 146 by threonine, was documented in Case CS-32. Genotyping of parental DNAs demonstrated the de novo origin of mutation in all cases. Genotyping of markers D3S1258, D5S818, D7S820, D8S1179, D13S317, D18S51, and D21S11 proved paternity in all families. In four of the nine subjects (Cases CS-01, CS-03, CS-05, and CS-06), fibroblast DNA was also available for molecular analysis; the c.34G?A mutation was present in each, supporting their germline origin.
To explore the effects of the p.Ala146Thr substitution on protein function, we exploited the crystallographic information available for HRAS. p.Ala146 is located within the G3 motif and is totally conserved in the RAS superfamily [Mitin et al., 2005]. According to the three dimensional structure of guanosine diphosphate (GDP)- or guanosine triphosphate (GTP)-bound HRAS (Protein Data Bank codes: GDP-HRAS, 4Q21; GTPHRAS, 5P21; www.wwpdb.org), this residue is located in a hydrophobic pocket involved in binding to the purine ring of GTP/GDP (Fig. 1A), and contributes to this bonding network by interacting directly with the guanine base [Wittinghofer and Waldmann, 2000]. Of note, a missense mutations (c.350A4G), affecting p.Lys117, which is also involved in binding to the GTP/ GDP purine ring, has recently documented in one subject with CS [Kerr et al., 2006]. These substitutions are predicted to destabilize HRAS binding to either GTP or GDP.
No mutation was observed among the PTPN11 and KRAS mutation-negative subjects diagnosed with NS or the PTPN11, KRAS, BRAF, MEK1, and MEK2 mutation-negative subjects with CFCS. Similarly, no lesion was identified in subjects with clinical features suggestive of those conditions but who did not meet criteria for diagnosis. To exclude somatic mosaicism in three cases exhibiting features at the interface with CS, genomic DNA was obtained from their cultured fibroblasts, hair bulbs, and/or buccal epithelial cells and mutation analysis demonstrated absence of any defect within the HRAS coding sequence.
Parental Germline Origin of HRAS Mutations
To trace the parental origin of the de novo germline HRAS mutations in CS, common and rare polymorphisms flanking the c.34G4A and c.436G4A changes were used. To that end, portions of the genomic region flanking the disease-causative lesion were analyzed for the presence of polymorphic sites in the nine affected individuals with an inherited HRAS mutation. Eight additional subjects with CS and a de novo c.34G4A substitution (see Materials and Methods) were also included in the study. Short overlapping stretches, located upstream and downstream to exons 2 and 4, were PCR amplified, analyzed by DHPLC, and those having abnormal denaturing profiles were sequenced bidirectionally. Six different potentially informative exonic or intronic polymorphic sites were identified in 13 subjects, and genotyping of their parents indicated that nine families were informative (Table 1). Amplification and cloning of the genomic fragments encompassing the exonic mutation and the intronic/exonic polymorphic site allowed determination of haplotypes in affected individuals, and segregation analysis demonstrated the paternal germline origin of mutation in all cases. This nonrandom distribution was statistically significant (X2 = 5.67; P=0.017). Figure 1B shows results from four representative families. The identification of only two haplotypes among six sequenced clones for each PCR product further argued against the presence of somatic mosaicism in these individuals.
Comparison of the ages of the Italian fathers to the Italian population data (Office of Population Census, Italy; http:// demo.istat.it/altridati/natid1d2/index.html), indicated that the unaffected fathers transmitting the mutation tended to have an advanced age at conception, exhibiting an average of 37.2 years, which was 5.1 years older than the population average for their average year of birth.
Phenotypic Spectrum of HRAS 34G4AChange
A detailed clinical characterization of Cases CS-01 to CS-08 was carried out. Constant findings in the prenatal/neonatal period included polyhydramnios (between the 20th and 28th weeks) and birth weight over the mean (mean =1.9 standard deviation [SD]; range=0.7�2.9 SD). Absolute macrocephaly at birth was observed in five cases (mean = +2.4 SD; range =1.6�3.4 SD) (Supplementary Table S2). Among the dysmorphic and ectodermal features (Supplementary Table S3), a ��coarse�� face was observed in all cases. Dysmorphisms included wide forehead, epicanthal folds, wide and depressed nasal bridge, bulbous tip of nose, posteriorly rotated and/or low set ears with fleshy and cocked forward lobe, full cheeks, large mouth with thick lips, and macroglossia (Fig. 2).Worsening of these features with age was documented in adolescent and adult patients. The most striking cutaneous signs consisted of deep palmar and plantar creases and ridges, redundant and loose skin around the neck and over the hands and feet, and increased pigmentation. Velvet skin was common, while hyperkeratosis did not represent a constant feature and appeared later.
Feeding disorders have been considered as a hallmark of CS. Indeed, HRAS c.34G?A-positive newborns showed extreme failure to thrive, due to impaired swallowing in all cases. Gastroesophageal reflux requiring pharmacological treatment occurred in four subjects. Newborns presented with such poor oral intake that nasogastric feeding was required in all subjects (1�30 months), which resulted in only a mild improvement in weight gain. Gastrostomy tubes were placed in two children, and another two received total parenteral nutrition. With resolution of feeding difficulties, patients slowly and spontaneously started to gain weight with the persistence of linear growth deficiency (mean= -4.4 SD; range = -7.5 to -3.2 SD) (Supplementary Table S2).
Neurologic impairment was documented in all HRAS mutationpositive patients (Supplementary Table S3). Head control was achieved at a mean age of 9.6 months (range 6�13 months) and patients sat unassisted at a mean age of 12.5 months (range= 8�16 months). Walking started between the age of 14 and 36 months (mean=24.1 months), and expressive language was initiated between the age of 18�30 months (mean =23.7 months). Patients showed mild-to-moderate psychomotor delay with intelligence quotient (IQ) ranging from 43 to 77 (mean= 58). Sleep disorders, most frequently dyssomnia, and mild-tomoderate obstructive sleep apnea (OSA) were documented in almost all subjects.
Joint and skeletal anomalies included hyperextensibility of interphalangeal joints and ulnar deviation of the third, fourth, and fifth fingers (Supplementary Table S3). Limitation of dorsiflexion of feet extension of the elbows were common. Of note, all the patients developed a distinctive posture with anteroflexion of the trunk.
Cardiac involvement was present in all patients (Supplementary Table S4). The most common defect was hypertrophic cardiomyopathy (HCM), characteristically asymmetric (with anterior septal hypertrophy usually being predominant), with heterogeneity in age of onset, progression, severity, and clinical significance. Unlike HCM, congenital heart defects were not frequently observed among these subjects. ECG Holter recording showed benign forms of monomorphic nonrepetitive premature atrial and/or ventricular contraction(s) in the majority of the subjects. Finally, perinasal and perianal papillomata were observed in all subjects, while three distinct neoplasms (ganglioneuroblastoma, vesicular papilloma, and gastric leiomyoma) were documented in three cases during childhood.
Besides the classic CS signs, Subject CS-32 (p.Ala146Thr) presented with unusual features and natural history. Specifically, neonatal axometric parameters were within the normal range, and growth, despite the swallowing difficulty that required a nasogastric tube until 6 years of age, was less compromised. Minor involvement of skin and joints was also observed. Of note, she was microcephalic with a flat occiput, hairs were sparse and thin but not curly, and ears were normally located without the distinctive fleshy and forward-cocked lobes.
DISCUSSION
In the present report, we investigated the molecular diversity, phenotypic spectrum, and parental germline origin of de novo HRAS mutations. Our analysis of a clinically well-characterized CS cohort allowed the identification of a heterozygous missense mutation in all affected subjects. The recurrent c.34G4A change (p.Gly12Ser) was observed in eight subjects, while a novel c.436G?A transition (p.Ala146Thr) was identified in one individual. Mutations occurred de novo, and analysis of available fibroblast DNA of affected individuals confirmed the presence of mutations in all cases, supporting their germline origin. Detailed clinical characterization of the individuals carrying the c.34G?A change indicated a substantially homogeneous phenotype. By analyzing the genomic region flanking the disease causing HRAS lesions, we demonstrated the paternal germline origin of de novo mutations in 9 out of 9 informative families. Finally, no HRAS mutation was identified in subjects with a phenotype suggestive of CS but lacking sufficient signs for a definitive diagnosis or exhibiting a phenotype evolving with time. Similarly, no HRAS defect was observed among subjects with NS or CFCS, indicating a solid genotype�phenotype correlation.
HRAS encodes for a member of a family of small monomeric GTPases that participate in multiple signal transduction pathways controlling cell proliferation, differentiation, and survival [Mitin et al., 2005]. These proteins function as GDP/GTP-regulated molecular switches to control intracellular signal flow. Like the other RAS members, HRAS cycles from a GDP-bound inactive state to a GTP-bound active state, the latter allowing interaction with multiple effectors. According to the available data, the identity of affected residues and type of amino acid substitutions of CS-causing HRAS lesions partially overlap the distribution of defects documented as somatic events in cancer [Adjei, 2001]. While a large amount of experimental data supports the view that mutations affecting residues p.Gly12 and p.Gly13 render the protein insensitive to GTPase activating protein (GAP)-promoted GTP hydrolysis, resulting in a constitutively GTP-bound, active state, the c.436G?A change (p.Ala146Thr) described in this study and the previously reported c.350A?G substitution (p.His117Arg) [Kerr et al., 2006] would represent a novel gain-of-function mechanism. In the inactive complex, RAS binding to GDP is stable, requiring interaction with a guanylyl exchanging factor (GEF) for its dissociation. Both these mutations involve residues placed in the purine ring binding pocket of the protein, and are predicted to affect the stability of HRAS binding to GDP/GTP. By destabilizing the GDP-bound state, these mutations are expected to favor spontaneous dissociation of this complex, and HRAS binding to GTP, since GDP and GTP have similar dissociation constants [Sigal et al., 1986] but the latter has a significantly higher concentration in the cytoplasm [Proud, 1986].
This would shift the equilibrium toward the GTP-bound, active form, bypassing the requirement for a GEF. Biochemical and functional data on similar HRAS mutants support this model [Clanton et al., 1986; Der et al., 1986; Feig et al., 1986; Walter et al., 1986]. Of note, similar consequences on protein function have recently been proposed for two KRAS mutations (p.Val142Gly and p.Asp153Val) in NS and CFCS [Carta et al., 2006].
The present and previously published data indicate that the p.Gly12Ser amino acid substitution is the most recurrent mutation in CS, accounting for approximately 85% of affected individuals with mutated HRAS. This change recurs with considerably lower prevalence in human cancers, accounting for approximately 5% of total HRAS amino acid substitutions, according to the Sanger Catalogue of Somatic Mutations in Cancer (COSMIC; www. sanger.ac.uk/genetics/CGP/cosmic). Experimental data indicate that p.Gly12Ser has a lower transformation potential than the p.Gly12Val mutant, which is the most common somatic HRAS defect in human tumors (approximately 45% of total somatic HRAS mutations according to the COSMIC database). The latter has been documented in one individual with CS who died of severe cardiomyopathy during infancy (1% of cases with an HRAS mutation). Consistently no mutation affecting residue p.Gln61, which constitutes approximately 33% of total HRAS lesions in human cancer (COSMIC Database), has been documented in individuals with CS to date.
The occurrence of such a striking difference in prevalence and spectrum of HRAS amino acid substitutions in individuals with CS (germline origin) or malignancies (somatic origin) suggest that CS-causative mutations might have less potency for deregulating HRAS function than cancer-contributing ones and that the latter might exert a stronger perturbing effect on development when inherited. Our groups recently documented a similar phenomenon for the PTPN11 gene, having two nearly mutually exclusive classes of lesions with differential gain-of-function consequences that are either inherited germline (causing NS) or somatically acquired (contributing to leukemogenesis). Such a striking correlation has also been documented for germline and somatic KRAS mutations causing NS and CFCS or hematologic malignancies and solid tumors (COSMIC Database) [Carta et al., 2006; Niihori et al., 2006; Schubbert et al., 2006]. On the basis of the available genetic and functional data, distinct activating properties of individual substitutions would seem to explain the observed differences in prevalences among germline and somatic mutations. We cannot exclude the possibility that diverse mechanisms involved in mutagenesis might also contribute to this differential spectrum and mutation prevalence [Pfeifer, 2000].
In the present study, neoplasms were documented in 3 out of 8 cases, while perinasal or perianal papillomata occurred in all cases. Although recent published studies suggested a higher risk for malignancies in subjects carrying the c.35G?C change (p.Gly12Ala) larger numbers are required to confirm this genotype�phenotype relationship.
Available data from previous reports indicated that HRAS mutations accounted for 83 to 92% of subjects with a confirmed diagnosis of CS. This consistent finding was explained as either locus heterogeneity, as documented for the clinically related NS and CFCS, or by the difficulties of diagnosis in cases exhibiting overlapping features with clinically related disorders or a phenotype that evolves with time. The present study documented occurrence of a HRAS gene mutation in all the subjects undoubtedly diagnosed as having CS; while no mutation was observed in a few subjects with phenotype suggestive of CS at birth or infancy, a changing phenotype with time toward clinically related disorders (NS or CFCS) was documented in these subjects. These observations support the idea that mutations in the HRAS gene would account for the totality of CS cases, and that absence of a HRAS mutation in a subject with phenotype apparently fitting CS might be predictive of a clinically related but nosologically distinct condition. Clinical reevaluation of these cases would help considerably to confirm or reject diagnosis. The present data also document that a relatively homogeneous phenotype is associated with the p.Gly12Ser change.
Detailed clinical characterization on eight mutation positive subjects indicated HCM as a constant feature, in addition to polyhydramnios, macrocephaly, failure to thrive due to swallowing impairment, distinctive facial appearance, hoarse voice, delayed neurological development, interphalangeal hyperlaxity and limitation of some joints, deep palmar and plantar creases, and papillomata. Echocardiographic follow-up in the present cohort might account for the difference in prevalence of cardiac involvement previously reported for this disorder.
In the present study, no HRAS lesion was identified among subjects with NS or CFCS, further supporting the phenotypic homogeneity resulting from activating HRAS mutations. Notably, both NS and CFCS have recently been associated with germline defects of KRAS. Similar to HRAS mutations, KRAS missense changes are predicted to promote upregulation of RAS mediated intracellular signal flow. Even though functional redundancy among RAS proteins does exist, biological differences among these signal transducers are well documented both among and within cells. The identification of clinically diverse disorders associated with mutations in distinct RAS genes further supports a unique role of each of the three members of the family in cell signaling. Consistent with this view, a preferential association of human cancers with a particular RAS gene is well documented, bolstering the cell-context specificity of their function. Indeed, somatic HRAS gene mutations occur in a restricted group of solid tumors, most commonly bladder cancer, and are rarely observed in hematologic malignancies. Consistently, rhabdomyosarcoma and bladder carcinoma occur in children and young adults with CS, while myeloproliferative disorders or leukemias, which represent the most frequent malignancies in children with NS and are associated with KRAS mutations, do not.
We provided evidence for a paternal origin of de novo HRAS mutations in CS. This finding confirms previous studies indicating a predominance of paternal origin of point mutations among autosomal dominant diseases, including retinoblastoma, neurofibromatosis type I, Apert, Crouzon, and Pfeiffer syndromes, multiple endocrine neoplasia 2A and 2B, achondroplasia, Rett syndrome, and NS. This phenomenon has been ascribed in part to the increased opportunity for mitotic errors in spermatogonia, which cycle continuously throughout the reproductive life of a male, compared to oogonia, which do not. Consistently, advanced paternal age had been documented in CS [Lurie, 1994], and was confirmed in this study. Additional contributing mechanisms are likely to contribute to this phenomenon, such as selective advantage of spermatogonial cells carrying the mutation, cell-specific DNA repair efficiency and/or decreased apoptotic control with age.
ACKNOWLEDGMENTS
We are indebted to the patients and families who participated in the study. We thank the ��Associazione Italiana Sindrome di Costello�� for support. This work was supported by the Telethon- Italy grant GGP04172 and ��Programma di Collaborazione Italia-USA/malattie rare�� (to M.T.), RC 2006 grants from the Italian Ministry of Health (to B.D.), and NIH grants HL71207 and HD01294 (to B.D.G.) and HD048502 (to K.A.R.).