Alexsandra C. Malaquias

Noonan Syndrome and Related Disorders: A Review of Clinical Features and Mutations in Genes of the RAS/MAPK Pathway

Noonan syndrome (NS) is one of the most common syndromes transmitted by a mendelian mode. In recent years, germline mutations that affect components of the RAS-MAPK (mitogen-activated protein kinase) pathway were shown to be involved in the pathogenesis of NS and four rare syndromes with clinical features overlapping with NS: Leopard syndrome, cardio-facio-cutaneous syndrome, Costello syndrome and neurofibromatosis type 1. Several hormones act through receptors that stimulate the RAS-MAPK pathway, and therefore, NS and related disorders represent a remarkable opportunity to study the implication of the RAS-MAPK pathway in different endocrine systems. Additionally, children with NS frequently are referred to the endocrinologist because of short stature, delayed puberty and/or undescended testes in males. In this paper, we review the diagnostic, clinical and molecular aspects of NS and NS-related disorders.

Rare variants in SOS2 and LZTR1 are associated with Noonan syndrome

Background Noonan syndrome is an autosomal dominant, multisystemic disorder caused by dysregulation of the RAS/mitogen activated protein kinase (MAPK) pathway. Heterozygous, pathogenic variants in 11 known genes account for approximately 80% of cases. The identification of novel genes associated with Noonan syndrome has become increasingly challenging, since they might be responsible for very small fractions of the cases. Methods A cohort of 50 Brazilian probands negative for pathogenic variants in the known genes associated with Noonan syndrome was tested through whole-exome sequencing along with the relatives in the familial cases. Families from the USA and Poland with mutations in the newly identified genes were included subsequently. Results We identified rare, segregating or de novo missense variants in SOS2 and LZTR1 in 4% and 8%, respectively, of the 50 Brazilian probands. SOS2 and LZTR1 variants were also found to segregate in one American and one Polish family. Notably, SOS2 variants were identified in patients with marked ectodermal involvement, similar to patients with SOS1 mutations. Conclusions We identified two novel genes, SOS2 and LZTR1, associated with Noonan syndrome, thereby expanding the molecular spectrum of RASopathies. Mutations in these genes are responsible for approximately 3% of all patients with Noonan syndrome. While SOS2 is a natural candidate, because of its homology with SOS1, the functional role of LZTR1 in the RAS/MAPK pathway is not known, and it could not have been identified without the large pedigrees. Additional functional studies are needed to elucidate the role of LZTR1 in RAS/MAPK signalling and in the pathogenesis of Noonan syndrome.

Further evidence of the importance of RIT1 in Noonan syndrome

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.

The sitting height/height ratio for age in healthy and short individuals and its potential role in selecting short children for SHOX analysis.

Aims: To determine the presence of abnormal body proportion, assessed by sitting height/height ratio for age and sex (SH/H SDS) in healthy and short individuals, and to estimate its role in selecting short children for SHOX analysis. Methods: Height, sitting height and weight were evaluated in 1,771 healthy children, 128 children with idiopathic short stature (ISS), 58 individuals with SHOX defects (SHOX-D) and 193 females with Turner syndrome (TS). Results: The frequency of abnormal body proportion, defined as SH/H SDS >2, in ISS children was 16.4% (95% CI 10-22%), which was higher than in controls (1.4%, 95% CI 0.8-1.9%, p < 0.001). The SHOX gene was evaluated in all disproportionate ISS children and defects in this gene were observed in 19%. Among patients with SHOX-D, 88% of children (95% CI 75-100%) and 96% of adults had body disproportion. In contrast, SH/H SDS >2 were less common in children (48%, 95% CI 37-59%) and in adults (28%, 95% CI 20-36%) with TS. Conclusion: Abnormal body proportions were observed in almost all individuals with SHOX-D, 50% of females with TS and 16% of children considered ISS. Defects in SHOX gene were identified in 19% of ISS children with SH/H SDS >2, suggesting that SH/H SDS is a useful tool to select children for undergoing SHOX molecular studies.

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.

Genome-wide screening of copy number variants in children born small for gestational age reveals several candidate genes involved in growth pathways

BACKGROUND The etiology of prenatal-onset short stature with postnatal persistence is heterogeneous. Submicroscopic chromosomal imbalances, known as copy number variants (CNVs), may play a role in growth disorders. OBJECTIVE To analyze the CNVs present in a group of patients born small for gestational age (SGA) without a known cause. PATIENTS AND METHODS A total of 51 patients with prenatal and postnatal growth retardation associated with dysmorphic features and/or developmental delay, but without criteria for the diagnosis of known syndromes, were selected. Array-based comparative genomic hybridization was performed using DNA obtained from all patients. The pathogenicity of CNVs was assessed by considering the following criteria: inheritance; gene content; overlap with genomic coordinates for a known genomic imbalance syndrome; and overlap with CNVs previously identified in other patients with prenatal-onset short stature. RESULTS In 17 of the 51 patients, 18 CNVs were identified. None of these imbalances has been reported in healthy individuals. Nine CNVs, found in eight patients (16%), were categorized as pathogenic or probably pathogenic. Deletions found in three patients overlapped with known microdeletion syndromes (4q, 10q26, and 22q11.2). These imbalances are de novo, gene rich and affect several candidate genes or genomic regions that may be involved in the mechanisms of growth regulation. CONCLUSION Pathogenic CNVs in the selected patients born SGA were common (at least 16%), showing that rare CNVs are probably among the genetic causes of short stature in SGA patients and revealing genomic regions possibly implicated in this condition.

Genetic Predictors of Long-Term Response to Growth Hormone (GH) Therapy in Children With GH Deficiency and Turner Syndrome: The Influence of a SOCS2 Polymorphism

BACKGROUND There is great interindividual variability in the response to GH therapy. Ascertaining genetic factors can improve the accuracy of growth response predictions. Suppressor of cytokine signaling (SOCS)-2 is an intracellular negative regulator of GH receptor (GHR) signaling. OBJECTIVE The objective of the study was to assess the influence of a SOCS2 polymorphism (rs3782415) and its interactive effect with GHR exon 3 and -202 A/C IGFBP3 (rs2854744) polymorphisms on adult height of patients treated with recombinant human GH (rhGH). DESIGN AND PATIENTS Genotypes were correlated with adult height data of 65 Turner syndrome (TS) and 47 GH deficiency (GHD) patients treated with rhGH, by multiple linear regressions. Generalized multifactor dimensionality reduction was used to evaluate gene-gene interactions. RESULTS Baseline clinical data were indistinguishable among patients with different genotypes. Adult height SD scores of patients with at least one SOCS2 single-nucleotide polymorphism rs3782415-C were 0.7 higher than those homozygous for the T allele (P < .001). SOCS2 (P = .003), GHR-exon 3 (P= .016) and -202 A/C IGFBP3 (P = .013) polymorphisms, together with clinical factors accounted for 58% of the variability in adult height and 82% of the total height SD score gain. Patients harboring any two negative genotypes in these three different loci (homozygosity for SOCS2 T allele; the GHR exon 3 full-length allele and/or the -202C-IGFBP3 allele) were more likely to achieve an adult height at the lower quartile (odds ratio of 13.3; 95% confidence interval of 3.2-54.2, P = .0001). CONCLUSION The SOCS2 polymorphism (rs3782415) has an influence on the adult height of children with TS and GHD after long-term rhGH therapy. Polymorphisms located in GHR, IGFBP3, and SOCS2 loci have an influence on the growth outcomes of TS and GHD patients treated with rhGH. The use of these genetic markers could identify among rhGH-treated patients those who are genetically predisposed to have less favorable outcomes.

The Interactive Effect of GHR-Exon 3 and −202 A/C IGFBP3 Polymorphisms on rhGH Responsiveness and Treatment Outcomes in Patients with Turner Syndrome

CONTEXT There is great interindividual variability in the response to recombinant human (rh) GH therapy in patients with Turner syndrome (TS). Ascertaining genetic factors can improve the accuracy of growth response predictions. OBJECTIVE The objective of the study was to assess the individual and combined influence of GHR-exon 3 and -202 A/C IGFBP3 polymorphisms on the short- and long-term outcomes of rhGH therapy in patients with TS. DESIGN AND PATIENTS GHR-exon 3 and -202 A/C IGFBP3 genotyping (rs2854744) was correlated with height data of 112 patients with TS who remained prepubertal during the first year of rhGH therapy and 65 patients who reached adult height after 5 ± 2.5 yr of rhGH treatment. MAIN OUTCOME MEASURES First-year growth velocity and adult height were measured. RESULTS Patients carrying at least one GHR-d3 or -202 A-IGFBP3 allele presented higher mean first-year growth velocity and achieved taller adult heights than those homozygous for GHR-fl or -202 C-IGFBP3 alleles, respectively. The combined analysis of GHR-exon 3 and -202 A/C IGFBP3 genotypes showed a clear nonadditive epistatic influence on adult height of patients with TS treated with rhGH (GHR-exon 3 alone, R² = 0.27; -202 A/C IGFBP3, R² = 0.24; the combined genotypes, R² = 0.37 at multiple linear regression). Together with clinical factors, these genotypes accounted for 61% of the variability in adult height of patients with TS after rhGH therapy. CONCLUSION Homozygosity for the GHR-exon3 full-length allele and/or the -202C-IGFBP3 allele are associated with less favorable short- and long-term growth outcomes after rhGH treatment in patients with TS.