Amanda Salem Brasil

Further evidence of genetic heterogeneity in Costello syndrome: involvement of the KRAS gene

AbstractCostello syndrome is an autosomal dominant disorder comprising growth deficiency, mental retardation, curly hair, coarse facial features, nasal papillomata, low-set ears with large lobes, cardiac anomalies, redundant skin in palms and soles with prominent creases, dark skin, and propensity to certain solid tumors. HRAS mutations have been implicated in approximately 85% of the affected cases. The clinical overlap among Costello, Noonan, and cardiofaciocutaneous syndromes is now better understood given their common molecular background, such that all these syndromes constitute a class of disorders caused by deregulated RAS-MAPK signaling. We report on a novel KRAS gene mutation in a patient presenting the clinical features typical of Costello syndrome and the additional findings seen in Noonan syndrome. This description emphasizes that a subset of patients with Costello syndrome could harbor mutations in other genes involved in the RAS-MAPK signaling.

Growth Standards of Patients With Noonan and Noonan-Like Syndromes With Mutations in the RAS/MAPK Pathway

Noonan syndrome (NS) and Noonan‐like syndromes (NLS) are autosomal dominant disorders caused by heterozygous mutations in genes of the RAS/MAPK pathway. The aim of the study was to construct specific growth charts for patients with NS and NLS. Anthropometric measurements (mean of 4.3 measurements per patient) were obtained in a mixed cross‐sectional and longitudinal mode from 127 NS and 10 NLS patients with mutations identified in PTPN11 (n = 90), SOS1 (n = 14), RAF1 (n = 10), KRAS (n = 8), BRAF (n = 11), and SHOC2 (n = 4) genes. Height, weight, and body mass index (BMI) references were constructed using the lambda, mu, sigma (LMS) method. Patients had birth weight and length within normal ranges for gestational age although a higher preterm frequency (16%) was observed. Mean final heights were 157.4 cm [−2.4 standard deviation score (SDS)] and 148.4 cm (−2.2 SDS) for adult males and females, respectively. BMI SDS was lower when compared to Brazilian standards (BMI SDS of −0.9 and −0.5 SDS for males and females, respectively). Patients harboring mutations in RAF1 and SHOC2 gene were shorter than other genotypes, whereas patients with SOS1 and BRAF mutations had more preserved postnatal growth. In addition, patients with RAF1 and BRAF had the highest BMI whereas patients with SHOC2 and KRAS mutations had the lowest BMI. The present study established the first height, weight, and BMI reference curves for NS and NLS patients, based only on patients with a proven molecular cause. These charts can be useful for the clinical follow‐up of patients with NS and NLS.

Autoimmune disease and multiple autoantibodies in 42 patients with RASopathies.

The association of RASopathies [Noonan syndrome (NS) and Noonan‐related syndromes] and autoimmune disorders has been reported sporadically. However, a concomitant evaluation of autoimmune diseases and an assessment of multiple autoantibodies in a large population of patients with molecularly confirmed RASopathy have not been performed. The clinical and laboratory features were analyzed in 42 RASopathy patients, the majority of whom had NS and five individuals had Noonan‐related disorders. The following autoantibodies were measured: Anti‐nuclear antibodies, anti‐double stranded DNA, anti‐SS‐A/Ro, anti‐SS‐B/La, anti‐Sm, anti‐RNP, anti‐Scl‐70, anti‐Jo‐1, anti‐ribosomal P, IgG and IgM anticardiolipin (aCL), thyroid, anti‐smooth muscle, anti‐endomysial (AE), anti‐liver cytosolic protein type 1 (LC1), anti‐parietal cell (APC), anti‐mitochondrial (AM) antibodies, anti‐liver‐kidney microsome type 1 antibodies (LKM‐1), and lupus anticoagulant. Six patients (14%) fulfilled the clinical criteria for autoimmune diseases [systemic lupus erythematous, polyendocrinopathy (autoimmune thyroiditis and celiac disease), primary antiphospholipid syndrome (PAPS), autoimmune hepatitis, vitiligo, and autoimmune thyroiditis]. Autoimmune antibodies were observed in 52% of the patients. Remarkably, three (7%) of the patients had specific gastrointestinal and liver autoantibodies without clinical findings. Autoimmune diseases and autoantibodies were frequently present in patients with RASopathies. Until a final conclusion of the real incidence of autoimmunity in Rasopathy is drawn, the physicians should be alerted to the possibility of this association and the need for a fast diagnosis, proper referral to a specialist and ultimately, adequate treatment.

PTPN11 and KRAS gene analysis in patients with Noonan and Noonan-like syndromes.

Noonan and Noonan-like syndromes are disorders of dysregulation of the rat sarcoma viral oncogene homolog (RAS)-mitogen-activated protein kinase signaling pathway. In Noonan syndrome (NS), four genes of this pathway (PTPN11, SOS1, RAF1, and KRAS) are responsible for roughly 70% of the cases. We analyzed PTPN11 and KRAS genes by bidirectional sequencing in 95 probands with NS and 29 with Noonan-like syndromes, including previously reported patients already screened for PTPN11 gene mutations. In the new patients with NS, 20/46 (43%) showed a PTPN11 gene mutation, two of them novel. In our total cohort, patients with NS and a PTPN11 mutation presented significantly higher prevalence of short stature (p = 0.03) and pulmonary valve stenosis (p = 0.01), and lower prevalence of hypertrophic cardiomyopathy (p = 0.01). Only a single gene alteration, of uncertain role, was found in the KRAS gene in an NS patient also presenting a PTPN11 gene mutation. We further analyzed the influence in clinical variability of three frequent polymorphisms found in the KRAS gene and no statistically significant difference was observed among the frequency of clinical findings regarding the studied polymorphisms.

Co-occurring PTPN11 and SOS1 gene mutations in Noonan syndrome: does this predict a more severe phenotype?

Noonan syndrome (NS) is an autosomal dominant disorder, with variable phenotypic expression, characterized by short stature, facial dysmorphisms and heart disease. Different genes of the RAS/MAPK signaling pathway are responsible for the syndrome, the most common are: PTPN11, SOS1, RAF1, and KRAS. The objective of this study was to report a patient with Noonan syndrome presenting mutations in two genes of RAS/MAPK pathway in order to establish whether these mutations lead to a more severe expression of the phenotype. We used direct sequencing of the PTPN11, SOS1, RAF1, and KRAS genes. We have identified two described mutations in heterozygosity: p.N308D and p.R552G in the genes PTPN11 and SOS1, respectively. The patient has typical clinical features similar to the ones with NS and mutation in only one gene, even those with the same mutation identified in this patient. A more severe or atypical phenotype was not observed, suggesting that these mutations do not exhibit an additive effect.

Ocular manifestations of Noonan syndrome

Purpose: To describe the ophthalmological characteristics in a group of Noonan syndrome patients with proven mutations in the PTPN11 gene. Methods: Thirty-five Noonan syndrome patients with PTPN11 gene mutations underwent ophthalmological exams, which consisted of external inspection, slit-lamp biomicroscopy examination and an ophthalmoscopic examination after instillation of 1.0% tropicamide or 1.0% cyclopentolate. Results: All 35 patients had at least one abnormality upon ophthalmological examination. The eyelid and external eye abnormalities were the prevailing features, followed by prominent corneal nerves on slit-lamp exam. Fundus changes were detected in 8% of the subjects, mainly associated with high myopia. No statistically significant differences were observed among the patients presenting specific mutations in the PTPN11 gene. Conclusions: The current study further supports the finding that ocular symptoms account for a large fraction of the clinical manifestations of NS. Additional characteristics are described here. The roles for the various mutations of PTPN11 in ocular development are yet to be established.

Multiple, diffuse schwannomas in a RASopathy phenotype patient with germline KRAS mutation: a causal relationship?

To the Editor : RAS proto-oncogenes (KRAS, HRAS and NRAS ) encode GTPases, and their role in cancer, as somatic mutations, is well known (1, 2). Germline mutations in genes within the RAS/MAPK signaling pathway are responsible for a group of phenotypically overlapping developmental disorders (RASopathies) (3), including Noonan syndrome (NS) and Noonan-related disorders: NS with multiple lentigines, cardio-facio-cutaneous syndrome and Costello syndrome (CS) (4). Tumor risk in RASopathies is not yet fully established, and more studies are needed (5, 6). NS patients harboring PTPN11 germline mutations have a 3.5-fold increased risk compared to the general population (6). CS patients carrying HRAS mutations have a higher incidence (approximately 15%) of certain solid tumors, especially rhabdomyosarcomas, and a screening surveillance protocol has been proposed (7, 8). Here, we describe the presence of multiple diffuse schwannomas in a patient harboring a germline KRAS mutation (p.K5E), previously reported on (9). The proband is a 23-year-old female with clinical features compatible with a RASopathy phenotype (Fig. 1a). She was initially evaluated in our service at 8 years old. At the age of 22, she started to experience difficulty in relaxation of the hand muscles, fatigue, abdominal pain and nausea. Six months later, a superficial nodule in her forth right intercostal space was palpable. An exploratory magnetic resonance imaging (MRI) scan of her cranium, thorax and abdomen showed diffuse enlargement of all nerve roots of the spine, brachial plexus, radial, ulnar and median nerves, as well as masses on the retroperitoneum (Fig. 1b), suggesting an overgrowth of peripheral nerve sheaths, compatible with a schwannoma or neurofibroma. No brain abnormalities were found by MRI scanning. Histopathological and immunohistochemical analysis of an excised thoracic nodule were compatible with the diagnosis of schwannoma (Fig. 1c). Combining histological and MRI findings, our final diagnosis was multiple diffuse schwannomas. In otherwise healthy patients, benign tumors of the peripheral nerve sheath typically occur singly. In rare cases malignant transformation occurs. The presence of multiple schwannomas in an individual suggests an underlying tumor predisposition syndrome, more particularly neurofibromatosis type II (NF2) or schwannomatosis (SMARCB1 ) (10). Comprehensive NF2 and SMARCB1 mutation analysis was performed on DNA extracted from blood and tumor. Analysis included sequencing of the entire coding region, dosage analysis by Multiplex Ligation Probe Assay and Loss of Heterozygosity analysis using 15 microsatellite markers along chromosome 22q. No pathogenic NF2/SMARCB1 mutations were identified. A novel NF2 benign intronic variant was identified in blood and tumor: c.88615C>T. This alteration replaces a non-evolutionary conserved pyrimidine by another pyrimidine at the splice acceptor of exon 10 and was proven not to affect splicing through RNA analysis in blood, in agreement with results from in silico splice predictions. Thus, the co-occurrence of a KRAS -associated phenotype and NF2/schwannomatosis caused by mutations in NF2 or SMARCB1 genes is highly unlikely. Therefore, KRAS could be in itself the tumor predisposing gene leading to diffuse schwannomas in our patient. As it is known that SMARCB1 mutations are only found in a minority of the NF2 -negative schwannomatosis patients (11, 12), we further performed sequence analysis of all KRAS exons in eight NF2/SMARCB1 negative schwannomatosis patients, but no mutations were found, indicating that KRAS might not be a major contributing gene in NF2/SMARCB1 -negative schwannomatosis patients. However, the presence of schwannoma has been reported previously in an NS and a CS patient, though this was prior to the identification of the genes causing these disorders (13, 14). Exacerbated growth of peripheral nerve sheaths (neurofibromas) is also observed in neurofibromatosis type 1 patients carrying NF1 mutations, another tumor suppressor gene of the RAS/MAPK pathway. Activation of this pathway regulates mTOR which is phosphoinositol 3′ kinase (PI3K) dependent, a critical effector pathway involved in disease pathogenesis (15). Although functional studies are not available for the mutation p.K5E found in our patient, a different gene

KRAS gene mutations in Noonan syndrome familial cases cluster in the vicinity of the switch II region of the G-domain: Report of another family with metopic craniosynostosis

Noonan syndrome (NS) and Noonan‐related disorders [cardio‐facio‐cutaneous (CFC), Costello, Noonan syndrome with multiple lentigines (NS‐ML), and neurofibromatosis‐Noonan syndromes (NFNS)] are a group of developmental disorders caused by mutations in genes of the RAS/MAPK pathway. Mutations in the KRAS gene account for only a small proportion of affected Noonan and CFC syndrome patients that present an intermediate phenotype between these two syndromes, with more frequent and severe intellectual disability in NS and less ectodermal involvement in CFC syndrome, as well as atypical clinical findings such as craniosynostosis. Recently, the first familial case with a novel KRAS mutation was described. We report on a second vertical transmission (a mother and two siblings) with a novel mutation (p.M72L), in which the proband has trigonocephaly and the affected mother and sister, prominent ectodermal involvement. Metopic suture involvement has not been described before, expanding the main different cranial sutures which can be affected in NS and KRAS gene mutations. The gene alteration found in the studied family is in close proximity to the one reported in the other familial case (close to the switch II region of the G‐domain), suggesting that this specific region of the gene could have less severe effects on intellectual ability than the other KRAS gene mutations found in NS patients and be less likely to hamper reproductive fitness.

Tegumentary manifestations of Noonan and Noonan-related syndromes

OBJECTIVES: Noonan and Noonan-related syndromes are common autosomal dominant disorders with neuro-cardio-facial-cutaneous and developmental involvement. The objective of this article is to describe the most relevant tegumentary findings in a cohort of 41 patients with Noonan or Noonan-related syndromes and to detail certain aspects of the molecular mechanisms underlying ectodermal involvement. METHODS: A standard questionnaire was administered. A focused physical examination and a systematic review of clinical records was performed on all patients to verify the presence of tegumentary alterations. The molecular analysis of this cohort included sequencing of the following genes in all patients: PTPN1, SOS1, RAF1, KRAS, SHOC2 and BRAF. RESULTS: The most frequent tegumentary alterations were xeroderma (46%), photosensitivity (29%), excessive hair loss (24%), recurrent oral ulcers (22%), curly hair (20%), nevi (17%), markedly increased palmar and plantar creases (12%), follicular hyperkeratosis (12%), palmoplantar hyperkeratosis (10%), café-au-lait spots (10%) and sparse eyebrows (7%). Patients with mutations in PTPN11 had lower frequencies of palmar and plantar creases and palmar/plantar hyperkeratosis compared with the other patients. CONCLUSIONS: We observed that patients with mutations in genes directly involved in cell proliferation kinase cascades (SOS1, BRAF, KRAS and RAF1) had a higher frequency of hyperkeratotic lesions compared with patients with mutations in genes that have a more complex interaction with and modulation of cell proliferation kinase cascades (PTPN11).

A possible role of different PTPN genes in immune regulation.

To the Editor: We have recently evaluated the presence of autoimmunity in 42 molecularly confirmed patients diagnosed with RASopathies [1], a class of common autosomaldominant syndromes with neurofaciocutaneous and developmental involvement that include Noonan syndrome and Noonan-related disorders [2]. We were surprised with the high frequency (14%) of the patients presenting an association with autoimmune diseases, including systemic lupus erythematosus, thyroiditis, autoimmune hepatitis and others, in a such young population (average age of 19 years). This represents a twoto threefold increase in frequency when compared with the normal population (5–8%) [3]. All of the patients with such association presented a pathogenic mutation in the PTPN11 gene, which encodes a tyrosine phosphorylation enzyme that belongs to the same family of PTPN22, widely known for its participation in autoimmunity [4]. Additionally, we also found alterations in the levels of immunoglobulins (Igs) in 14 (52%) of the 27 patients with mutations in the PTPN11 gene in whom the Igs were measured (Table 1). These patients had at least one immunoglobulin out of the normal range: IgA and IgM levels were below normal limits for age in, respectively, one patient each (respectively, 38.1 and 35 mg ⁄ dl) patients. IgG, IgM and IgE levels were elevated, respectively, in three (1607–1740 mg ⁄ dl), one (237.6 mg ⁄ dl) and 11 (188–160 mg ⁄ dl) patients. None of them presented clinical criteria for the diagnosis of an immunodeficiency, although this inquiry was not the primary focus of our study. We were not able to establish a genotype–phenotype correlation considering the level of the Igs nor a statistically relevant (P < 0.05) difference between the groups of patients because the number of patients, though considerable for a genetic disorder, was small to reach a statistical significance. More than 60% of the 107 known PTPNs are expressed in the leucocytes and play a key role in regulating many signalling pathways and processes, including signalling, activation and tolerance [5, 6]. The high frequency of autoimmunity and alterations in the levels of Igs in patients harbouring mutations in PTPN11 may suggest the involvement of other PTPNs, besides PTPN22, in the regulation of the immunity. While we wait for more studies to clarify the participations of other PTPNs in the regulation of immunity and to determine the real extent in the clinical outcomes, it is important for the clinicians to be aware of the possible association of immune dysregulation when dealing with RASopathic patients.