Genotypic Findings in Noonan and Non-Noonan RASopathies and Patient Eligibility for Growth Hormone Treatment.

Molecular study has become an invaluable tool in the field of RASopathies. Treatment with recombinant human growth hormone is approved in Noonan syndrome but not in the other RASopathies. The aim of this study was to learn about the molecular base of a large cohort of patients with RASopathies, with particular emphasis on patients with pathogenic variants in genes other than PTPN11, and its potential impact on rGH treatment indication. We reviewed the clinical diagnosis and molecular findings in 451 patients with a genetically confirmed RASopathy. HRAS alterations were detected in only 2 out of 19 patients referred with a Costello syndrome suspicion, whereas pathogenic variants in RAF1 and SHOC2 were detected in 3 and 2, respectively. In 22 patients referred with a generic suspicion of RASopathy, including cardiofaciocutaneous syndrome, pathogenic alterations in classic Noonan syndrome genes (PTPN11, SOS1, RAF1, LZTR1, and RIT1) were found in 7 patients and pathogenic variants in genes associated with other RASopathies (HRAS, SHOC2, and PPPCB1) in 4. The correct nosological classification of patients with RASopathies is critical to decide whether they are candidates for treatment with rhGH. Our data illustrate the complexity of differential diagnosis in RASopathies, as well as the importance of genetic testing to guide the diagnostic orientation in these patients.

Impact of COVID-19 pandemic on HbA1c management and results in pediatric and adult outpatients with diabetes.

Objectives: Diabetes mellitus intensify the risks and complications related to COVID-19 infection. A major effect of the pandemic has been a drastic reduction of in-person visits. The aim of this study was to evaluate the impact of the COVID-19 pandemic on HbA1c management and results among pediatric and adult outpatients with diabetes, considering the laboratory and point-of-care testing (POCT) HbA1c measurements. Methods: Observational retrospective study including patients from pediatric and adult diabetes units was conducted. HbA1c results obtained in the laboratory and POCT over 3 years (2019-2021) were collected from the laboratory information system. Results: After the lockdown, the number of HbA1c plummeted. Children returned soon to routine clinical practice. The number of HbA1c increased gradually in adults, especially in POCT. Globally, HbA1c results were lower in children compared with adults (p<0.001). HbA1c values in children (p<0.001) and adults (p=0.002) decreased between pre-pandemic and post-pandemic periods, though lower than the HbA1c reference change value. The percentage of HbA1c results above 8% remained stable during the study period. Conclusions: Continuous glucose monitoring and a telemedicine have been crucial, even allowing for improvements in HbA1c results. During the lockdown, patients with better metabolic control were managed in the laboratory whereas patients with poorer control or a severe clinical situation were attended in diabetes units by POCT. Adults returned to pre-pandemic management slowly because they were more susceptible to morbidity and mortality due to COVID-19. Coordination among all health professionals has been essential to offering the best management, especially in difficult scenarios such as the COVID-19 pandemic.

High prevalence of variants in skeletal dysplasia associated genes in individuals with short stature and minor skeletal anomalies.

OBJECTIVE: Next generation sequencing (NGS) has expanded the diagnostic paradigm turning the focus to the growth plate. The aim of the study was to determine the prevalence of variants in genes implicated in skeletal dysplasias in probands with short stature and mild skeletal anomalies. DESIGN: Clinical and radiological data were collected from 108 probands with short stature and mild skeletal anomalies. METHODS: A customized skeletal dysplasia NGS panel was performed. Variants were classified using ACMG recommendations and Sherloc. Anthropometric measurements and skeletal anomalies were subsequently compared in those with or without an identified genetic defect. RESULTS: Heterozygous variants were identified in 21/108 probands (19.4%). Variants were most frequently identified in ACAN (n = 10) and IHH (n = 7) whilst one variant was detected in COL2A1, CREBBP, EXT1, and PTPN11. Statistically significant differences (P < 0.05) were observed for sitting height/height (SH/H) ratio, SH/H ratio standard deviation score (SDS), and the SH/H ratio SDS >1 in those with an identified variant compared to those without. CONCLUSIONS: A molecular defect was elucidated in a fifth of patients. Thus, the prevalence of mild forms of skeletal dysplasias is relatively high in individuals with short stature and mild skeletal anomalies, with variants in ACAN and IHH accounting for 81% of the cases. An elevated SH/H ratio appears to be associated with a greater probability in detecting a variant, but no other clinical or radiological feature has been found determinant to finding a genetic cause. Currently, we cannot perform extensive molecular studies in all short stature individuals so detailed clinical and radiological phenotyping may orientate which are the candidate patients to obtain worthwhile results. In addition, detailed phenotyping of probands and family members will often aid variant classification.

[Noonan syndrome: genetic and clinical update and treatment options]. / Síndrome de Noonan: actualización genética, clínica y de opciones terapéuticas.

Noonan syndrome (NS) is a relatively common genetic condition characterised by short stature, congenital heart defects, and distinctive facial features. NS and other clinically overlapping conditions such as NS with multiple lentigines (formerly called LEOPARD syndrome), cardiofaciocutaneous syndrome, or Costello syndrome, are caused by mutations in genes encoding proteins of the RAS-MAPKinases pathway. Because of this shared mechanism, these conditions have been collectively termed «RASopathies¼. Despite the recent advances in molecular genetics, nearly 20% of patients still lack a genetic cause, and diagnosis is still made mainly on clinical grounds. NS is a clinically and genetically heterogeneous condition, with variable expressivity and a changing phenotype with age, and affects multiple organs and systems. Therefore, it is essential that physicians involved in the care of these patients are familiarised with their manifestations and the management recommendations, including management of growth and development. Data on growth hormone treatment efficacy are sparse, and show a modest response in height gains, similar to that observed in Turner syndrome. The role of RAS/MAPK hyper-activation in the pathophysiology of this group of disorders offers a unique opportunity for the development of targeted approaches.

Body Composition as a Mediator between Cardiorespiratory Fitness and Bone Mass during Growth.

INTRODUCTION AND PURPOSE: To examine the effect of cardiorespiratory fitness (CRF) and muscle power output (MPO) on bone mass of prepubertal and pubertal children using lean mass (LM) and percentage of fat mass (%FM) as mediator variables. The hypothesis was that both LM and %FM would be independent mediators of the relationships during the sexual maturation period. METHODS: We analyzed 200 children (88 boys and 112 girls [11.5 ± 2.0 yr]). Body composition was analyzed by bone densitometry, and indirect calorimetry and cycle ergometer were used to calculate V˙O2peak (mL·kg·min) and MPO (W) during an incremental exercise test. Sample was divided by pubertal status. RESULTS: In the prepubertal group, LM and %FM acted independently as mediators in the relationship between bone mass and CRF or MPO (22%-25% for LM and 37%-50% for %FM, respectively). In pubertal children, LM acted as mediator at 37%. CONCLUSIONS: Although the independent mediator role of LM and %FM in the associations between CRF or MPO and bone mass was present during the prepubertal stage, only LM remain its mediator role in these associations during the postpubertal period. Therefore, with growth and sexual maturation, the full effect of LM seems to increase, whereas the influence of %FM seems to disappear.

[Cardiofaciocutaneous syndrome, a Noonan syndrome related disorder: clinical and molecular findings in 11 patients]. / Síndrome cardiofaciocutáneo, un trastorno relacionado con el síndrome de Noonan: hallazgos clínicos y moleculares en 11 pacientes.

OBJECTIVES: To describe 11 patients with cardiofaciocutaneous syndrome (CFC) and compare them with 130 patients with other RAS-MAPK syndromes (111 Noonan syndrome patients [NS] and 19 patients with LEOPARD syndrome). PATIENTS AND METHODS: Clinical data from patients submitted for genetic analysis were collected. Bidirectional sequencing analysis of PTPN11, SOS1, RAF1, BRAF, and MAP2K1 focused on exons carrying recurrent mutations, and of all KRAS exons were performed. RESULTS: Six different mutations in BRAF were identified in 9 patients, as well as 2 MAP2K1 mutations. Short stature, developmental delay, language difficulties and ectodermal anomalies were more frequent in CFC patients when compared with other neuro-cardio-faciocutaneous syndromes (P<.05). In at least 2 cases molecular testing helped reconsider the diagnosis. DISCUSSION: CFC patients showed a rather severe phenotype but at least one patient with BRAF mutation showed no developmental delay, which illustrates the variability of the phenotypic spectrum caused by BRAF mutations. Molecular genetic testing is a valuable tool for differential diagnosis of CFC and NS related disorders.

LEOPARD syndrome: a variant of Noonan syndrome strongly associated with hypertrophic cardiomyopathy.

INTRODUCTION AND OBJECTIVES: LEOPARD syndrome is an autosomal dominant condition related to Noonan syndrome, although it occurs less frequently. The aim of this study was to characterize the clinical and molecular features of a large series of LEOPARD syndrome patients. METHODS: We collected clinical data from 19 patients in 10 hospitals. Bidirectional sequencing analysis of PTPN11, RAF1, and BRAF focused on exons carrying recurrent mutations. RESULTS: After facial dysmorphism, structural heart defects (88%) were the most common feature described. Hypertrophic cardiomyopathy (71%) was diagnosed more often than pulmonary valve stenosis (35%). Multiple lentigines or café au lait spots were found in 84% of the series, and deafness was diagnosed in 3 patients. Mutations in PTPN11 were identified in 16 (84%) patients (10 patients had the recurrent LEOPARD syndrome mutation, p.Thr468Met) (NP_002825.3T468M). Two other patients had a mutation in RAF, and 1 patient had a mutation in BRAF. When compared with other neurocardiofaciocutaneous syndromes, LEOPARD syndrome patients showed a higher prevalence of hypertrophic cardiomyopathy and cutaneous abnormalities, and a lower prevalence of pulmonary valve stenosis and short stature. CONCLUSIONS: LEOPARD syndrome patients display distinctive features apart from multiple lentigines, such as a higher prevalence of hypertrophic cardiomyopathy and lower prevalence of short stature. Given its clinical implications, active search for hypertrophic cardiomyopathy is warranted in Noonan syndrome spectrum patients, especially in LEOPARD syndrome patients.

Alterations in RAS-MAPK genes in 200 Spanish patients with Noonan and other neuro-cardio-facio-cutaneous syndromes. Genotype and cardiopathy.

INTRODUCTION AND OBJECTIVES: Molecular characterization of congenital heart diseases now includes the not infrequent dysmorphic Noonan syndrome. A study of 6 genes of the RAS-MAPK pathway in Spanish patients is presented: the impact of heart disease, clinical expressivity, and diagnostic yield are investigated. METHODS: The study included 643 patients (and 182 family members) diagnosed by dysmorphologists, cardiologists, and pediatric endocrinologists from 74 tertiary hospitals. Bidirectional sequencing analysis of PTPN11, SOS1, RAF1, BRAF, KRAS and HRAS focused on exons carrying recurrent mutations accounting for 80% to 95% of previously described mutations. RESULTS: Mutations were detected in 230 patients (91 women and 139 men) in 200 (31%) families (172 PTPN11+, 14 SOS1+, 9 RAF1+, 5 BRAF+). There was specific reference to the heart defect suffered in 156 index cases: 103 patients had shown pulmonary stenosis, 12 pulmonary stenosis with hyperthrophic cardiomyopathy, 18 hypertrophic cardiomiopathy, and 14 other cardiopathies; heart disease was absent in 9 index cases. Heart disease had not been documented in 23 of 30 family members with positive genotype and compatible clinical signs. Diagnostic yield was higher (P=.016) for samples from some centers (53%; 14/32) and even from certain professionals (64%; 9/14; P=.019). Characterization rate was 18% in patients for whom clinical data were not available. Genotyping led to a more precise diagnosis in 26 patients. CONCLUSIONS: Most patients (94%) with a positive genotype had known congenital heart disease, 79% pulmonary stenosis and 12% hyperthrophic cardiomyopathy. Cardiopathy had not been documented in 76% of family members carrying the mutation. Molecular study is a useful tool in these syndromes but a more rigorous clinical diagnosis should be intended as well.

Long-term benefits in lung function and nutritional status of strict metabolic control of cystic fibrosis-related diabetes.

Cystic fibrosis related diabetes (CFRD) is a strong determinant for lung function decline and increased mortality. Insulin treatment of CFRD is reportedly beneficial for this situation. We report on the long-term impact of insulin treatment of CFRD on pulmonary function and nutritional status in a CF male patient since diagnosis of diabetes. We report the case of a patient diagnosed with CF at the age of 16. Two years later, he experienced a rapidly evolving decrease in pulmonary function, some months later criteria were met warranting lung transplantation. Concomitantly, he was diagnosed with CFRD and insulin therapy was started. Lung function (spirometry), nutritional status (body mass index) and metabolic control (HbA(1c)) were determined every 3 months. After the introduction of insulin treatment, pulmonary function and nutritional status progressively improved and good glycemic control was achieved. The significant and sustained improvement in pulmonary function allowed for the patient's withdrawal from the lung transplantation program within 4 months, a situation which has been maintained until now, 8 years later. The long follow-up of our patient documents the rapid and prolonged beneficial effect of proper metabolic control of CFRD on the respiratory deterioration in CF.

LEOPARD syndrome (PTPN11, T468M) in three boys fulfilling neurofibromatosis type 1 clinical criteria.

Noonan syndrome (NS) and neurofibromatosis type 1 (NF1) are well-defined entities. The association of both disorders is called neurofibromatosis-Noonan syndrome (NFNS), a disorder that has been related to mutations in the NF1 gene. Both NS and NFNS display phenotypic overlapping with LEOPARD syndrome (LS), and differential diagnosis between these two entities often represents a challenge for clinicians. We report on three patients (two brothers and a not-related patient) diagnosed as having NFNS. They fulfilled NF1 diagnostic criteria and had some features of NS. The three of them had hypertophic cardiomyopathy while neurofibromas, Lisch nodules, and unidentified bright objects on MRI were absent. PTPN11 gene assays revealed a T468M mutation, typical of LS. Thorough clinical examinations of the patients revealed multiple lentigines, which were considered to be freckling in the initial evaluation. We conclude that NF1 clinical criteria should be used with caution in patients with features of NS. Patients with hyperpigmented cutaneous spots associated with cardiac anomalies, even if fulfilling the minimal NF1 criteria for diagnosis, should be strongly considered for LS diagnosis.

Two cases of LEOPARD syndrome--RAF1 mutations firstly described in children.

. LEOPARD syndrome 2 (LS-2) (OMIM #611554) is a rare, dominantly inherited genetic disorder affecting multiple organ systems. We report two unrelated females of different ages whose phenotype fits best in the category of LEOPARD syndrome, both with proven mutations in the RAF1 gene not previouslyreported in pediatric patients. In our 10-year-old patient, who was negative in the PTPN11 gene analysis but involving the RAF1 gene in a complementary analysis, the sequence variant Ser257Leu (770C > T, exon 7) was detected, which previously had been reported in only one 35-year-old woman with LS. The second patient was a two-year-old female infant with Ser259Leu mutation in the same gene, described in several patients with Noonan syndrome (NS) but not in LS patients of any age. The first girl had ventricular and supraventricular extrasystoles, and the second had episodes of paroxysmal supraventricular tachycardia. Echocardiographic examination revealed biventricular obstructive hypertrophic cardiomyopathy in both patients.

Insulin dose adjustment when changing from multiple daily injections to continuous subcutaneous insulin infusion in the pediatric age group.

The aim of this study is to determine the proper initial dose adjustment when switching from multiple daily injections to continuous subcutaneous insulin infusion for type-1 diabetic pediatric patients. Our hypothesis is that the insulin adjustment varies depending on the pubertal status and the previous long-acting insulin used. Charts of 60 patients were reviewed. Data regarding insulin dose, type of insulin administrated, HbA1c, BMI, severe hypoglycemia and DKA events were collected during the previous year and after 6 weeks of pump therapy. In the prepubertal patients the reduction was 19% (26% if the previous insulin used was detemir). Pubertal patients experienced a decrease of 26%, and the detemir group 33%. The ratio long acting-basal/short acting-bolus insulin changed from 1.26 ± 0.84 to 0.93 ± 0.46 (P < 0.05). The total daily insulin dose needs to be decreased. Basal insulin constitutes 40-45% in prepubertal and 45-50% in pubertal patients. The reduction is different depending on the previous long-acting insulin used; being greater if the insulin is detemir.

An XX male with an intratubular undifferentiated germ cell neoplasia.

OBJECTIVE: To report a case of a 46,XX male with an intratubular undifferentiated germ cell neoplasia within an extra-abdominal gonad. DESIGN: Case report. SETTING: Molecular, cytogenetic, pathologic, and clinical units of three tertiary hospitals. PATIENT(S): A male with ambiguous genitalia at birth and descended testes observed in a pediatric endocrinology setting. INTERVENTION(S): Physical examination, hormonal assays, cytogenetic investigation, molecular analysis, surgical intervention for biopsies and bilateral orchiectomy, and pathologic evaluation. MAIN OUTCOME MEASURE(S): Pathologic evaluation with immunostaining for placental alkaline phosphatase and C-kit. RESULT(S): Conventional chromosome analysis revealed a 46,XXq- karyotype, and fluorescence in situ hybridization experiments with the SRY probe found a signal at the short arm of the deleted X chromosome. Molecular analysis indicated the presence of a portion of the short arm of the Y chromosome including the proto-oncogene TSPY. Pathologic evaluation of the gonads revealed an intratubular undifferentiated germ cell neoplasia. CONCLUSION(S): This is the first case of a 46,XX male with descended testes in whom an intratubular undifferentiated germ cell neoplasia developed. When proposals of management in this subgroup of disorders of sexual differentiation are formulated, the risk of germ cell malignancy must be taken into account.

New mutation type in pseudohypoparathyroidism type Ia.

CONTEXT: The GNAS gene encodes the alpha-subunit of the stimulatory G proteins, which play a crucial role in intracellular signal transduction of peptide and neurotransmitter receptors. Heterozygous inactivating maternally inherited mutations of GNAS (including translation initiation mutations, amino acid substitutions, nonsense mutations, splice site mutations and small insertions or deletions) lead to a phenotype in which Albright hereditary osteodystrophy is associated with pseudohypoparathyroidism type Ia. OBJECTIVE: We sought to identify the molecular defect in a patient who was thought to have PHP-Ia. METHODS AND RESULTS: The GNAS gene of a 5-year-old boy with brachydactily, mental retardation, pseudohypoparathyroidism and congenital hypothyroidism was investigated. We found a heterozygous inversion of exon 2 and part of intron 1 of de novo origin. Molecular studies of cDNA from blood RNA demonstrated that both the normal and the mutant variants were stable and that new splice-sites were generated. CONCLUSION: This report demonstrates the first evidence for an inversion at the GNAS gene responsible of pseudohypoparathyroidism type Ia.