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Mosaic RASopathies are a heterogeneous group of diseases characterized by the presence at birth or early onset of congenital anomalies, cutaneous and vascular anomalies, segmental overgrowth, and increased cancer risk. They are caused by somatic pathogenic variants of the genes belonging the RAt Sarcoma Mitogen-activated protein kinase (RAS/MAPK) pathway causing its hyperactivation. Here, we review the clinical and molecular characteristics of this heterogeneous group of diseases, including the possibilities of molecular diagnosis and new therapeutic perspectives.

Different scoring systems for the clinical diagnosis of the Beckwith-Wiedemann spectrum (BWSp) have been developed over time, the most recent being the international consensus score. Here we try to validate and provide data on the performance metrics of these scoring systems of the 2018 international consensus and the previous ones, relating them to BWSp features, molecular tests, and the probability of cancer development in a cohort of 831 patients. The consensus scoring system had the best performance (sensitivity 0.85 and specificity 0.43). In our cohort, the diagnostic yield of tests on blood-extracted DNA was low in patients with a low consensus score (similar to 20% with a score = 2), and the score did not correlate with cancer development. We observed hepatoblastoma (HB) in 4.3% of patients with UPD(11)pat and Wilms tumor in 1.9% of patients with isolated lateralized overgrowth (ILO). We validated the efficacy of the currently used consensus score for BWSp clinical diagnosis. Based on our observation, a first-tier analysis of tissue-extracted DNA in patients with

Despite major technical and genetic advances, more than half of the neurodevelopmental disorders (NDDs) cases remain undiagnosed. We explored the frequency of non-random XCI in the mothers of male patients and in affected females from a clinically heterogeneous cohort of unsolved NDD cases, negative at FRAXA, chromosomal microarray analysis and Trio Exome Sequencing. We hypothesize that an unbalanced XCI could unmask previously discarded genetic variants on the X chromosome connected both to XCI and NDD. A multiplex fluorescent-PCR-based assay was used to screen the XCI pattern after methylation sensitive HhaI digestion. Trio-based ES re-analysis was performed in families with skewed XCI occurrence. Linkage analysis and RT-PCR were used to further study the X-chromosome inactive allele. X-drop was used to define the chromosome deletion boundaries. We found a skewed XCI (>90%) in 16/186 mothers of affected NDD males (8.6%) and 12/90 female patients (13.3%), far beyond the expected XCI in normal population (3.6%, OR=4.10; OR=2.51). Reanalyzing ES and clinical data, we solved 7/28 cases (25%). These included variants in the KDM5C, PDZD4, PHF6, TAF1, OTUD5, and ZMYM3, and a genomic deletion spanning exons 3-4 of the ATRX gene. The identification of a skewed XCI is an easy assay that can help selecting a subgroup of patients for the re-evaluation of X-linked variants, improving the diagnostic yield in NDD patients, and allowing the identification of new X-linked disorders.

Kaposiform hemangioendothelioma (KHE) is a rare locally aggressive mixed vascular tumor, with typical onset in early childhood and characterized by progressive angio- and lymphangiogenesis. Its etiopathogenesis and molecular bases are still unclear. Here, we report the first case of congenital KHE harboring a PIK3CA mosaic pathogenic variant (c.323G > A, p.Arg108His) in a boy with very subtle PIK3CA-related overgrowth spectrum (PROS) features. This finding provides insights into the pathophysiology of KHE, offering targeted therapeutic options by inhibition of the PI3K/Akt/mTOR pathway. We propose the inclusion of this mixed lymphatic and vascular anomaly within the PROS.

Lateralized/segmental overgrowth disorders (LOs) encompass a heterogeneous group of congenital conditions with excessive body tissue growth. Documented molecular alterations in LOs mostly consist of somatic variants in genes of the PI3KCA/AKT/mTOR pathway or of chromosome band 11p15.5 imprinted region anomalies. In some cases, somatic pathogenic variants in genes of the RAS/MAPK pathway have been reported. We present the first case of a somatic pathogenic variant (T507K) in PTPN11 causing a LO phenotype characterized by severe lateralized overgrowth, vascular proliferation, and cerebral astrocytoma. The T507K variant was detected in DNA from overgrown tissue in a leg with capillary malformation. The astrocytoma tissue showed a higher PTPN11 variant allele frequency. A pathogenic variant in FGFR1 was also found in tumor tissue, representing a second hit on the RAS/MAPK pathway. These findings indicate that RAS/MAPK cascade overactivation can cause mosaic overgrowth phenotypes resembling PIK3CA-related overgrowth disorders (PROS) with cancer predisposition and are consistent with the hypothesis that RAS/MAPK hyperactivation can be involved in the pathogenesis of astrocytoma. This observation raises the issue of cancer predisposition in patients with RAS/MAPK pathway gene variants and expands genotype spectrum of LOs and the treatment options for similar cases through inhibition of the RAS/MAPK oversignaling.

BackgroundMost cases of Beckwith-Wiedemann spectrum (BWSp) are diagnosed after birth and few studies evaluated the prenatal phenotype; here, we investigate these aspects in a large series of patients with BWSp.MethodsEighty-nine patients with BWSp recruited through the BWSp Internal Registry of the Pediatric Genetics Unit of the Regina Margherita Children’s Hospital of Torino and through the Italian Association of Patients with BWSp. Data collection was conducted through administration of a personalised questionnaire, interview to patients’ parents, review of the clinical records, including prenatal ultrasound (US) and biochemical screening tests, physical examination and review of clinical and molecular data of the patients.ResultsSeventeen patients (19.1%) were conceived through assisted reproductive techniques (ART). Twinning occurred in nine pregnancies (three from ART). Pregnancy biochemical screening tests showed increased alpha-fetoprotein (1.52±0.79 multiples of median (MoM), p=0.001), uEstriol (1.37±0.38 MoM, pConclusionWe propose a clinical scoring system for prenatal molecular investigations defining major, minor and supportive criteria among the several features often observed prenatally in BWSp.

Noonan syndrome (NS) and related disorders encompass a phenotypically heterogeneous group of conditions due to mutations in the Ras/Mitogen-activated protein kinase pathway. The main objective of this study was to assess the presence and characteristics of epilepsy in children and adolescents affected by NS and related disorders. The study included all the patients aged 5-21 years who had been diagnosed with NS or of one of three Noonan-like syndromes (i.e., cardio-facio-cutaneous syndrome, Noonan syndrome with multiple lentigines, and Noonan-like syndrome with loose anagen hair) at a university pediatric hospital. Clinical, EEGs, brain MRIs, and genotype data were extracted from the medical records, and follow-up telephone interviews were conducted to obtain updated information about epilepsy and its course. Out of a total of 75 patients (38 [50.7%] males, median age at assessment 12.0 years [q1 9.0-q3 17.0]; 61 [81.3%] with NS; and 14 [18.7%] with a Noonan-like syndrome), 13 (17.3%) had epilepsy, with median age at onset of 4.0 years (q1 2.0-q3 8.0, min 0.1-max 17.0). Epilepsy was more common among Noonan-like patients (50.0%) than in NS (9.8%, p 0.001), and its presence was associated with neurodevelopmental delay (p 0.001, OR 14.6 95% CI 3.6-59.4), cognitive impairment (p = 0.002, OR 11.2 95% CI 2.5-51.0), need for educational support (p 0.001, OR 21.8, 95% CI 2.6-179.1), and lower adaptive functioning (median [q1-q3]: 54.0 [q1 40.0-q3 77.5] vs 97.0 [q1 76.5-q3 107.0] of the non-epileptic subgroup, p = 0.004). In 10 out of 13 cases (76.9%), the epilepsy outcome was good (i.e., seizure-free for more than 12 months with or without anti-seizure medication).Epilepsy was more common in NS than reported in the general population, with a significantly higher rate in Noonan-like syndromes. Epilepsy was associated with neurodevelopmental delay, cognitive impairment, and lower adaptive functioning.• Neurological abnormalities have been reported in NS and related disorders. • There is evidence of a phenotype-genotype relationship for neurological abnormalities.• Epilepsy was found to be more common in NS and related disorders than typically reported in the general population and associated with neurodevelopmental delay, cognitive, and functional impairment. • The Noonan-like phenotype had a higher frequency of epilepsy than typical NS.

The prevalence of Beckwith-Wiedemann spectrum (BWSp) is tenfold increased in children conceived through assisted reproductive techniques (ART). More than 90% of ART-BWSp patients reported so far display imprinting center 2 loss-of-methylations (IC2-LoM), versus 50% of naturally conceived BWSp patients. We describe a cohort of 74 ART-BWSp patients comparing their features with a cohort of naturally conceived BWSp patients, with the ART-BWSp patients previously described in literature, and with the general population of children born from ART. We found that the distribution of UPD(11)pat was not significantly different in ART and naturally conceived patients. We observed 68.9% of IC2-LoM and 16.2% of mosaic UPD(11)pat in our ART cohort, that strongly differ from the figure reported in other cohorts so far. Since UPD(11)pat likely results from post-fertilization recombination events, our findings allows to hypothesize that more complex molecular mechanisms, besides methylation disturbances, may underlie BWSp increased risk in ART pregnancies. Moreover, comparing the clinical features of ART and non-ART BWSp patients, we found that ART-BWSp patients might have a milder phenotype. Finally, our data show a progressive increase in the prevalence of BWSp over time, paralleling that of ART usage in the last decades.

Esophageal atresia/tracheoesophageal fistula (EA/TEF) is a life-threatening birth defect that often occurs with other major birth defects (EA/TEF+). Despite advances in genetic testing, a molecular diagnosis can only be made in a minority of EA/TEF+ cases. Here, we analyzed clinical exome sequencing data and data from the DECIPHER database to determine the efficacy of exome sequencing in cases of EA/TEF+ and to identify phenotypic expansions involving EA/TEF. Among 67 individuals with EA/TEF+ referred for clinical exome sequencing, a definitive or probable diagnosis was made in 11 cases for an efficacy rate of 16% (11/67). This efficacy rate is significantly lower than that reported for other major birth defects, suggesting that polygenic, multifactorial, epigenetic, and/or environmental factors may play a particularly important role in EA/TEF pathogenesis. Our cohort included individuals with pathogenic or likely pathogenic variants that affect TCF4 and its downstream target NRXN1, and FANCA, FANCB, and FANCC, which are associated with Fanconi anemia. These cases, previously published case reports, and comparisons to other EA/TEF genes made using a machine learning algorithm, provide evidence in support of a potential pathogenic role for these genes in the development of EA/TEF.

Simple Summary Congenital lateralized or segmental overgrowth (LO) disorders are conditions characterized by excessive tissue growth of a body region often associated with a predisposition to cancer. LOs are caused by mosaic DNA anomalies, that is, they are present only in a part of the cells making up the body. LOs have an extremely heterogeneous clinical presentation: they widely overlap in presentation, are difficult to frame from a clinical point of view and have a diagnostic complexity representing a challenge for the clinician who approaches them. Here we review the key features of the various LOs, expose their molecular causes, and detail the implications for each of them, such as the need for specific cancer screening or the possibility of treatment. The latter represents a recent scientific achievement in medicine, allowed by the development of precision drugs finely tuning cellular pathways involved in growth and tumorigenesis deranged in LO. Abstract Congenital disorders of lateralized or segmental overgrowth (LO) are heterogeneous conditions with increased tissue growth in a body region. LO can affect every region, be localized or extensive, involve one or several embryonic tissues, showing variable severity, from mild forms with minor body asymmetry to severe ones with progressive tissue growth and related relevant complications. Recently, next-generation sequencing approaches have increased the knowledge on the molecular defects in LO, allowing classifying them based on the deranged cellular signaling pathway. LO is caused by either genetic or epigenetic somatic anomalies affecting cell proliferation. Most LOs are classifiable in the Beckwith–Wiedemann spectrum (BWSp), PI3KCA/AKT-related overgrowth spectrum (PROS/AROS), mosaic RASopathies, PTEN Hamartoma Tumor Syndrome, mosaic activating variants in angiogenesis pathways, and isolated LO (ILO). These disorders overlap over common phenotypes, making their appraisal and distinction challenging. The latter is crucial, as specific management strategies are key: some LO is associated with increased cancer risk making imperative tumor screening since childhood. Interestingly, some LO shares molecular mechanisms with cancer: recent advances in tumor biological pathway druggability and growth downregulation offer new avenues for the treatment of the most severe and complicated LO.

β-thalassemia is an inherited disease causing an impaired hemoglobin production, eventually leading to a severe chronic anemia. Resolutive treatments are still limited to a small number of patients and blood transfusions still represent the standard of care. In this scenario, luspatercept is the first approved drug able to significantly modify the disease phenotype. Developed as a fusion protein, it binds TGF-β ligands, contributing to a reduction of ineffective erythropoiesis. As shown by clinical trials in thalassemia, this effect determines an increase in mean hemoglobin levels and/or a decrease in transfusion burden. While some potential indications are still being evaluated in trials, luspatercept has recently entered the clinical practice for transfusion-dependent thalassemia patients.

International audience; We present the largest exome sequencing study of autism spectrum disorder (ASD) to date (n = 35,584 total samples, 11,986 with ASD). Using an enhanced analytical framework to integrate de novo and case-control rare variation, we identify 102 risk genes at a false discovery rate of 0.1 or less. Of these genes, 49 show higher frequencies of disruptive de novo variants in individuals ascertained to have severe neurodevelopmental delay, whereas 53 show higher frequencies in individuals ascertained to have ASD; comparing ASD cases with mutations in these groups reveals phenotypic differences. Expressed early in brain development, most risk genes have roles in regulation of gene expression or neuronal communication (i.e., mutations effect neurodevelopmental and neurophysiological changes), and 13 fall within loci recurrently hit by copy number variants. In cells from the human cortex, expression of risk genes is enriched in excitatory and inhibitory neuronal lineages, consistent with multiple paths to an excitatory-inhibitory imbalance underlying ASD.

BackgroundPostzygotic activatingPIK3CAvariants cause several phenotypes within thePIK3CA-related overgrowth spectrum (PROS). Variant strength, mosaicism level, specific tissue involvement and overlapping disorders are responsible for disease heterogeneity. We explored these factors in 150 novel patients and in an expanded cohort of 1007PIK3CA-mutated patients, analysing our new data with previous literature to give a comprehensive picture.MethodsWe performed ultradeep targeted next-generation sequencing (NGS) on DNA from skin biopsy, buccal swab or blood using a panel including phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin pathway genes andGNAQ,GNA11,RASA1andTEK. Additionally, 914 patients previously reported were systematically reviewed.Results93 of our 150 patients hadPIK3CApathogenetic variants. The merged PROS cohort showed thatPIK3CAvariants span thorough all gene domains, some were exclusively associated with specific PROS phenotypes: weakly activating variants were associated with central nervous system (CNS) involvement, and strongly activating variants with extra-CNS phenotypes. Among the 57 with a wild-typePIK3CAallele, 11 patients with overgrowth and vascular malformations overlapping PROS had variants inGNAQ,GNA11,RASA1orTEK.ConclusionWe confirm that (1) molecular diagnostic yield increases when multiple tissues are tested and by enriching NGS panels with genes of overlapping ‘vascular’ phenotypes; (2) strongly activatingPIK3CAvariants are found in affected tissue, rarely in blood: conversely, weakly activating mutations more common in blood; (3) weakly activating variants correlate with CNS involvement, strong variants are more common in cases without; (4) patients with vascular malformations overlapping those of PROS can harbour variants in genes other thanPIK3CA.

Purpose: Beckwith-Wiedemann syndrome (BWS) is a developmental disorder caused by dysregulation of the imprinted gene cluster of chromosome 11p15.5 and often associated with loss of methylation (LOM) of the imprinting center 2 (IC2) located in KCNQ1 intron 10. To unravel the etiological mechanisms underlying these epimutations, we searched for genetic variants associated with IC2 LOM.Methods: We looked for cases showing the clinical features of both BWS and long QT syndrome (LQTS), which is often associated with KCNQ1 variants. Pathogenic variants were identified by genomic analysis and targeted sequencing. Functional experiments were performed to link these pathogenic variants to the imprinting defect.Results: We found three rare cases in which complete IC2 LOM is associated with maternal transmission of KCNQ1 variants, two of which were demonstrated to affect KCNQ1 transcription upstream of IC2. As a consequence of KCNQ1 haploinsufficiency, these variants also cause LQTS on both maternal and paternal transmission.Conclusion: These results are consistent with the hypothesis that, similar to what has been demonstrated in mouse, lack of transcription across IC2 results in failure of methylation establishment in the female germline and BWS later in development, and also suggest a new link between LQTS and BWS that is important for genetic counseling.

β-thalassaemia is a rare genetic condition caused by mutations in the β-globin gene that result in severe iron-loading anaemia, maintained by a detrimental state of ineffective erythropoiesis (IE). The role of multiple mechanisms involved in the pathophysiology of the disease has been recently unravelled. The unbalanced production of α-globin is a major source of oxidative stress and membrane damage in red blood cells (RBC). In addition, IE is tightly linked to iron metabolism dysregulation, and the relevance of new players of this pathway, i.e., hepcidin, erythroferrone, matriptase-2, among others, has emerged. Advances have been made in understanding the balance between proliferation and maturation of erythroid precursors and the role of specific factors in this process, such as members of the TGF-β superfamily, and their downstream effectors, or the transcription factor GATA1. The increasing understanding of IE allowed for the development of a broad set of potential therapeutic options beyond the current standard of care. Many candidates of disease-modifying drugs are currently under clinical investigation, targeting the regulation of iron metabolism, the production of foetal haemoglobin, the maturation process, or the energetic balance and membrane stability of RBC. Overall, they provide tools and evidence for multiple and synergistic approaches that are effectively moving clinical research in β-thalassaemia from bench to bedside.

De novo variants in the WDR26 gene leading to haploinsufficiency have recently been associated with Skraban-Deardorff syndrome. This condition is an ultra-rare autosomal dominant neurodevelopmental disorder characterized by a broad range of clinical signs, including intellectual disability (ID), developmental delay (DD), seizures, abnormal facial features, feeding difficulties, and minor skeletal anomalies. Currently, 18 cases have been reported in the literature and for only 15 of them a clinical description is available. Here, we describe a child with Skraban-Deardorff syndrome associated with the WDR26 pathogenic de novo variant NM_025160.6:c.69dupC, p.(Gly24ArgfsTer48), and an adult associated with the pathogenic de novo variant c.1076G > A, p.(Trp359Ter). The adult patient was a 29-year-old female with detailed information on clinical history and pharmacological treatments since birth, providing an opportunity to map disease progression and patient management. By comparing our cases with published reports of Skraban-Deardorff syndrome, we provide a genetic and clinical summary of this ultrarare condition, describe the clinical management from childhood to adult age, and further expand on the clinical phenotype.

Smith-Kingsmore syndrome (SKS) is a rare autosomal dominant disorder caused by heterozygous germline activating pathogenic variants in mammalian target of rapamycin (MTOR) on chromosome 1p36. A few patients with disseminated mosaicism have been described so far and they seem to display a different phenotype when compared to germline cases. Here we report the sixth case with a disseminated mosaic MTOR pathogenic variant, a 7-year-old boy with hemimegalencephaly, epilepsy, developmental delay, hypomelanosis of Ito, and lateralized overgrowth. Genetic testing revealed a pathogenic variant (c.4448G > A, p.Cys1483Tyr) in MTOR with a frequency of 32% in the DNA extracted from a skin sample, 3% in saliva and 0.46% in blood. The clinical features observed in our patient further corroborate the existence of differences in phenotypic presentation of germline and mosaic SKS cases. Moreover, lateralized overgrowth, a finding never described so far in SKS, further expands the phenotypic spectrum of SKS and allows the inclusion of MTOR pathogenic variants among the several causes of asymmetric body overgrowth.

Objective To provide information on evolution over time of leg length discrepancy in patients with syndromic and isolated lateralized overgrowth. Study design This retrospective study investigates leg length discrepancy longitudinally in 105 patients with lateralized overgrowth either isolated (n = 37) or associated with Beckwith-Wiedemann spectrum (n = 56) or PIK3CA-related overgrowth spectrum (n = 12). Discrepancy was measured by standard methods and categorized as minor, mild, severe, and critical, based on the thresholds of 1, 2 and 5, respectively. Results The period of observation from diagnosis was 1.7 ± 2.6 to 9.0 ± 6.0 years. Leg length discrepancy was 11.0 ± 7.2 mm at diagnosis and 17.1 ± 14.4 mm at last visit. Both final leg length discrepancy and change over time were correlated with discrepancy at diagnosis (r2 = 0.45, P Conclusions Leg length discrepancy associated with isolated or syndromic lateralized overgrowth tends to worsen with growth and correlates with discrepancy at first observation. Among the genotypic groups, isolated lateralized overgrowth tends to have a milder evolution, whereas Beckwith-Wiedemann spectrum predisposes to a more severe outcome, especially if associated with paternal chromosome 11 uniparental disomy genotype.

OBJECTIVE The objective of this study was to examine psychopathology and its impact on adaptive functioning in a sample of patients affected by Noonan syndrome (NS), a genetically heterogeneous condition with systemic manifestations. METHOD Forty-two subjects affected by NS (23 males and 19 females), aged 5 to 21 years (mean 12.6 ± SD 5.1), were assessed for nonverbal cognitive abilities, with dimensional measures of psychopathology, adaptive functioning, and family quality of life. RESULTS The nonverbal intelligence quotient (IQ) mean was 99.4 ± SD 22.2, with 3 subjects (8%, 95% confidence interval [CI], 1.6%-20.9%) showing cognitive impairment (IQ

Macrocephaly, defined as head circumference ≥ 2 SDs, is a cardinal feature of Sotos syndrome (SS) and generally persists in adulthood. Subdural fluid collection, typically associated with macrocephaly, is described in children due to anatomical conformation, and in adulthood due to brain atrophy and ex-vacuo hydrocephalus. On the other hand, a true, symptomatic, chronic subdural hematoma (CSH) is a previously unreported complication of SS in adulthood. Here we describe the first SS patient presenting symptomatic CSH, leading to frequent hospitalizations for surgical evacuations that consistently recurred. Middle meningeal artery (MMA) embolization and epidural blood patch (EBP) allowed to resolve the CSH with complete resolution of clinical signs and symptoms. We hypothesize that appearance and recurrences of CSH may be related to pathological biomechanics of brain, cerebro-spinal fluid and skull, secondary to anatomical features of SS. In this context, surgical evacuation may be less efficient than usual to cure CSH. Alternative treatment to avoid blood extravasation, as MMA embolization, or to cure concurrent causes of the pathology, as EBP, may be considered.

The Tousled-like kinases 1 and 2 (TLK1 and TLK2) are involved in many fundamental processes, including DNA replication, cell cycle checkpoint recovery and chromatin remodelling. Mutations inWe re-evaluate whole exome sequencing and array-CGH data from a large cohort of patients affected by neurodevelopmental disorders. Using spatial proteomics (BioID) and single-cell gel electrophoresis, we investigated the proximity interaction landscape ofWe identified three new unrelated MRD57 families. Two were sporadic and caused by a missense change (c.1652AG; p.(Asp551Gly)) or a 39 kb deletion encompassingOur study identified novel

Germ line mutations in H3F3A and H3F3B cause a previously unidentified neurodevelopmental syndrome., Although somatic mutations in Histone 3.3 (H3.3) are well-studied drivers of oncogenesis, the role of germline mutations remains unreported. We analyze 46 patients bearing de novo germline mutations in histone 3 family 3A (H3F3A) or H3F3B with progressive neurologic dysfunction and congenital anomalies without malignancies. Molecular modeling of all 37 variants demonstrated clear disruptions in interactions with DNA, other histones, and histone chaperone proteins. Patient histone posttranslational modifications (PTMs) analysis revealed notably aberrant local PTM patterns distinct from the somatic lysine mutations that cause global PTM dysregulation. RNA sequencing on patient cells demonstrated up-regulated gene expression related to mitosis and cell division, and cellular assays confirmed an increased proliferative capacity. A zebrafish model showed craniofacial anomalies and a defect in Foxd3-derived glia. These data suggest that the mechanism of germline mutations are distinct from cancer-associated somatic histone mutations but may converge on control of cell proliferation.

Radial head prostheses can be implanted using different fixation approaches, and cemented stems are broadly described. The best method for stem fixation is still under debate, and there are pros and cons to every option. A cemented stem may be chosen in order to avoid/reduce the risk of an intraoperative radius fracture, in cases of revision surgery and in cases where rigid fixation is not sufficiently obtained during surgery using a press-fit method but is strictly required (i.e., when an anatomic prosthesis is used). Some surgeons prefer not to cement in order to avoid problems should a revision surgery become necessary. All the basic principles for a proper cement fixation should be followed. In particular, a small bone plug is inserted into the radius shaft to restrict excessive cement migration, the medullary canal is carefully cleaned using saline to allow for proper cement grip, and the extra cement is carefully removed after the stem is inserted. It is also helpful to add a small amount of methylene blue to the cement to make it easier to distinguish from the endosteal bone should a revision be needed later on.

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.

ObjectiveTo estimate the prevalence of Joubert syndrome (JS) in Italy applying standards of descriptive epidemiology and to provide a molecular characterization of the described patient cohort.MethodsWe enrolled all patients with a neuroradiologically confirmed diagnosis of JS who resided in Italy in 2018 and calculated age and sex prevalence, assuming a Poisson distribution. We also investigated the correlation between proband chronological age and age at diagnosis and performed next-generation sequencing (NGS) analysis on probands' DNA when available.ResultsWe identified 284 patients with JS: the overall, female- and male-specific population-based prevalence rates were 0.47 (95% confidence interval [CI] 0.41–0.53), 0.41 (95% CI 0.32–0.49), and 0.53 (95% CI 0.45–0.61) per 100,000 population, respectively. When we considered only patients in the age range from 0 to 19 years, the corresponding population-based prevalence rates rose to 1.7 (95% CI 1.49–1.97), 1.62 (95% CI 1.31–1.99), and 1.80 (95% CI 1.49–2.18) per 100,000 population. NGS analysis allowed identifying the genetic cause in 131 of 219 screened probands. Age at diagnosis was available for 223 probands, with a mean of 6.67 ± 8.10 years, and showed a statistically significant linear relationship with chronological age (r2 = 0.79; p < 0.001).ConclusionsWe estimated for the first time the age and sex prevalence of JS in Italy and investigated the patients’ genetic profile. The obtained population-based prevalence rate was ≈10 times higher than that available in literature for children population.

IntroductionThe Tousled-like kinases 1 and 2 (TLK1 and TLK2) are involved in many fundamental processes, including DNA replication, cell cycle checkpoint recovery and chromatin remodelling. Mutations in TLK2 were recently associated with ‘Mental Retardation Autosomal Dominant 57’ (MRD57, MIM# 618050), a neurodevelopmental disorder characterised by a highly variable phenotype, including mild-to-moderate intellectual disability, behavioural abnormalities, facial dysmorphisms, microcephaly, epilepsy and skeletal anomalies.MethodsWe re-evaluate whole exome sequencing and array-CGH data from a large cohort of patients affected by neurodevelopmental disorders. Using spatial proteomics (BioID) and single-cell gel electrophoresis, we investigated the proximity interaction landscape of TLK2 and analysed the effects of p.(Asp551Gly) and a previously reported missense variant (c.1850C>T; p.(Ser617Leu)) on TLK2 interactions, localisation and activity.ResultsWe identified three new unrelated MRD57 families. Two were sporadic and caused by a missense change (c.1652A>G; p.(Asp551Gly)) or a 39 kb deletion encompassing TLK2, and one was familial with three affected siblings who inherited a nonsense change from an affected mother (c.1423G>T; p.(Glu475Ter)). The clinical phenotypes were consistent with those of previously reported cases. The tested mutations strongly impaired TLK2 kinase activity. Proximal interactions between TLK2 and other factors implicated in neurological disorders, including CHD7, CHD8, BRD4 and NACC1, were identified. Finally, we demonstrated a more relaxed chromatin state in lymphoblastoid cells harbouring the p.(Asp551Gly) variant compared with control cells, conferring susceptibility to DNA damage.ConclusionOur study identified novel TLK2 pathogenic variants, confirming and further expanding the MRD57-related phenotype. The molecular characterisation of missense variants increases our knowledge about TLK2 function and provides new insights into its role in neurodevelopmental disorders.