CLINICAL REPORT
Further Evidence of the Importance of RIT1 in Noonan Syndrome De´bora R. Bertola,1,2* Guilherme L. Yamamoto,1,2 Tatiana F. Almeida,2 Michelle Buscarilli,1 Alexander A. L. Jorge,3 Alexsandra C. Malaquias,3 Chong A. Kim,1 Vanessa N. V. Takahashi,2 Maria Rita Passos-Bueno,2 and Alexandre C. Pereira4 1
Unidade de Gene´tica do Instituto da Crianc¸a, Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil 2 Instituto de Biocieˆncias, Universidade de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil 3
Endocrinologia, LIM/25, Faculdade de Medicina da Universidade de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil Instituto do Corac¸˜ao, Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil
4
Manuscript Received: 17 March 2014; Manuscript Accepted: 8 July 2014
Noonan syndrome (NS) is an autosomal dominant disorder consisting of short stature, short and/or webbed neck, distinctive facial features, cardiac abnormalities, cryptorchidism, and coagulation defects. NS exhibits genetic heterogeneity, associated with mutated genes that participate in RAS-mitogen-activated protein kinase signal transduction. Recently, a new gene (RIT1) was discovered as the causative gene in 17 of 180 Japanese individuals who were negative for the previously known genes for NS and were studied using exome sequencing (four patients), followed by Sanger sequencing (13 patients). The present study used the same technique in 70 Brazilian patients with NS and identified six with RIT1 missense mutations. Thus, we confirm that RIT1 is responsible for approximately 10% of the patients negative for mutations in the previously known genes. The phenotype includes a high frequency of high birth weight, relative macrocephaly, left ventricular hypertrophy, and ectodermal findings, such as curly hair, hyperpigmentation, and wrinkled palms and soles. Short stature and pectus deformity were less frequent. The majority of patients with a RIT1 mutation did not show apparent intellectual disability. Because of the relatively high frequency of mutations in RIT1 among patients with NS and its occurrence in different populations, we suggest that it should be added to the list of genes included in panels for the molecular diagnosis of NS through targeted next-generation sequencing. Ó 2014 Wiley Periodicals, Inc.
Key words: left ventricular hypertrophy; noonan syndrome; RAS-MAPK signaling; RIT1; whole-exome sequencing
INTRODUCTION Noonan syndrome (NS) consists of short stature, distinctive facial features, short and/or webbed neck, cardiac abnormalities (mainly pulmonary stenosis and left ventricular hypertrophy), cryptorchidism in males and coagulation defects [Romano et al., 2010]. It is considered to be a relatively common autosomal dominant
Ó 2014 Wiley Periodicals, Inc.
How to Cite this Article: Bertola DR, Yamamoto GL, Almeida TF, Buscarilli M, Jorge AAL, Malaquias AC, Kim CA, Takahashi VNV, Passos-Bueno MR, Pereira AC. 2014. Further evidence of the importance of RIT1 in noonan syndrome. Am J Med Genet Part A. 9999:1–6.
disorder, although its prevalence has not been determined accurately to date [Tartaglia et al., 2011]. This disorder exhibits great genetic heterogeneity, caused by heterozygous mutations in PTPN11, KRAS, SOS1, RAF1, MAP2K1, BRAF, SHOC2, NRAS, and CBL, genes which participate in RASmitogen-activated protein kinase (MAPK) signal transduction, generally causing activation of this pathway [Tartaglia et al., 2001; Schubbert et al., 2006; Nava et al., 2007; Pandit et al., 2007; Razzaque et al., 2007; Roberts et al., 2007; Cordeddu et al., 2009; Sarkozy et al., 2009; Cirstea et al., 2010; Martinelli et al., 2010]. Among patients with NS, a causative gene mutation can be identified in approximately 75% of the patients, in which PTPN11 is by far the most prevalent, accounting for 50% of those affected. The rest of the genes are significantly less commonly involved [Tartaglia et al., Grant sponsor: FAPESP; Grant number: 2011/17299-3; Grant sponsor: CNPq. Correspondence to: De´bora R. Bertola, Unidade de Gene´tica do Instituto da Crianc¸a, Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Av. Dr. Ene´as Carvalho de Aguiar, 647, Cerqueira Ce´sar – Sa˜o Paulo, SP, Brazil. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 00 Month 2014 DOI 10.1002/ajmg.a.36722
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FIG. 1. RIT1 structure and mutations identified. In the upper row, the mutations found in the study by Aoki et al. [2013] and in the lower row, mutations identified in this study. Recurrent mutations are depicted in residues 57 and 95.
TABLE I. Clinical Findings in the Presented Cases with RIT1 Mutations, in the ones Described by Aoki et al. [2013] and in both Cohorts
Clinical findings Sex Age Prenatal abnormalities Birth weight (BW) Growth parameters Short stature Height Craniofacial abnormalities Relative macrocephaly Typical facies Short/webbed neck Cardiac abnormalities Pulmonic stenosis Left ventricular hypertrophy Patent ductus arteriosus ASD and/or VSD Pectus deformity Cryptorchidism Coagulation defects Ectodermal findings Curly hair Hyperpigmentation Hyperelastic skin Wrinkled palms and soles Tumors Intellectual disability Other findings RIT1 mutation a
c
Total frequency (N ¼ 23)
Patient 1 Male 2y
Patient 2 Female 16 y polyhydramnios
Patient 3 Female 27 y nd
Patient 4 Male 27 y nd
Patient 5 Male 28 y
4,020 g
1,800 g (7 m)
4,500 g
5,000 g
3,910 g
Patient 6 Male 10 y cystic hygroma 4,280 g
94.5 cm
þd 144 cm
þ 155 cm
þ 138.5 cm
171 cm
þ 124.5 cm
3 (14)
0.35
þ þ þ
þ þ þ
þ þ
þ þ þ
þ þ
þ þ þ
7 (13) 14 (16) 9 (15)
0.58 0.91 0.71
þ
þ þ ASD NA
þ þ NA
þ þ þ
þ þ þ þ
þ ASD þ VSD þ þ þ
11(17) 12 (17) 2 (16) 8 (16) 2 (13) 6 (9) 1 (11)
0.70 0.61 0.14 0.45 0.26 0.69 0.18
nd nd nd nd
þ
þ þ þ
þ þ þ
þ
5 (15) 6 (12) 4 (12) 5 (11) 1 (17) e
0.45 0.35 0.35 0.50 0.09
nd
Dandy–Walker anomaly/ systemic lupus eritythematosus p.G95A
Graves disease
severe scoliosis
þ Giant cell lesion
4 (13)
0.22
p.A57G
p.G95A
p.A57G
p.F82L
p.S35T
Number of affected individuals (Number of evaluated individuals). Polyhydramnios (4 patients); pleural effusion/chylothorax (3); increased nuchal translucency (3). BW >90th centile. d GH treatment without success; ASD: ostium secundum atrial septal defect; VSD: ventricular septal defect; NA: not applicable; nd: no data. e acute lymphoblastic leukemia. b
Aoki et al. (2013) a (N ¼ 17) 8F/9M mean ¼ 6.0 y 7 (16) b 7 (16)
0.45
c
BERTOLA ET AL. 2011]. A genotype-phenotype correlation for NS lacks a strong relationship, but some relevant clinical associations have been noted which influence the choice of genes to be studied during a molecular-genetic investigation using Sanger based sequencing. Recently, in an attempt to identify new genes responsible for NS, one technique of next-generation sequencing (NGS), namely exome sequencing (ES) was applied to 14 out of 180 Japanese individuals with NS who had tested negative for the known genes, and detected missense mutations in RIT1 in four of them. Sanger analysis was performed on the rest of the cohort identifying 13 additional patients, resulting in a total of 17 (9%) individuals showing heterozygous missense mutations in RIT1. These patients had a high frequency of left ventricular hypertrophy, in addition to the typical facial features and relative macrocephaly [Aoki et al., 2013]. We report on six additional individuals with NS from Brazil harboring mutations in RIT1, with the aim to further delineate the clinical and molecular aspects of patients with RIT1 mutations.
PATIENTS AND METHODS In this study, the same technique ES was performed in 70 Brazilian probands with NS, previously screened negative for PTPN11 (exons 2–15), SOS1 (exons 1–23), KRAS (exons 2–6), RAF1 (exons 7, 14, and 17), SHOC2 (exon 2), and CBL (exons 8 and 9). The patients were only included after informed consent was obtained from them or from the parental guardian. Exome sequencing of genomic DNA obtained from the peripheral blood of the affected individuals
3 was performed with Illumina’s TruSeq Exome Enrichment kits for library preparation and exome capture, and the Illumina HiSeq sequencer. Alignments were made with the Burrows-Wheeler Aligner (BWA) [Li and Durbin, 2009] and the Genome Analysis Tool Kit (GATK) [McKenna et al., 2010] was used for data processing and variant calling. Variant annotation was performed with ANNOVAR [Wang et al., 2010]. Statistical comparisons between the clinical findings of RIT1 positive patients and the rest of our cohort presenting mutations in PTPN11 (74 individuals), SOS1 (17), RAF1 (9), and KRAS (9) were made by Fisher’s exact test. Statistical comparison between the mean age of the patients harboring RIT1 mutations from Japan and Brazil was made by Mann–Whitney U test.
RESULTS In six of the 70 (9%) patients, heterozygous mutations in RIT1 were detected (OMIM 609591; RefSeq accession number NM_006912.5), all of them previously reported by Aoki et al. [2013] (Fig. 1). DNA from the parents was available only for Individuals five and six, which showed that it was an apparently de novo event in both patients. The clinical findings of these six patients are described in detail in Table I. Of note, Patient 4 developed a severe and progressive scoliosis (Fig. 2 A2-A3-A4) and Patient 5 developed multiple giant cell lesions in the mandible at the age of 15 (Fig. 2 C2). Biochemical analysis of calcium, phosphorus, and parathyroid hormone yielded normal results. The course of this tumor was very aggressive, with recurrence
FIG. 2. Photographs of three individuals with RIT1 mutations (A1, A2, B, C1). Note the severe scoliosis in Individual 4 (A2, A3, A4) and the maxillary lesion in Patient 5 (C2).
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and development of new lesions, requiring seven surgical interventions and the loss of several teeth. Patient 5 also presented with factor XI deficiency with a tendency for profuse bleeding during the procedures. Although a formal IQ evaluation was not done in our patients, they all performed well in normal schools. A comparison between the clinical findings of patients harboring RIT1 mutations and the ones from our cohort presenting PTPN11, SOS1, RAF1, and KRAS mutations showed a statistically significant difference in the frequency of prenatal ultrasound findings, short stature, relative macrocephaly, type of cardiac abnormalities, pectus deformity, ectodermal findings, and intellectual disability (Table II).
DISCUSSION Some of the well-known genes involved in NS were discovered based on their role in the RAS-MAPK pathway. The recently application of NGS emerged as a very capable tool in identifying genes responsible for monogenic disorders. RIT1 mutations were found in 9% of the probands studied by Aoki et al. [2013], as well as
in our cohort (6/70), indicating that RIT1 is an important gene in the etiology of NS, probably worldwide. Recurrent mutations were observed involving the residues 57 and 95 in the protein, suggesting hotspots (Fig. 1). Although the number (23) of patients described thus far with RIT1 mutations is still small, a pattern of clinical findings is starting to emerge. Several characteristics were observed to present a statistically significant difference from at least one of the other group of genes, while none of them could be attributed solely to the RIT1 group (Tables I and II). We observed that in the prenatal period, nine out of 20 (45%) individuals had an abnormal ultrasound examination, mainly polyhydramnios. Other findings included chylothorax/pleural effusion and cystic hygroma in the neck, which in combination, characterize fetal hydrops. As a consequence, a high birth weight in neonates born at term was a constant finding (mean of 4,342 g), and resembles other patients with RASopathy syndromes [Nava et al., 2007; Tartaglia et al., 2011]. The facial features were typical of NS (Fig. 2 A1-B-C1). Regarding the main clinical findings in NS, RIT1 positive patients
TABLE II. Comparison of the Clinical Findings Between RIT1 Positive Patients and the Other Genes Responsible for Noonan Syndrome from our Center
Clinical findings Prenatal abnormalities Mean birth weight (g)b (range) Growth parameters Short stature Craniofacial abnormalities Relative macrocephaly Typical facies Short/webbed neck Cardiac abnormalities Pulmonic stenosis Left ventricular hypertrophy Patent ductus arteriosus ASD and/or VSD Pectus deformity Cryptorchidism Coagulation defects Ectodermal findings Curly hair Hyperpigmentation Hyperelastic skin Wrinkled palms and soles Hyperkeratosis Tumors Intellectual disability a
RIT1 (N ¼ 23) 9/20 (0.45)
RIT1 vs. other genes P < 0.05 0.0090 (PTPN11)
PTPN11 (N ¼ 74) 8/59 (0.14)
SOS1 (N ¼ 17) 2/10 (0.2)
RAF1 (N ¼ 9) 5/8 (0.625)
KRAS (N ¼ 9) 3/9 (0.33)
4342 (3910–5000)
3250 (2140–4400)
3070 (2000–3950)
3174 (2500–3800)
3180 (3050–3455)
7/20 (0.35)
55/67 (0.82)
8/14 (0.57)
8/8 (1)
6/9 (0.67)
0.0001 (PTPN11) 0.0025 (RAF1)
11/19 20/22 15/21 22/23 16/23 14/23
(0.58) (0.91) (0.71) (0.96) (0.70) (0.61)
9/62 (0.15) 73/74 (0.99) 58/65 (0.89) 58/69 (0.84) 39/58 (0.67) 11/58 (0.19)
4/13 (0.31) 17/17 (1) 14/14 (1) 14/17 (0.82) 12/14 (0.86) 0/14 (0)
7/8 (0.875) 9/9 (1) 8/8 (1) 9/9 (1) 1/9 (0.11) 8/9 (0.89)
7/9 (0.78) 9/9 (1) 9/9 (1) 8/9 (0.89) 6/8 (0.75) 2/8 (0.25)
0.0004 (PTPN11)
3/22 (0.14) 10/22 (0.45) 5/19 (0.26)
2/58 (0.034) 24/58 (0.41) 36/64 (0.56)
1/14 (0.07) 4/14 (0.29) 11/14 (0.79)
0/9 (0) 2/9 (0.22) 3/8 (0.375)
1/8 (0.125) 2/8 (0.25) 3/8 (0.375)
9/13 (0.70) 3/17 (0.18)
23/41 (0.56) 11/60 (0.18)
5/10 (0.50) 2/14 (0.14)
2/4 (0.50) 0/6 (0)
1/5 (0.20) 1/7 (0.14)
9/21 6/19 6/18 8/17
12/68 (0.18) 0/68 (0) nd 6/68 (0.09)
8/14 (0.57) 1/14 (0.07) nd 5/14 (0.36)
2/8 (0.25) 0/8 (0) nd 1/8 (0.125)
4/9 (0.44) 1/9 (0.11) nd 5/9 (0.55)
7/68 (0.10) Chiari I; GCL(2); JMML(2) 35/60 (0.58)
6/14 (0.43) GCL (2) 5/9 (0.56)
3/8 (0.375) cerebellar lesion 1/4 (0.25)
2/9 (0.22) NP/S 4/5 (0.8)
a
(0.43) (0.32) (0.33) (0.47)
0/5 (0) GCL; ALL 4/18 (0.22)
0.0049 (RAF1) 0.0004 (PTPN11) 0.0002 (SOS1) 0.0352 (PTPN11) 0.0049 (SOS1)
0.0362 (PTPN11) 0.0001 (PTPN11) 0.0008 (PTPN11)
0.0141 (PTPN11) 0.0329 (KRAS)
Number of affected individuals/number of evaluated individuals. Neonates born at term; ASD: ostium secundum atrial septal defect; VSD: ventricular septal defect; nd: no data; GCL: giant cell lesion tumor; ALL: acute lymphoblastic leukemia; JMML: juvenile myelomonocytic leukemia; NP/S: nasal papilloma þ schwannomatosis. b
BERTOLA ET AL. showed less frequency of short stature, pectus deformity and learning problems. On the other hand, relative macrocephaly, left ventricular hypertrophy, and ectodermal findings, such as curly hair, hyperpigmentation, and wrinkled palms and soles were more frequently observed. Increased risk for autoimmune disorders and neoplasia has been associated with NS [Kratz et al., 2011; Quaio et al., 2012]. One patient reported by Aoki et al. [2013] developed acute lymphoblastic leukemia and one of our patients showed giant cell lesion tumors. Moreover, two out of six patients in our cohort showed autoimmune disorders, namely Graves disease and systemic lupus erythematosus. It is too early to determine if RIT1 shows a greater predisposition to neoplasias and auto-immune disorders. Further descriptions of patients harboring mutations in this gene, as well as the long-term follow-up of the ones already described, especially the ones described by Aoki et al. [2013] who showed a lower mean age compared to our cohort (6.0 y vs. 18.3 y; p ¼ 0.0158), are necessary for a better definition of these risks. In summary, we described six additional patients with NS harboring RIT1 mutations identified by exome-sequencing, contributing to the phenotypic description of patients harboring mutations in this gene. It is interesting to note that in the approach to identifying the genes responsible for the remaining 25% of unknown genetic causes for NS, RIT1 emerged as an important gene and should be added to the list of genes included in a panel for the diagnosis of NS through targeted NGS, a powerful methodology now used for monogenic disorders with great heterogeneity. We hypothesize that we are approaching the end of the era of detecting additional genes responsible for a large proportion of NS patients, since no further genes were immediately found using WES in both studies. It is possible that additional genes discovered in the future will be each responsible for a very small number of cases.
ACKNOWLEDGMENTS
5 Zenker M, Merlo D, Dallapiccola B, Iyengar R, Bazzicalupo P, Gelb BD, Tartaglia M. 2009. Mutation of SHOC2 promotes aberrant protein Nmyristoylation and causes Noonan-like syndrome with loose anagen hair. Nat Genet 41:1022–U95. Kratz CP, Rapisuwon S, Reed H, Hasle H, Rosenberg PS. 2011. Cancer in Noonan, Costello cardiofaciocutaneous and LEOPARD syndromes. Am J Med Genet C Semin Med Genet 157:83–89. Li H, Durbin R. 2009. Fast and accurate short read alignment with BurrowsWheeler transform. Bioinformatics 25:1754–1760. Martinelli S, De Luca A, Stellacci E, Rossi C, Checquolo S, Lepri F, Caputo V, Silvano M, Buscherini F, Consoli F, Ferrara G, Digilio MC, Cavaliere ML, van Hagen JM, Zampino G, van der Burgt I, Ferrero GB, Mazzanti L, Screpanti I, Yntema HG, Nillesen WM, Savarirayan R, Zenker M, Dallapiccola B, Gelb BD, Tartaglia M. 2010. Heterozygous germline mutations in the CBL tumor-suppressor gene cause a Noonan syndromelike phenotype. Am J Hum Genet 87:250–257. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA. 2010. The Genome Analysis Toolkit: A MapReduce framework for analyzing nextgeneration DNA sequencing data. Genome Res 20:1297–1303. Nava C, Hanna N, Michot C, Pereira S, Pouvreau N, Niihori T, Aoki Y, Matsubara Y, Arveiler B, Lacombe D, Pasmant E, Parfait B, Baumann C, Heron D, Sigaudy S, Toutain A, Rio M, Goldenberg A, Leheup B, Verloes A, Cave H. 2007. 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 44:763– 771. Pandit B, Sarkozy A, Pennacchio LA, Carta C, Oishi K, Martinelli S, Pogna EA, Schackwitz W, Ustaszewska A, Landstrom A, Bos JM, Ommen SR, Esposito G, Lepri F, Faul C, Mundel P, Siguero JPL, Tenconi R, Selicorni A, Rossi C, Mazzanti L, Torrente I, Marino B, Digilio MC, Zampino G, Ackerman MJ, Dallapiccola B, Tartaglia M, Gelb BD. 2007. Gain-offunction RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy. Nat Genet 39:1007–1012. Quaio CRDC, Dutra RL, Brasil AS, Pereira AC, Kim CA, Bertola DR. 2012. A possible role of different PTPN genes in immune regulation. Scand J Immunol 75:540–541.
The authors thank the families for their cooperation in this study. This work was financially supported by FAPESP 2011/17299-3 and CNPq.
Razzaque MA, Nishizawa T, Komoike Y, Yagi H, Furutani M, Amo R, Kamisago M, Momma K, Katayama H, Nakagawa M, Fujiwara Y, Matsushima M, Mizuno K, Tokuyama M, Hirota H, Muneuchi J, Higashinakagawa T, Matsuoka R. 2007. Germline gain-of-function mutations in RAF1 cause Noonan syndrome. Nat Genet 39:1013–1017.
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Further Evidence of the Importance of RIT1 in Noonan Syndrome De´bora R. Bertola,1,2* Guilherme L. Yamamoto,1,2 Tatiana F. Almeida,2 Michelle Buscarilli,1 Alexander A. L. Jorge,3 Alexsandra C. Malaquias,3 Chong A. Kim,1 Vanessa N. V. Takahashi,2 Maria Rita Passos-Bueno,2 and Alexandre C. Pereira4 1
Unidade de Gene´tica do Instituto da Crianc¸a, Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil 2 Instituto de Biocieˆncias, Universidade de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil 3
Endocrinologia, LIM/25, Faculdade de Medicina da Universidade de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil Instituto do Corac¸˜ao, Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil
4
Manuscript Received: 17 March 2014; Manuscript Accepted: 8 July 2014
Noonan syndrome (NS) is an autosomal dominant disorder consisting of short stature, short and/or webbed neck, distinctive facial features, cardiac abnormalities, cryptorchidism, and coagulation defects. NS exhibits genetic heterogeneity, associated with mutated genes that participate in RAS-mitogen-activated protein kinase signal transduction. Recently, a new gene (RIT1) was discovered as the causative gene in 17 of 180 Japanese individuals who were negative for the previously known genes for NS and were studied using exome sequencing (four patients), followed by Sanger sequencing (13 patients). The present study used the same technique in 70 Brazilian patients with NS and identified six with RIT1 missense mutations. Thus, we confirm that RIT1 is responsible for approximately 10% of the patients negative for mutations in the previously known genes. The phenotype includes a high frequency of high birth weight, relative macrocephaly, left ventricular hypertrophy, and ectodermal findings, such as curly hair, hyperpigmentation, and wrinkled palms and soles. Short stature and pectus deformity were less frequent. The majority of patients with a RIT1 mutation did not show apparent intellectual disability. Because of the relatively high frequency of mutations in RIT1 among patients with NS and its occurrence in different populations, we suggest that it should be added to the list of genes included in panels for the molecular diagnosis of NS through targeted next-generation sequencing. Ó 2014 Wiley Periodicals, Inc.
Key words: left ventricular hypertrophy; noonan syndrome; RAS-MAPK signaling; RIT1; whole-exome sequencing
INTRODUCTION Noonan syndrome (NS) consists of short stature, distinctive facial features, short and/or webbed neck, cardiac abnormalities (mainly pulmonary stenosis and left ventricular hypertrophy), cryptorchidism in males and coagulation defects [Romano et al., 2010]. It is considered to be a relatively common autosomal dominant
Ó 2014 Wiley Periodicals, Inc.
How to Cite this Article: Bertola DR, Yamamoto GL, Almeida TF, Buscarilli M, Jorge AAL, Malaquias AC, Kim CA, Takahashi VNV, Passos-Bueno MR, Pereira AC. 2014. Further evidence of the importance of RIT1 in noonan syndrome. Am J Med Genet Part A. 9999:1–6.
disorder, although its prevalence has not been determined accurately to date [Tartaglia et al., 2011]. This disorder exhibits great genetic heterogeneity, caused by heterozygous mutations in PTPN11, KRAS, SOS1, RAF1, MAP2K1, BRAF, SHOC2, NRAS, and CBL, genes which participate in RASmitogen-activated protein kinase (MAPK) signal transduction, generally causing activation of this pathway [Tartaglia et al., 2001; Schubbert et al., 2006; Nava et al., 2007; Pandit et al., 2007; Razzaque et al., 2007; Roberts et al., 2007; Cordeddu et al., 2009; Sarkozy et al., 2009; Cirstea et al., 2010; Martinelli et al., 2010]. Among patients with NS, a causative gene mutation can be identified in approximately 75% of the patients, in which PTPN11 is by far the most prevalent, accounting for 50% of those affected. The rest of the genes are significantly less commonly involved [Tartaglia et al., Grant sponsor: FAPESP; Grant number: 2011/17299-3; Grant sponsor: CNPq. Correspondence to: De´bora R. Bertola, Unidade de Gene´tica do Instituto da Crianc¸a, Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Av. Dr. Ene´as Carvalho de Aguiar, 647, Cerqueira Ce´sar – Sa˜o Paulo, SP, Brazil. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 00 Month 2014 DOI 10.1002/ajmg.a.36722
1
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AMERICAN JOURNAL OF MEDICAL GENETICS PART A
FIG. 1. RIT1 structure and mutations identified. In the upper row, the mutations found in the study by Aoki et al. [2013] and in the lower row, mutations identified in this study. Recurrent mutations are depicted in residues 57 and 95.
TABLE I. Clinical Findings in the Presented Cases with RIT1 Mutations, in the ones Described by Aoki et al. [2013] and in both Cohorts
Clinical findings Sex Age Prenatal abnormalities Birth weight (BW) Growth parameters Short stature Height Craniofacial abnormalities Relative macrocephaly Typical facies Short/webbed neck Cardiac abnormalities Pulmonic stenosis Left ventricular hypertrophy Patent ductus arteriosus ASD and/or VSD Pectus deformity Cryptorchidism Coagulation defects Ectodermal findings Curly hair Hyperpigmentation Hyperelastic skin Wrinkled palms and soles Tumors Intellectual disability Other findings RIT1 mutation a
c
Total frequency (N ¼ 23)
Patient 1 Male 2y
Patient 2 Female 16 y polyhydramnios
Patient 3 Female 27 y nd
Patient 4 Male 27 y nd
Patient 5 Male 28 y
4,020 g
1,800 g (7 m)
4,500 g
5,000 g
3,910 g
Patient 6 Male 10 y cystic hygroma 4,280 g
94.5 cm
þd 144 cm
þ 155 cm
þ 138.5 cm
171 cm
þ 124.5 cm
3 (14)
0.35
þ þ þ
þ þ þ
þ þ
þ þ þ
þ þ
þ þ þ
7 (13) 14 (16) 9 (15)
0.58 0.91 0.71
þ
þ þ ASD NA
þ þ NA
þ þ þ
þ þ þ þ
þ ASD þ VSD þ þ þ
11(17) 12 (17) 2 (16) 8 (16) 2 (13) 6 (9) 1 (11)
0.70 0.61 0.14 0.45 0.26 0.69 0.18
nd nd nd nd
þ
þ þ þ
þ þ þ
þ
5 (15) 6 (12) 4 (12) 5 (11) 1 (17) e
0.45 0.35 0.35 0.50 0.09
nd
Dandy–Walker anomaly/ systemic lupus eritythematosus p.G95A
Graves disease
severe scoliosis
þ Giant cell lesion
4 (13)
0.22
p.A57G
p.G95A
p.A57G
p.F82L
p.S35T
Number of affected individuals (Number of evaluated individuals). Polyhydramnios (4 patients); pleural effusion/chylothorax (3); increased nuchal translucency (3). BW >90th centile. d GH treatment without success; ASD: ostium secundum atrial septal defect; VSD: ventricular septal defect; NA: not applicable; nd: no data. e acute lymphoblastic leukemia. b
Aoki et al. (2013) a (N ¼ 17) 8F/9M mean ¼ 6.0 y 7 (16) b 7 (16)
0.45
c
BERTOLA ET AL. 2011]. A genotype-phenotype correlation for NS lacks a strong relationship, but some relevant clinical associations have been noted which influence the choice of genes to be studied during a molecular-genetic investigation using Sanger based sequencing. Recently, in an attempt to identify new genes responsible for NS, one technique of next-generation sequencing (NGS), namely exome sequencing (ES) was applied to 14 out of 180 Japanese individuals with NS who had tested negative for the known genes, and detected missense mutations in RIT1 in four of them. Sanger analysis was performed on the rest of the cohort identifying 13 additional patients, resulting in a total of 17 (9%) individuals showing heterozygous missense mutations in RIT1. These patients had a high frequency of left ventricular hypertrophy, in addition to the typical facial features and relative macrocephaly [Aoki et al., 2013]. We report on six additional individuals with NS from Brazil harboring mutations in RIT1, with the aim to further delineate the clinical and molecular aspects of patients with RIT1 mutations.
PATIENTS AND METHODS In this study, the same technique ES was performed in 70 Brazilian probands with NS, previously screened negative for PTPN11 (exons 2–15), SOS1 (exons 1–23), KRAS (exons 2–6), RAF1 (exons 7, 14, and 17), SHOC2 (exon 2), and CBL (exons 8 and 9). The patients were only included after informed consent was obtained from them or from the parental guardian. Exome sequencing of genomic DNA obtained from the peripheral blood of the affected individuals
3 was performed with Illumina’s TruSeq Exome Enrichment kits for library preparation and exome capture, and the Illumina HiSeq sequencer. Alignments were made with the Burrows-Wheeler Aligner (BWA) [Li and Durbin, 2009] and the Genome Analysis Tool Kit (GATK) [McKenna et al., 2010] was used for data processing and variant calling. Variant annotation was performed with ANNOVAR [Wang et al., 2010]. Statistical comparisons between the clinical findings of RIT1 positive patients and the rest of our cohort presenting mutations in PTPN11 (74 individuals), SOS1 (17), RAF1 (9), and KRAS (9) were made by Fisher’s exact test. Statistical comparison between the mean age of the patients harboring RIT1 mutations from Japan and Brazil was made by Mann–Whitney U test.
RESULTS In six of the 70 (9%) patients, heterozygous mutations in RIT1 were detected (OMIM 609591; RefSeq accession number NM_006912.5), all of them previously reported by Aoki et al. [2013] (Fig. 1). DNA from the parents was available only for Individuals five and six, which showed that it was an apparently de novo event in both patients. The clinical findings of these six patients are described in detail in Table I. Of note, Patient 4 developed a severe and progressive scoliosis (Fig. 2 A2-A3-A4) and Patient 5 developed multiple giant cell lesions in the mandible at the age of 15 (Fig. 2 C2). Biochemical analysis of calcium, phosphorus, and parathyroid hormone yielded normal results. The course of this tumor was very aggressive, with recurrence
FIG. 2. Photographs of three individuals with RIT1 mutations (A1, A2, B, C1). Note the severe scoliosis in Individual 4 (A2, A3, A4) and the maxillary lesion in Patient 5 (C2).
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AMERICAN JOURNAL OF MEDICAL GENETICS PART A
and development of new lesions, requiring seven surgical interventions and the loss of several teeth. Patient 5 also presented with factor XI deficiency with a tendency for profuse bleeding during the procedures. Although a formal IQ evaluation was not done in our patients, they all performed well in normal schools. A comparison between the clinical findings of patients harboring RIT1 mutations and the ones from our cohort presenting PTPN11, SOS1, RAF1, and KRAS mutations showed a statistically significant difference in the frequency of prenatal ultrasound findings, short stature, relative macrocephaly, type of cardiac abnormalities, pectus deformity, ectodermal findings, and intellectual disability (Table II).
DISCUSSION Some of the well-known genes involved in NS were discovered based on their role in the RAS-MAPK pathway. The recently application of NGS emerged as a very capable tool in identifying genes responsible for monogenic disorders. RIT1 mutations were found in 9% of the probands studied by Aoki et al. [2013], as well as
in our cohort (6/70), indicating that RIT1 is an important gene in the etiology of NS, probably worldwide. Recurrent mutations were observed involving the residues 57 and 95 in the protein, suggesting hotspots (Fig. 1). Although the number (23) of patients described thus far with RIT1 mutations is still small, a pattern of clinical findings is starting to emerge. Several characteristics were observed to present a statistically significant difference from at least one of the other group of genes, while none of them could be attributed solely to the RIT1 group (Tables I and II). We observed that in the prenatal period, nine out of 20 (45%) individuals had an abnormal ultrasound examination, mainly polyhydramnios. Other findings included chylothorax/pleural effusion and cystic hygroma in the neck, which in combination, characterize fetal hydrops. As a consequence, a high birth weight in neonates born at term was a constant finding (mean of 4,342 g), and resembles other patients with RASopathy syndromes [Nava et al., 2007; Tartaglia et al., 2011]. The facial features were typical of NS (Fig. 2 A1-B-C1). Regarding the main clinical findings in NS, RIT1 positive patients
TABLE II. Comparison of the Clinical Findings Between RIT1 Positive Patients and the Other Genes Responsible for Noonan Syndrome from our Center
Clinical findings Prenatal abnormalities Mean birth weight (g)b (range) Growth parameters Short stature Craniofacial abnormalities Relative macrocephaly Typical facies Short/webbed neck Cardiac abnormalities Pulmonic stenosis Left ventricular hypertrophy Patent ductus arteriosus ASD and/or VSD Pectus deformity Cryptorchidism Coagulation defects Ectodermal findings Curly hair Hyperpigmentation Hyperelastic skin Wrinkled palms and soles Hyperkeratosis Tumors Intellectual disability a
RIT1 (N ¼ 23) 9/20 (0.45)
RIT1 vs. other genes P < 0.05 0.0090 (PTPN11)
PTPN11 (N ¼ 74) 8/59 (0.14)
SOS1 (N ¼ 17) 2/10 (0.2)
RAF1 (N ¼ 9) 5/8 (0.625)
KRAS (N ¼ 9) 3/9 (0.33)
4342 (3910–5000)
3250 (2140–4400)
3070 (2000–3950)
3174 (2500–3800)
3180 (3050–3455)
7/20 (0.35)
55/67 (0.82)
8/14 (0.57)
8/8 (1)
6/9 (0.67)
0.0001 (PTPN11) 0.0025 (RAF1)
11/19 20/22 15/21 22/23 16/23 14/23
(0.58) (0.91) (0.71) (0.96) (0.70) (0.61)
9/62 (0.15) 73/74 (0.99) 58/65 (0.89) 58/69 (0.84) 39/58 (0.67) 11/58 (0.19)
4/13 (0.31) 17/17 (1) 14/14 (1) 14/17 (0.82) 12/14 (0.86) 0/14 (0)
7/8 (0.875) 9/9 (1) 8/8 (1) 9/9 (1) 1/9 (0.11) 8/9 (0.89)
7/9 (0.78) 9/9 (1) 9/9 (1) 8/9 (0.89) 6/8 (0.75) 2/8 (0.25)
0.0004 (PTPN11)
3/22 (0.14) 10/22 (0.45) 5/19 (0.26)
2/58 (0.034) 24/58 (0.41) 36/64 (0.56)
1/14 (0.07) 4/14 (0.29) 11/14 (0.79)
0/9 (0) 2/9 (0.22) 3/8 (0.375)
1/8 (0.125) 2/8 (0.25) 3/8 (0.375)
9/13 (0.70) 3/17 (0.18)
23/41 (0.56) 11/60 (0.18)
5/10 (0.50) 2/14 (0.14)
2/4 (0.50) 0/6 (0)
1/5 (0.20) 1/7 (0.14)
9/21 6/19 6/18 8/17
12/68 (0.18) 0/68 (0) nd 6/68 (0.09)
8/14 (0.57) 1/14 (0.07) nd 5/14 (0.36)
2/8 (0.25) 0/8 (0) nd 1/8 (0.125)
4/9 (0.44) 1/9 (0.11) nd 5/9 (0.55)
7/68 (0.10) Chiari I; GCL(2); JMML(2) 35/60 (0.58)
6/14 (0.43) GCL (2) 5/9 (0.56)
3/8 (0.375) cerebellar lesion 1/4 (0.25)
2/9 (0.22) NP/S 4/5 (0.8)
a
(0.43) (0.32) (0.33) (0.47)
0/5 (0) GCL; ALL 4/18 (0.22)
0.0049 (RAF1) 0.0004 (PTPN11) 0.0002 (SOS1) 0.0352 (PTPN11) 0.0049 (SOS1)
0.0362 (PTPN11) 0.0001 (PTPN11) 0.0008 (PTPN11)
0.0141 (PTPN11) 0.0329 (KRAS)
Number of affected individuals/number of evaluated individuals. Neonates born at term; ASD: ostium secundum atrial septal defect; VSD: ventricular septal defect; nd: no data; GCL: giant cell lesion tumor; ALL: acute lymphoblastic leukemia; JMML: juvenile myelomonocytic leukemia; NP/S: nasal papilloma þ schwannomatosis. b
BERTOLA ET AL. showed less frequency of short stature, pectus deformity and learning problems. On the other hand, relative macrocephaly, left ventricular hypertrophy, and ectodermal findings, such as curly hair, hyperpigmentation, and wrinkled palms and soles were more frequently observed. Increased risk for autoimmune disorders and neoplasia has been associated with NS [Kratz et al., 2011; Quaio et al., 2012]. One patient reported by Aoki et al. [2013] developed acute lymphoblastic leukemia and one of our patients showed giant cell lesion tumors. Moreover, two out of six patients in our cohort showed autoimmune disorders, namely Graves disease and systemic lupus erythematosus. It is too early to determine if RIT1 shows a greater predisposition to neoplasias and auto-immune disorders. Further descriptions of patients harboring mutations in this gene, as well as the long-term follow-up of the ones already described, especially the ones described by Aoki et al. [2013] who showed a lower mean age compared to our cohort (6.0 y vs. 18.3 y; p ¼ 0.0158), are necessary for a better definition of these risks. In summary, we described six additional patients with NS harboring RIT1 mutations identified by exome-sequencing, contributing to the phenotypic description of patients harboring mutations in this gene. It is interesting to note that in the approach to identifying the genes responsible for the remaining 25% of unknown genetic causes for NS, RIT1 emerged as an important gene and should be added to the list of genes included in a panel for the diagnosis of NS through targeted NGS, a powerful methodology now used for monogenic disorders with great heterogeneity. We hypothesize that we are approaching the end of the era of detecting additional genes responsible for a large proportion of NS patients, since no further genes were immediately found using WES in both studies. It is possible that additional genes discovered in the future will be each responsible for a very small number of cases.
ACKNOWLEDGMENTS
5 Zenker M, Merlo D, Dallapiccola B, Iyengar R, Bazzicalupo P, Gelb BD, Tartaglia M. 2009. Mutation of SHOC2 promotes aberrant protein Nmyristoylation and causes Noonan-like syndrome with loose anagen hair. Nat Genet 41:1022–U95. Kratz CP, Rapisuwon S, Reed H, Hasle H, Rosenberg PS. 2011. Cancer in Noonan, Costello cardiofaciocutaneous and LEOPARD syndromes. Am J Med Genet C Semin Med Genet 157:83–89. Li H, Durbin R. 2009. Fast and accurate short read alignment with BurrowsWheeler transform. Bioinformatics 25:1754–1760. Martinelli S, De Luca A, Stellacci E, Rossi C, Checquolo S, Lepri F, Caputo V, Silvano M, Buscherini F, Consoli F, Ferrara G, Digilio MC, Cavaliere ML, van Hagen JM, Zampino G, van der Burgt I, Ferrero GB, Mazzanti L, Screpanti I, Yntema HG, Nillesen WM, Savarirayan R, Zenker M, Dallapiccola B, Gelb BD, Tartaglia M. 2010. Heterozygous germline mutations in the CBL tumor-suppressor gene cause a Noonan syndromelike phenotype. Am J Hum Genet 87:250–257. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA. 2010. The Genome Analysis Toolkit: A MapReduce framework for analyzing nextgeneration DNA sequencing data. Genome Res 20:1297–1303. Nava C, Hanna N, Michot C, Pereira S, Pouvreau N, Niihori T, Aoki Y, Matsubara Y, Arveiler B, Lacombe D, Pasmant E, Parfait B, Baumann C, Heron D, Sigaudy S, Toutain A, Rio M, Goldenberg A, Leheup B, Verloes A, Cave H. 2007. 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 44:763– 771. Pandit B, Sarkozy A, Pennacchio LA, Carta C, Oishi K, Martinelli S, Pogna EA, Schackwitz W, Ustaszewska A, Landstrom A, Bos JM, Ommen SR, Esposito G, Lepri F, Faul C, Mundel P, Siguero JPL, Tenconi R, Selicorni A, Rossi C, Mazzanti L, Torrente I, Marino B, Digilio MC, Zampino G, Ackerman MJ, Dallapiccola B, Tartaglia M, Gelb BD. 2007. Gain-offunction RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy. Nat Genet 39:1007–1012. Quaio CRDC, Dutra RL, Brasil AS, Pereira AC, Kim CA, Bertola DR. 2012. A possible role of different PTPN genes in immune regulation. Scand J Immunol 75:540–541.
The authors thank the families for their cooperation in this study. This work was financially supported by FAPESP 2011/17299-3 and CNPq.
Razzaque MA, Nishizawa T, Komoike Y, Yagi H, Furutani M, Amo R, Kamisago M, Momma K, Katayama H, Nakagawa M, Fujiwara Y, Matsushima M, Mizuno K, Tokuyama M, Hirota H, Muneuchi J, Higashinakagawa T, Matsuoka R. 2007. Germline gain-of-function mutations in RAF1 cause Noonan syndrome. Nat Genet 39:1013–1017.
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