Loss-of-Function Variants in CUL3 Cause a Syndromic Neurodevelopmental Disorder.

OBJECTIVE: De novo variants in cullin-3 ubiquitin ligase (CUL3) have been strongly associated with neurodevelopmental disorders (NDDs), but no large case series have been reported so far. Here, we aimed to collect sporadic cases carrying rare variants in CUL3, describe the genotype-phenotype correlation, and investigate the underlying pathogenic mechanism. METHODS: Genetic data and detailed clinical records were collected via multicenter collaboration. Dysmorphic facial features were analyzed using GestaltMatcher. Variant effects on CUL3 protein stability were assessed using patient-derived T-cells. RESULTS: We assembled a cohort of 37 individuals with heterozygous CUL3 variants presenting a syndromic NDD characterized by intellectual disability with or without autistic features. Of these, 35 have loss-of-function (LoF) and 2 have missense variants. CUL3 LoF variants in patients may affect protein stability leading to perturbations in protein homeostasis, as evidenced by decreased ubiquitin-protein conjugates in vitro. Notably, we show that 4E-BP1 (EIF4EBP1), a prominent substrate of CUL3, fails to be targeted for proteasomal degradation in patient-derived cells. INTERPRETATION: Our study further refines the clinical and mutational spectrum of CUL3-associated NDDs, expands the spectrum of cullin RING E3 ligase-associated neuropsychiatric disorders, and suggests haploinsufficiency via LoF variants is the predominant pathogenic mechanism. ANN NEUROL 2024.

Differential effects of iron chelators on iron burden and long-term morbidity and mortality outcomes in a large cohort of transfusion-dependent ß-thalassemia patients who remained on the same monotherapy over 10 years.

We conducted a retrospective cohort study on 663 transfusion-dependent ß-thalassemia patients receiving the same iron chelation monotherapy with deferoxamine, deferiprone, or deferasirox for up to 10 years (median age 31.8 years, 49.9 % females). Patients on all three iron chelators had a steady and significant decline in serum ferritin over the 10 years (median deferoxamine: -170.7 ng/mL, P = 0.049, deferiprone: -236.7 ng/mL, P = 0.001; deferasirox: -323.7 ng/mL, P < 0.001) yet had no significant change in liver iron concentration or cardiac T2*; while noting that patients generally had low hepatic and cardiac iron levels at study start. Median absolute, relative, and normalized changes were generally comparable between the three iron chelators. Patients receiving deferasirox had the highest morbidity and mortality-free survival probability among the three chelators, although the difference was only statistically significant when compared with deferoxamine (P = 0.037). On multivariate Cox regression analysis, there was no significant association between iron chelator type and the composite outcome of morbidity or mortality. In a real-world setting, there is comparable long-term iron chelation effectiveness between the three available iron chelators for patients with mild-to-moderate iron overload.

Molecular Basis and Diagnostic Approach to Isolated and Syndromic Lateralized Overgrowth in Childhood.

OBJECTIVE: To demonstrate a high-yield molecular diagnostic workflow for lateralized overgrowth (LO), a congenital condition with abnormal enlargement of body parts, and to classify it by molecular genetics. STUDY DESIGN: We categorized 186 retrospective cases of LO diagnosed between 2003 and 2023 into suspected Beckwith-Wiedemann spectrum, PIK3CA-related overgrowth spectrum (PROS), vascular overgrowth, or isolated LO, based on initial clinical assessments, to determine the appropriate first-tier molecular tests and tissue for analysis. Patients underwent testing for 11p15 epigenetic abnormalities or somatic variants in genes related to PI3K/AKT/mTOR, vascular proliferation, and RAS-MAPK cascades using blood or skin DNA. For cases with negative initial tests, a sequential cascade molecular approach was employed to improve diagnostic yield. RESULTS: This approach led to a molecular diagnosis in 54% of cases, 89% of cases consistent with initial clinical suspicions, and 11% reclassified. Beckwith-Wiedemann spectrum was the most common cause, with 43% of cases exhibiting 11p15 abnormalities. PIK3CA-related overgrowth spectrum had the highest confirmation rate, with 74% of clinically diagnosed patients showing a PIK3CA variant. Vascular overgrowth demonstrated significant clinical overlap with other syndromes. A molecular diagnosis of isolated LO proved challenging, with only 21% of cases classifiable into a specific condition. CONCLUSIONS: LO is underdiagnosed from a molecular viewpoint and to date has had no diagnostic guidelines, which is crucial for addressing potential cancer predisposition, enabling precision medicine treatments, and guiding management. This study sheds light on the molecular etiology of LO, highlighting the importance of a tailored diagnostic approach and of selecting appropriate testing to achieve the highest diagnostic yield.

Revisiting iron overload status and change thresholds as predictors of mortality in transfusion-dependent ß-thalassemia: a 10-year cohort study.

Data on iron overload status and change thresholds that can predict mortality in patients with transfusion-dependent ß-thalassemia (TDT) are limited. This was a retrospective cohort study of 912 TDT patients followed for up to 10 years at treatment centers in Italy (median age 32 years, 51.6% female). The crude mortality rate was 2.9%. Following best-predictive threshold identification through receiver operating characteristic curve analyses, data from multivariate Cox-regression models showed that patients with Period Average Serum Ferritin (SF) > 2145 vs ≤ 2145 ng/mL were 7.1-fold (P < 0.001) or with Absolute Change SF > 1330 vs ≤ 1330 ng/mL increase were 21.5-fold (P < 0.001) more likely to die from any cause. Patients with Period Average Liver Iron Concentration (LIC) > 8 vs ≤ 8 mg/g were 20.2-fold (P < 0.001) or with Absolute Change LIC > 1.4 vs ≤ 1.4 mg/g increase were 27.6-fold (P < 0.001) more likely to die from any cause. Patients with Index (first) cardiac T2* (cT2*) < 27 vs ≥ 27 ms were 8.6-fold (P < 0.001) more likely to die from any cause. Similarly, results at varying thresholds were identified for death from cardiovascular disease. These findings should support decisions on iron chelation therapy by establishing treatment targets, including safe iron levels and clinically meaningful changes over time.

'Phenoconversion' in adult patients with ß-thalassemia.

Rate and risk factors for phenoconversion from non-transfusion-dependent ß-thalassemia (NTDT) to transfusion-dependent ß-thalassemia (TDT) during a 10-year follow up of adult patients in Italy.

CAPRIN1 haploinsufficiency causes a neurodevelopmental disorder with language impairment, ADHD and ASD.

We describe an autosomal dominant disorder associated with loss-of-function variants in the Cell cycle associated protein 1 (CAPRIN1; MIM*601178). CAPRIN1 encodes a ubiquitous protein that regulates the transport and translation of neuronal mRNAs critical for synaptic plasticity, as well as mRNAs encoding proteins important for cell proliferation and migration in multiple cell types. We identified 12 cases with loss-of-function CAPRIN1 variants, and a neurodevelopmental phenotype characterized by language impairment/speech delay (100%), intellectual disability (83%), attention deficit hyperactivity disorder (82%) and autism spectrum disorder (67%). Affected individuals also had respiratory problems (50%), limb/skeletal anomalies (50%), developmental delay (42%) feeding difficulties (33%), seizures (33%) and ophthalmologic problems (33%). In patient-derived lymphoblasts and fibroblasts, we showed a monoallelic expression of the wild-type allele, and a reduction of the transcript and protein compatible with a half dose. To further study pathogenic mechanisms, we generated sCAPRIN1+/- human induced pluripotent stem cells via CRISPR-Cas9 mutagenesis and differentiated them into neuronal progenitor cells and cortical neurons. CAPRIN1 loss caused reduced neuronal processes, overall disruption of the neuronal organization and an increased neuronal degeneration. We also observed an alteration of mRNA translation in CAPRIN1+/- neurons, compatible with its suggested function as translational inhibitor. CAPRIN1+/- neurons also showed an impaired calcium signalling and increased oxidative stress, two mechanisms that may directly affect neuronal networks development, maintenance and function. According to what was previously observed in the mouse model, measurements of activity in CAPRIN1+/- neurons via micro-electrode arrays indicated lower spike rates and bursts, with an overall reduced activity. In conclusion, we demonstrate that CAPRIN1 haploinsufficiency causes a novel autosomal dominant neurodevelopmental disorder and identify morphological and functional alterations associated with this disorder in human neuronal models.

Genotypes and phenotypes heterogeneity in PIK3CA-related overgrowth spectrum and overlapping conditions: 150 novel patients and systematic review of 1007 patients with PIK3CA pathogenetic variants.

BACKGROUND: Postzygotic activating PIK3CA variants cause several phenotypes within the PIK3CA-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 1007 PIK3CA-mutated patients, analysing our new data with previous literature to give a comprehensive picture. METHODS: We 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 and GNAQ, GNA11, RASA1 and TEK. Additionally, 914 patients previously reported were systematically reviewed. RESULTS: 93 of our 150 patients had PIK3CA pathogenetic variants. The merged PROS cohort showed that PIK3CA variants 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-type PIK3CA allele, 11 patients with overgrowth and vascular malformations overlapping PROS had variants in GNAQ, GNA11, RASA1 or TEK. CONCLUSION: We 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 activating PIK3CA variants 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 than PIK3CA.

The somatic p.T81dup variant in AKT3 gene underlies a mild cerebral phenotype and expands the spectrum including capillary malformation and lateralized overgrowth.

Heterozygous germline or somatic variants in AKT3 gene can cause isolated malformations of cortical development (MCDs) such as focal cortical dysplasia, megalencephaly (MEG), Hemimegalencephaly (HME), dysplastic megalencephaly, and syndromic forms like megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome, and megalencephaly-capillary malformation syndrome. This report describes a new case of HME and capillary malformation caused by a somatic AKT3 variant that differs from the common p.E17K variant described in literature. The patient's skin biopsy from the angiomatous region revealed an heterozygous likely pathogenic variant AKT3:c.241_243dup, p.(T81dup) that may affect the binding domain and downstream pathways. Compared to previously reported cases with a common E17K mosaic variant, the phenotype is milder and patients showed segmental overgrowth, an uncommon characteristic in AKT3 variant cases. These findings suggest that the severity of the disease may be influenced not only by the level of mosaicism but also by the type of variant. This report expands the phenotypic spectrum associated with AKT3 variants and highlights the importance of genomic analysis in patients with capillary malformation and MCDs.

Missense variants in RPH3A cause defects in excitatory synaptic function and are associated with a clinically variable neurodevelopmental disorder.

PURPOSE: RPH3A encodes a protein involved in the stabilization of GluN2A subunit of N-methyl-D-aspartate (NMDA)-type glutamate receptors at the cell surface, forming a complex essential for synaptic plasticity and cognition. We investigated the effect of variants in RPH3A in patients with neurodevelopmental disorders. METHODS: By using trio-based exome sequencing, GeneMatcher, and screening of 100,000 Genomes Project data, we identified 6 heterozygous variants in RPH3A. In silico and in vitro models, including rat hippocampal neuronal cultures, have been used to characterize the effect of the variants. RESULTS: Four cases had a neurodevelopmental disorder with untreatable epileptic seizures [p.(Gln73His)dn; p.(Arg209Lys); p.(Thr450Ser)dn; p.(Gln508His)], and 2 cases [p.(Arg235Ser); p.(Asn618Ser)dn] showed high-functioning autism spectrum disorder. Using neuronal cultures, we demonstrated that p.(Thr450Ser) and p.(Asn618Ser) reduce the synaptic localization of GluN2A; p.(Thr450Ser) also increased the surface levels of GluN2A. Electrophysiological recordings showed increased GluN2A-dependent NMDA ionotropic glutamate receptor currents for both variants and alteration of postsynaptic calcium levels. Finally, expression of the Rph3AThr450Ser variant in neurons affected dendritic spine morphology. CONCLUSION: Overall, we provide evidence that missense gain-of-function variants in RPH3A increase GluN2A-containing NMDA ionotropic glutamate receptors at extrasynaptic sites, altering synaptic function and leading to a clinically variable neurodevelopmental presentation ranging from untreatable epilepsy to autism spectrum disorder.

Occult ischemic bone lesions in children with sickle cell disease: A study of prevalence.

INTRODUCTION: Avascular necrosis (AVN) is a severe complication of sickle cell disease (SCD) and involves principally the femoral head. Few data exist about the prevalence of lesions in other segments. METHODS: In this cross-sectional study, 42 children (20 males and 22 females) underwent a magnetic resonance imaging (MRI) of the spine, upper arms, and femurs. The primary outcome was to define the prevalence and locations of bone infarcts. RESULTS: Forty-two patients completed the study; the total median age was 11.9 years (interquartile range = 9.5-13.9). Eleven patients (26.2%) were positive for altered bone findings for a total of 32 lesions. Most of the lesions were in the humerus 17 (53.1%), 11 (34.4%) in the femurs, and 4 (12.5%) in the vertebrae. The median number of vaso-occlusive crises (VOCs) was two and four for patients without and with bone lesions, respectively (p = .01). The annual rate of VOC is the best marker for the positive MRI (odds ratio = 82.6; p = .03), and it is correlated with the number of sites involved (p = .02). CONCLUSIONS: Our study highlights that the prevalence of skeletal lesions could be underestimated, and it provides the basis for clinical reasoning and tailored therapy in SCD children.