J Hum Genet (2008) 53:999–1006 DOI 10.1007/s10038-008-0343-6 ORIGINAL ARTICLE PTPN11, SOS1, KRAS, and RAF1 gene analysis, and genotype–phenotype correlation in Korean patients with Noonan syndrome Jung Min Ko Æ Jae-Min Kim Æ Gu-Hwan Kim Æ Han-Wook Yoo Received: 30 July 2008 / Accepted: 27 October 2008 / Published online: 20 November 2008 Ó The Japan Society of Human Genetics and Springer 2008 Abstract After 2006, germline mutations in the KRAS, KRAS (1.7%), and RAF1 (5.1%) genes. Three novel SOS1, and RAF1 genes were reported to cause Noonan mutations (T59A in PTPN11, K170E in SOS1, S259T in syndrome (NS), in addition to the PTPN11 gene, and now RAF1) were identified. The patients with PTPN11 muta- we can find the etiology of disease in approximately tions showed higher prevalences of patent ductus 60–70% of NS cases. The aim of this study was to assess arteriosus and thrombocytopenia. The patients with SOS1 the correlation between phenotype and genotype by mutations had a lower prevalence of delayed psychomotor molecular analysis of the PTPN11, SOS1, KRAS, and development. All patients with RAF1 mutations had RAF1 genes in 59 Korean patients with NS. We found hypertrophic cardiomyopathy. Typical facial features and disease-causing mutations in 30 (50.8%) patients, which auxological parameters were, on statistical analysis, not were located in the PTPN11 (27.1%), SOS1 (16.9%), significantly different between the groups. The molecular defects of NS are genetically heterogeneous and involve several genes other than PTPN11 related to the RAS- MAPK pathway. Electronic supplementary material The online version of this article (doi:10.1007/s10038-008-0343-6) contains supplementary material, which is available to authorized users. Keywords Noonan syndrome Á Genotype–phenotype correlation Á PTPN11 Á SOS1 Á KRAS Á RAF1 J. M. Ko and J.-M. Kim contributed equally to this study. J. M. Ko Department of Pediatrics, Asan Medical Center, Introduction University of Ulsan College of Medicine, Seoul, South Korea Noonan syndrome (NS; OMIM 163950) is an autosomal J.-M. Kim dominant disorder with variable phenotype, characterized Genome Research Center for Birth defects and Genetic Diseases, by short stature, congenital heart disease, and typical facial Asan Medical Center, University of Ulsan College of Medicine, features (Noonan 1994). The main facial findings of NS are Seoul, South Korea hypertelorism with down-slanting palpebral fissures, ptosis, G.-H. Kim and low-set posteriorly rotated ears. Other manifestations Medical Genetics Clinic and Laboratory, are webbed neck, chest wall deformity, mild mental Asan Medical Center, University of Ulsan retardation, cryptorchidism in males, feeding difficulties College of Medicine, Seoul, South Korea in infancy, bleeding diathesis, and lymphatic dysplasia H.-W. Yoo (&) (Allanson et al. 1985). Department of Pediatrics, Medical Genetics Clinic The genes that cause NS encode members of the and Laboratory, Asan Medical Center, RAS-MAPK pathway. Gain-of-function mutations in the University of Ulsan College of Medicine, PTPN11 gene, the first identified NS-associated gene 388-1, Pungnap-2dong, Songpa-gu, Seoul 138-736, South Korea (Tartaglia et al. 2001), account for 30–60% of NS cases. In e-mail: [email protected] 2006, germline mutations in the KRAS gene were identified 123 1000 J Hum Genet (2008) 53:999–1006 in five patients with NS (Schubbert et al. 2006), and children (Burch et al. 1993). Because objective evalua- germline mutations in the SOS1 and RAF1 genes were tions, including the intelligence quotient, were not reported to cause NS in 2007. Therefore, deregulated RAS- performed in all patients of this study, the diagnosis of MAPK signaling arising from PTPN11, SOS1, KRAS, and delayed development or mental retardation was exclu- RAF1 mutations causes approximately 60–70% of NS sively restricted to the patient who had required special cases (Pandit et al. 2007; Razzaque et al. 2007). education. IGF-1 and IGF binding protein-3 (IGFBP-3) Attempts to find genotype–phenotype correlations have levels were measured by immunoradiometric assays at been performed by several authors (Bertola et al. 2006; diagnosis to evaluate the effects of these materials on Musante et al. 2003; Sarkozy et al. 2003; Zenker et al. growth. The levels of IGF-1 and IGFBP-3 are presented 2004). Considering that approximately half of NS cases as standard deviation scores (SDS) with reference to arise because of mutations in PTPN11, many studies have normal Korean values (Lee and Kim 2007). attempted to find a method for distinguishing the pheno- type of PTPN11 NS cases from NS attributable to other Mutational analysis causes. In some studies (Bertola et al. 2006; Tartaglia et al. 2002; Zenker et al. 2004), PS and hematological abnor- Genomic DNA was isolated from peripheral blood lym- malities were more prevalent in the NS group with phocytes using the PUREGENE DNA isolation kit PTPN11 gene mutations. On the other hand, Hypertrophic (Gentra, Minneapolis, MN). Four genes in the RAS- cardiomyopathy (HCM) was less often present in the NS MAPK pathway associated with Noonan syndrome were group with PTPN11 gene mutations (Tartaglia et al. 2002). analyzed for mutations; the genes were PTPN11, SOS1, Recent reports have suggested that there might be an KRAS, and RAF1. PCR and sequence analysis explored all association between KRAS gene mutation and mental coding exons, and their intronic flanking regions, except retardation (Zenker et al. 2007b), or between RAF1 gene in the case of RAF1. For the RAF1 gene, PCR and mutation and HCM (Pandit et al. 2007; Razzaque et al. sequence analysis focused on exons 7, 14, and 17, which 2007). Unusual ectodermal features, including facial ker- are major mutation sites (Pandit et al. 2007). Amplifica- atosis pilaris and curly hair, and generally normal tions were performed over 30 cycles, and each cycle development and growth were associated with SOS1 gene consisted of denaturation at 94°C for 30 s, annealing at mutations in another study (Tartaglia et al. 2007). In the 55°C for 30 s, and extension at 72°C for 45 s. PCR was present study, we conducted a mutation analysis on the carried out in a reaction volume of 10 ll, containing PTPN11, SOS1, KRAS, and RAF1 genes in Korean patients 100 ng of genomic DNA template, 10 pmol of each pri- with NS. In addition, we investigated genotype–phenotype mer, 200 lM of each dNTP, 2.5 mM MgCl2, 2.5 llof correlations. 109 buffer, and 1 unit of Taq DNA polymerase (Pro- mega, Madison, WI). Primer sequences are available on request. Materials and methods Subsequently, DNA sequencing reactions were per- formed using the same primer pairs, and the BigDye Clinical evaluation Terminator V3.1 Cycle Sequencing kit (Applied Biosys- tems, Foster City, CA) according to the manufacturer’s Fifty-nine unrelated Korean patients were diagnosed with instructions. Electrophoresis and analysis of sequencing NS by a single medical geneticist at the Asan Medical reaction mixtures were achieved with an ABI3130xl Center, Seoul, South Korea, between January 2000 and Genetic Analyzer (Applied Biosystems). July 2007. This study was approved by institutional review To predict the functional impact of amino acid changes, boards, and written informed consent to our work was we assessed novel sequence alterations by two in silico obtained from all subjects or from their parents. All prediction algorithms, PolyPhen (Polymorphism Pheno- patients had normal karyotypes. Inclusion criteria were typing) and SIFT (Sorting Intolerant from Tolerance), and based on the van der Burgt system (van der Burgt et al. performed molecular analyses in the patients’ parents and 1994). 100 healthy controls additionally. On the PolyPhen pro- Electrocardiograms, simple chest radiographs, and gram, a position-specific independent counts (PSIC) score echocardiograms were obtained from all patients for of[2.0 indicates probably damaging to protein function, a evaluation of cardiac anomalies. HCM was diagnosed score of 1.5–2.0 as possibly damaging, and a score of\1.5 when the left ventricular maximal end-diastolic wall as benign or unknown (Ng and Henikoff 2003; Ramensky thickness was [2 SD above the mean for a given age in et al. 2002). 123 J Hum Genet (2008) 53:999–1006 1001 Statistical analysis Table 1 PTPN11, SOS1, KRAS1, and RAF1 gene mutations identi- fied in 59 patients with NS Statistical analysis was carried out using the two-tailed Exon Nucleotide Amino acid Domain N (%) Fischer’s exact test and the Mann–Whitney U-test for substitution substitution between-group comparisons. P values of B0.05 were PTPN11 (N = 16) considered statistically significant. 3 c.175A [ G T59Aa N-SH2 1 (6.3) 3 c.184T [ G Y62D N-SH2 1 (6.3) 3 c.215C [ G A72G N-SH2 2 (12.5) Results 3 c.236A [ G Q79R N-SH2 2 (12.5) Study population 4 c.417G [ C E139D C-SH2 1 (6.3) 8 c.854T [ C F285S PTP 1 (6.3) Fifty-nine unrelated Korean NS patients (41 boys and 18 8 c.922A [°G N308D PTP 1 (6.3) girls) who met the inclusion criteria were enrolled in this 8 c.923A [ G N308S PTP 1 (6.3) study. Their ages at diagnosis ranged from 0.1 to 12 c.1382C [ T A461T PTP 2 (12.5) 17.2 years (median 3.7 years). Three of 59 cases were 12 c.1391G [ C G464A PTP 1 (6.3) familial (5.1%), and transmission of NS was maternal in 12 c.1403C [ T T468M PTP 1 (6.3) one patient and paternal in two patients. 13 c.1505A [ G S502L PTP 1 (6.3) 13 c.1510A [ G M504V PTP 1 (6.3) Spectrum of PTPN11, SOS1, KRAS, and RAF1 SOS1 (N = 10) mutations (Table 1) 4 c.508A [ G K170Ea HF 1 (10) 6 c.797C [ A T266K DH 1 (10) In 59 patients with NS, we identified mutations in 30 6 c.806T [ G M269R DH 2 (20) (50.8%).