NBAS pathogenic variants: Defining the associated clinical and facial phenotype and genotype-phenotype correlations.

The pathogenic variants in the neuroblastoma-amplified sequence (NBAS) are associated with a clinical spectrum involving the hepatic, skeletal, ocular, and immune systems. Here, we report on two unrelated subjects with a complex phenotype solved by whole-exome sequencing, who shared a synonymous change in NBAS that was documented to affect the transcript processing and co-occurring with a truncating change. Starting from these two cases, we systematically assessed the clinical information available for all subjects with biallelic NBAS pathogenic variants (73 cases in total). We revealed a recognizable facial profile (hypotelorism, thin lips, pointed chin, and "progeroid" appearance) determined by using DeepGestalt facial recognition technology, and we provide evidence for the occurrence of genotype-phenotype correlations. Notably, severe hepatic involvement was associated with variants affecting the NBAS-Nter and Sec39 domains, whereas milder liver involvement and immunodeficiency were generally associated with variants located at the N-terminus and C-terminus of the protein. Remarkably, no patient was reported to carry two nonsense variants, suggesting lethality of complete NBAS loss-of-function.

Structural, Functional, and Clinical Characterization of a Novel PTPN11 Mutation Cluster Underlying Noonan Syndrome.

Germline mutations in PTPN11, the gene encoding the Src-homology 2 (SH2) domain-containing protein tyrosine phosphatase (SHP2), cause Noonan syndrome (NS), a relatively common, clinically variable, multisystem disorder. Here, we report on the identification of five different PTPN11 missense changes affecting residues Leu261 , Leu262 , and Arg265 in 16 unrelated individuals with clinical diagnosis of NS or with features suggestive for this disorder, specifying a novel disease-causing mutation cluster. Expression of the mutant proteins in HEK293T cells documented their activating role on MAPK signaling. Structural data predicted a gain-of-function role of substitutions at residues Leu262 and Arg265 exerted by disruption of the N-SH2/PTP autoinhibitory interaction. Molecular dynamics simulations suggested a more complex behavior for changes affecting Leu261 , with possible impact on SHP2's catalytic activity/selectivity and proper interaction of the PTP domain with the regulatory SH2 domains. Consistent with that, biochemical data indicated that substitutions at codons 262 and 265 increased the catalytic activity of the phosphatase, while those affecting codon 261 were only moderately activating but impacted substrate specificity. Remarkably, these mutations underlie a relatively mild form of NS characterized by low prevalence of cardiac defects, short stature, and cognitive and behavioral issues, as well as less evident typical facial features.

Constitutional bone impairment in Noonan syndrome.

Noonan syndrome (NS) is an autosomal dominant trait characterized by genotypic and phenotypic variability. It belongs to the Ras/MAPK pathway disorders collectively named Rasopathies or neurocardiofaciocutaneous syndromes. Phenotype is characterized by short stature, congenital heart defects, facial dysmorphisms, skeletal and ectodermal anomalies, cryptorchidism, mild to moderate developmental delay/learning disability, and tumor predisposition. Short stature and skeletal dysmorphisms are almost constant and several studies hypothesized a role for the RAS pathway in regulating bone metabolism. In this study, we investigated the bone quality assessed by phalangeal quantitative ultrasound (QUS) and the metabolic bone profiling in a group of patients with NS, to determine whether low bone mineralization is primary or secondary to NS characteristics. Thirty-five patients were enrolled, including 20 males (55.6%) and 15 females (44.5%) aged 1.0-17.8 years (mean 6.4 ± 4.5, median 4.9 years). Each patients was submitted to clinical examination, estimation of the bone age, laboratory assays, and QUS assessment. Twenty-five percent of the cohort shows reduced QUS values for their age based on bone transmission time. Bone measurement were adjusted for multiple factors frequently observed in NS patients, such as growth retardation, delayed bone age, retarded puberty, and reduced body mass index, potentially affecting bone quality or its appraisal. In spite of the correction attempts, QUS measurement indicates that bone impairment persists in nearly 15% of the cohort studied. Our results indicate that bone impairment in NS is likely primary and not secondary to any of the phenotypic traits of NS, nor consistent with metabolic disturbances. © 2017 Wiley Periodicals, Inc.

Cancer Risk in Beckwith-Wiedemann Syndrome: A Systematic Review and Meta-Analysis Outlining a Novel (Epi)Genotype Specific Histotype Targeted Screening Protocol.

OBJECTIVE: To compare tumor risk in the 4 Beckwith-Wiedemann syndrome (BWS) molecular subgroups: Imprinting Control Region 1 Gain of Methylation (ICR1-GoM), Imprinting Control Region 2 Loss of Methylation (ICR2-LoM), Chromosome 11p15 Paternal Uniparental Disomy (UPD), and Cyclin-Dependent Kinase Inhibitor 1C gene (CDKN1C) mutation. STUDY DESIGN: Studies on BWS and tumor development published between 2000 and 2015 providing (epi)genotype-cancer correlations with histotype data were reviewed and meta-analysed with cancer histotypes as measured outcome and (epi)genotype as exposure. RESULTS: A total of 1370 patients with BWS were included: 102 developed neoplasms (7.4%). Tumor prevalence was 2.5% in ICR2-LoM, 13.8% in UPD, 22.8% in ICR1-GoM, and 8.6% in patients with CDKN1C mutations. Cancer ORs were 12.8 in ICR1-GoM, 6.5 in UPD, and 2.9 in patients with CDKN1C mutations compared with patients with ICR2-LoM. Wilms tumor was associated with ICR1-GoM (OR 68.3) and UPD (OR 13.2). UPD also was associated with hepatoblastoma (OR 5.2) and adrenal carcinoma (OR 7.0), and CDKN1C mutations with neuroblastic tumors (OR 7.2). CONCLUSION: Cancer screening in BWS could be differentiated on the basis of (epi)genotype and target specific histotypes. Patients with ICR1-GoM and UPD should undergo renal ultrasonography scanning, given their risk of Wilms tumor. Alpha feto protein monitoring for heptaoblastoma is suggested in patients with UPD. Adrenal carcinoma may deserve screening in patients with UPD. Patients with CDKN1C mutations may deserve neuroblastoma screening based on urinary markers and ultrasonography scanning. Finally, screening appears questionable in cases of ICR2-LoM, given low tumor risk.

Genealogy of breastfeeding.

Decline and resurgence of breastfeeding (BF) characterized last century. Several factors influencing BF outcome were identified. Despite the huge literature on BF, no data on its matrilineal transmission are available. BF practice was prospectively followed in 2546 Italian mothers. Lactation and BF outcome were related, besides to known factors interfering with BF, to the occurrence of previous maternal and paternal BF. Recalls of grandmaternal and grand-grandmaternal BF behaviours allowed the construction of familiar pedigrees of BF across three generations. Having been breastfed was the strongest factor addressing successful BF establishment (odds ratio (OR) 9.33; 95% confidence interval (CI) 7.40-11.84; p < 0.0001) and BF duration (at 6 months: OR 3.79; 95% CI 3.11-4.64; p < 0.0001). The hazard ratio for breastfed vs non-breastfed mothers was 0.46 (95% CI 0.41-0.50; log-rank p < 0.0001). The rate of BF failures was fivefold higher in non-breastfed mothers, mostly occurring during lactogenesis when the let-down reflex becomes essential. CONCLUSION: At any generation, mothers are likely to have daughters repeating their BF experience. Differently from the intergenerational effects of environmental factors responsible for the BF secular trend, this trait is transgenerationally transmitted and reversible, with temporal and clinical features of lactation failure. Accordingly, we speculate that epigenetic mechanisms might alter offspring's oxytocinergic receptor signalling. WHAT IS KNOWN: Several cultural and socio-demographic factors are known to influence breastfeeding outcome. The generational effects of breastfeeding itself have not been investigated so far. WHAT IS NEW: Maternal breastfeeding is the most important factor addressing daughters' breastfeeding outcome. This behavior is transmitted transgenerationally, with features suggesting epigenetic mechanisms.

Phenotypic variability associated with the invariant SHOC2 c.4A>G (p.Ser2Gly) missense mutation.

Noonan-like syndrome with loose anagen hair (NS/LAH; OMIM 607721) is a developmental disorder clinically related to Noonan syndrome (NS) and characterized by facial dysmorphisms, postnatal growth retardation, cardiac anomalies (in particular dysplasia of the mitral valve and septal defects), variable neurocognitive impairment, and florid ectodermal features. A distinctive trait of NS/LAH is its association with easily pluckable, slow growing, sparse, and thin hair. This rare condition is due to the invariant c.4A > G missense (p.Ser2Gly) change in SHOC2, which encodes a regulatory protein that participate in RAS signaling. Here we report two patients with molecularly confirmed NS/LAH, with extremely different phenotypic expression, in particular concerning the severity of the cardiac phenotype and neurocognitive profile. While the first available clinical records outlined a relatively homogeneous phenotype in NS/LAH, the present data emphasize that the phenotype spectrum associated with this invariant mutation is wider than previously recognized.

α-Fetoprotein assay on dried blood spot for hepatoblastoma screening in children with overgrowth-cancer predisposition syndromes.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) and hemihyperplasia (HH) are overgrowth conditions with predisposition to hepatoblastoma for which early diagnosis patients undergo cancer screening based on determination of the tumor marker α-fetoprotein (αFP). Repeated blood draws are a burden for patients with consequent compliance issues and poor adherence to surveillance protocol. We sought to analyze feasibility and reliability of αFP dosage using an analytical micromethod based on blood dried on filter paper (DBS). METHODS: Overall 143 coupled αFP determinations on plasma and DBS collected simultaneously were performed, of which 31 were in patients with hepatoblastoma predisposition syndromes and 112 were in controls. The plasma αFP dosage method was adapted to DBS adsorbed on paper matrix for newborn screening. RESULTS: There was strong correlation between plasmatic and DBS αFP (r2 = 0.999, P < 0.001). Cohen's k coefficient for correlation was 0.96 for diagnostic cut-off of 10 U/ml (P < 0.001), commonly employed in clinical practice. The measurements on plasma and DBS were highly overlapping and consistent. CONCLUSION: The DBS method allowed to dose αFP reliably and consistently for the concentrations commonly employed in clinical settings for the screening of hepatoblastoma, opening new scenarios about conducting cancer screening in overgrowth syndromes.

Transcriptional hallmarks of Noonan syndrome and Noonan-like syndrome with loose anagen hair.

Noonan syndrome (NS) is among the most common nonchromosomal disorders affecting development and growth. NS is genetically heterogeneous, being caused by germline mutations affecting various genes implicated in the RAS signaling network. This network transduces extracellular signals into intracellular biochemical and transcriptional responses controlling cell proliferation, differentiation, metabolism, and senescence. To explore the transcriptional consequences of NS-causing mutations, we performed global mRNA expression profiling on peripheral blood mononuclear cells obtained from 23 NS patients carrying heterozygous mutations in PTPN11 or SOS1. Gene expression profiling was also resolved in five subjects with Noonan-like syndrome with loose anagen hair (NS/LAH), a condition clinically related to NS and caused by an invariant mutation in SHOC2. Robust transcriptional signatures were found to specifically discriminate each of the three mutation groups from 21 age- and sex-matched controls. Despite the only partial overlap in terms of gene composition, the three signatures showed a notable concordance in terms of biological processes and regulatory circuits affected. These data establish expression profiling of peripheral blood mononuclear cells as a powerful tool to appreciate differential perturbations driven by germline mutations of transducers involved in RAS signaling and to dissect molecular mechanisms underlying NS and other RASopathies.

Nephrological findings and genotype-phenotype correlation in Beckwith-Wiedemann syndrome.

Beckwith-Wiedemann syndrome (BWS), an overgrowth disorder with several congenital abnormalities, encompasses nephrourological anomalies. The objective of the report is to analyze the latter and related genotype-phenotype correlations. The study was a retrospective review of nephrourological investigations and genotype in 67 BWS patients. Imaging and laboratory studies have been correlated with the molecular anomalies typical of BWS. Thirty-eight (56.7%) patients had a total of 61 nonmalignant nephrourological findings, including nephromegaly (n = 24), collecting system abnormalities (n = 14), cryptorchidism (n = 11), nephrolithiasis (n = 5), cysts (n = 5), and dysplasia (n = 1). Four patients had Wilms' tumor, all associated with renal hyperplasia. Renal findings were almost consistent in the BWS(IC1) group, with nephromegaly in all patients and collecting system abnormalities in half of them. BWS(UPD) and negative patients also had frequent anomalies (63.6% and 61.9% respectively), whereas only 36.0% of BWS(IC2) had renal findings (p = 0.003). Cryptorchidism was associated with abdominal wall defects (p < 0.001) appearing more frequently in BWS(IC2) (p = 0.028). Urinary tract infections were observed in 17.9% of patients, with two resulting in life-threatening sepsis. Hypercalciuria was present in 10% of cases. 55.5% of BWS patients have renal findings. Although variegate, these anomalies disclose a genotype-phenotype correlation.

SOS1 mutations in Noonan syndrome: molecular spectrum, structural insights on pathogenic effects, and genotype-phenotype correlations.

Noonan syndrome (NS) is among the most common nonchromosomal disorders affecting development and growth. NS is caused by aberrant RAS-MAPK signaling and is genetically heterogeneous, which explains, in part, the marked clinical variability documented for this Mendelian trait. Recently, we and others identified SOS1 as a major gene underlying NS. Here, we explored further the spectrum of SOS1 mutations and their associated phenotypic features. Mutation scanning of the entire SOS1 coding sequence allowed the identification of 33 different variants deemed to be of pathological significance, including 16 novel missense changes and in-frame indels. Various mutation clusters destabilizing or altering orientation of regions of the protein predicted to contribute structurally to the maintenance of autoinhibition were identified. Two previously unappreciated clusters predicted to enhance SOS1's recruitment to the plasma membrane, thus promoting a spatial reorientation of domains contributing to inhibition, were also recognized. Genotype-phenotype analysis confirmed our previous observations, establishing a high frequency of ectodermal anomalies and a low prevalence of cognitive impairment and reduced growth. Finally, mutation analysis performed on cohorts of individuals with nonsyndromic pulmonic stenosis, atrial septal defects, and ventricular septal defects excluded a major contribution of germline SOS1 lesions to the isolated occurrence of these cardiac anomalies.

A heritable cause of cleft lip and palate--Van der Woude syndrome caused by a novel IRF6 mutation. Review of the literature and of the differential diagnosis.

BACKGROUND: Orofacial clefts are common congenital malformations usually characterized by a multifactorial etiology. These heterogeneous defects comprise cleft lip (CL), CL with cleft palate (CL/P), and cleft palate, sometimes observed in recognizable syndromes, with mendelian, chromosomal, or environmental pathogenesis. The Van der Woude syndrome is a mendelian CL/P, accounting for about 2% of all cases and caused by mutations in the interferon regulatory factor 6 (IRF6) gene, located on 1q32.2 chromosome. OBJECTIVE: Here, we describe a familial case with a novel IRF6 mutation segregating in the maternal line, displaying a highly intrafamilial variable clinical expression. CONCLUSION: This report emphasizes the role of the clinician in recognizing the clinical variability and the genetic heterogeneity of CL/P.

Atypical cardiac defects in patients with RASopathies: Updated data on CARNET study.

BACKGROUND: RASopathies are a set of relatively common autosomal dominant clinically and genetically heterogeneous disorders. Cardiac outcomes in terms of mortality and morbidity for common heart defects (such as pulmonary valve stenosis and hypertrophic cardiomyopathy) have been reported. Nevertheless, also Atypical Cardiac Defects (ACDs) are described. The aim of the present study was to report both prevalence and cardiac outcome of ACDs in patients with RASopathies. METHODS: A retrospective, multicentric observational study (CArdiac Rasopathy NETwork-CARNET study) was carried out. Clinical, surgical, and genetic data of the patients who were followed until December 2019 were collected. RESULTS: Forty-five patients out of 440 followed in CARNET centers had ACDs. Noonan Syndrome (NS), NS Multiple Lentigines (NSML) and CardioFacioCutaneous Syndrome (CFCS) were present in 36, 5 and 4 patients, respectively. Median age at last follow-up was 20.1 years (range 6.9-47 years). Different ACDs were reported, including mitral and aortic valve dysfunction, ascending and descending aortic arch anomalies, coronary arteries dilation, enlargement of left atrial appendage and isolated pulmonary branches diseases. Five patients (11%) underwent cardiac surgery and one of them underwent a second intervention for mitral valve replacement and severe pericardial effusion. No patients died in our cohort until December 2019. CONCLUSIONS: Patients with RASopathies present a distinct CHD spectrum. Present data suggest that also ACDs must be carefully investigated for their possible impact on the clinical outcome. A careful longitudinal follow up until the individuals reach an adult age is recommended.

Four new cases of PHACES syndrome: variable phenotypic expression and endocrine features.

AIM: PHACES syndrome is a neurocutaneous condition characterized by the coexistence of large facial haemangiomas and at least one feature among posterior fossa malformations, cardiac and arterial anomalies, eye defects and sternal clefting. We review and discuss the phenotypes and the endocrine aspects of PHACES syndrome, hypothesizing that endocrine anomalies, although rare, could be considered as feature of the disease. METHODS: We described four new cases representative of the wide variable phenotype of this syndrome, commenting on the possible phenotypic expression. RESULTS: Two children displayed endocrine anomalies, sporadically described among PHACES subjects. One of them developed a transient hyperthyreotropinemia induced by interferon alpha-2alpha treatment for a giant facial haemangioma, while the second presented with congenital hypothyroidism with an in situ thyroid gland, a trait previously unreported in the syndrome. CONCLUSION: PHACES syndrome has a wide variable phenotypic expression and endocrine anomalies, especially hypothyroidism, may represent a trait of the syndrome and should be always investigated.

Data on cardiac defects, morbidity and mortality in patients affected by RASopathies. CARNET study results.

A comprehensive description of morbidity and mortality in patients affected by mutations in genes encoding for signal transducers of the RAS-MAPK cascade (RASopathies) was performed in our study recently published in the International Journal of Cardiology. Seven European cardiac centres participating to the CArdiac Rasopathy NETwork (CARNET), collaborated in this multicentric, observational, retrospective data analysis and collection. In this study, clinical records of 371 patients with confirmed molecular diagnosis of RASopathy were reviewed. Cardiac defects, crude mortality, survival rate of patients with 1) hypertrophic cardiomyopathy (HCM) and age <2 years or young adults; 2) individuals with Noonan syndrome and pulmonary stenosis carrying PTPN11 mutations; 3) biventricular obstruction and PTPN11 mutations; 4) Costello syndrome or cardiofaciocutaneous syndrome were analysed. Mortality was described as crude mortality, cumulative survival and restricted estimated mean survival. In particular, with this Data In Brief (DIB) paper, the authors aim to report specific statistic highlights of the multivariable regression analysis that was used to assess the impact of mutated genes on number of interventions and overall prognosis.

Assisted Reproductive Techniques and Risk of Beckwith-Wiedemann Syndrome.

BACKGROUND AND OBJECTIVES: The emerging association of assisted reproductive techniques (ART) with imprinting disorders represents a major issue in the scientific debate on infertility treatment and human procreation. We studied the prevalence of Beckwith-Wiedemann syndrome (BWS) in children conceived through ART to define the specific associated relative risk. METHODS: Patients with BWS born in Piemonte, Italy, were identified and matched with the general demographic data and corresponding regional ART registry. RESULTS: Between 2005 and 2014, live births in Piemonte were 379 872, including 7884 from ART. Thirty-eight patients with BWS were born, 7 from ART and 31 naturally conceived. BWS birth prevalence in the ART group was significantly higher than that of the naturally conceived group (1:1126 vs 1:12 254, P < .001). The absolute live birth risk in the ART group was 887.9 per 1 000 000 vs 83.3 per 1 000 000 in the naturally conceived group, providing a relative risk of 10.7 (95% confidence interval 4.7-24.2). During the 1997-2014 period, 67 patients were diagnosed with BWS out of 663 834 newborns (1:9908 live births). Nine out of the 67 BWS patients were conceived through ART (13.4%), and 8 were molecularly tested, with 4 having an imprinting center 2 loss of methylation, 2 with 11p15.5 paternal uniparental disomy, and 2 negative results. CONCLUSIONS: ART entails a 10-fold increased risk of BWS and could be implicated in the pathogenesis of genomic events besides methylation anomalies. These data highlight the need for awareness of ART-associated health risk.

Cardiac defects, morbidity and mortality in patients affected by RASopathies. CARNET study results.

BACKGROUND: RASopathies are developmental disease caused by mutations in genes encoding for signal transducers of the RAS-MAPK cascade. The aim of the present study was to provide a comprehensive description of morbidity and mortality in patients with molecularly confirmed RASopathy. METHODS: A multicentric, observational, retrospective study was conducted in seven European cardiac centres participating to the CArdiac Rasopathy NETwork (CARNET). Clinical records of 371 patients with confirmed molecular diagnosis of RASopathy were reviewed. Mortality was described as crude mortality, cumulative survival and restricted estimated mean survival. Multivariable regression analysis was used to assess the impact of mutated genes on number of interventions and overall prognosis. RESULTS: Cardiac defects occurred in 80.3% of cases, almost half of them underwent at least one intervention. Overall, crude mortality was 0.29/100 patients-year. Cumulative survival was 98.8%, 98.2%, 97.7%, 94.3%, at 1, 5, 10, and 20years, respectively. Restricted estimated mean survival at 20years follow-up was 19.6years. Ten patients died (2.7% of the entire cohort; 3.4% of patients with cardiac defect). Patients with hypertrophic cardiomyopathy (HCM) and age <2years or young adults, as well as subjects with biventricular obstruction and PTPN11 mutations had a higher risk of cardiac death. CONCLUSIONS: The risk of intervention was higher in individuals with Noonan syndrome and pulmonary stenosis carrying PTPN11 mutations. Overall, mortality was relatively low, even though the specific association between HCM, biventricular outflow tract obstructions and PTPN11 mutations appeared to be associated with early mortality, including immediate post-operative events and sudden death.

(Epi)genotype-phenotype correlations in Beckwith-Wiedemann syndrome.

Beckwith-Wiedemann syndrome (BWS) is characterized by cancer predisposition, overgrowth and highly variable association of macroglossia, abdominal wall defects, nephrourological anomalies, nevus flammeus, ear malformations, hypoglycemia, hemihyperplasia, and organomegaly. BWS molecular defects, causing alteration of expression or activity of the genes regulated by two imprinting centres (IC) in the 11p15 chromosomal region, are also heterogeneous. In this paper we define (epi)genotype-phenotype correlations in molecularly confirmed BWS patients. The characteristics of 318 BWS patients with proven molecular defect were compared among the main four molecular subclasses: IC2 loss of methylation (IC2-LoM, n=190), IC1 gain of methylation (IC1-GoM, n=31), chromosome 11p15 paternal uniparental disomy (UPD, n=87), and cyclin-dependent kinase inhibitor 1C gene (CDKN1C) variants (n=10). A characteristic growth pattern was found in each group; neonatal macrosomia was almost constant in IC1-GoM, postnatal overgrowth in IC2-LoM, and hemihyperplasia more common in UPD (P<0.001). Exomphalos was more common in IC2/CDKN1C patients (P<0.001). Renal defects were typical of UPD/IC1 patients, uretheral malformations of IC1-GoM cases (P<0.001). Ear anomalies and nevus flammeus were associated with IC2/CDKN1C genotype (P<0.001). Macroglossia was less common among UPD patients (P<0.001). Wilms' tumor was associated with IC1-GoM or UPD and never observed in IC2-LoM patients (P<0.001). Hepatoblastoma occurred only in UPD cases. Cancer risk was lower in IC2/CDKN1C, intermediate in UPD, and very high in IC1 cases (P=0.009). In conclusion, (epi)genotype-phenotype correlations define four different phenotypic BWS profiles with some degree of clinical overlap. These observations impact clinical care allowing to move toward (epi) genotype-based follow-up and cancer screening.