Tiffany Busa

HUWE1 (HECT, UBA, and WWE Domain Containing E3 Ubiquitin Protein Ligase1, OMIM 300697), located at Xp11.22, encodes a ubiquitin ligase that is highly conserved across species. Genetic variants in HUWE1 described in multiple independent studies cause X‐linked intellectual disability, including in the patients identified by Juberg, Marsidi, and Brooks. This report describes 35 additional cases of individuals with variants in HUWE1 and suggested guidelines for clinical management. Our study includes several female cases, which have not been widely reported previously. Our findings confirm earlier reported clinical features including developmental delay, autism, hypotonia, short stature, and dysmorphic facial features as well as additional multisystemic findings. Intrauterine growth restriction (IUGR) and feeding difficulties were common in the neonatal period. It is notable that nearly all females had de novo variants, and males had de novo or inherited variants from clinically unaffected carrier mothers. Three genetic hotspots were identified in evolutionarily conserved regions of HUWE1 that have clinical impact. This report provides additional characterization of the spectrum of HUWE1‐related neurodevelopmental disorder (HNDD).

Congenital heart disease (CHD) occurs in over half of individuals with 22q11.2 deletion syndrome (22q11.2DS), but lesion type varies widely. We analyzed 3,016 unrelated postnatal individuals with 22q11.2DS from specialized centers in the United States, Canada, Europe, South America, Israel, and Australia, including 1,868 with whole-genome sequencing. The typical 3 Mb LCR22 A-D deletion was present in 2,788 individuals (92.4%), with smaller A-B (n=172, 5.7%) and A-C (n=56, 1.9%) deletions comprising the remaining cohort. In multivariable mixed-effects logistic regression adjusting for sex, deletion group, genome-wide principal components (PCs), and recruitment site, four non-intercept associations, to test relationships, met study-wide FDR statistical significance. Compared with the A-B deletion, the A-D deletion was associated with lower odds of persistent truncus arteriosus (OR=0.37, 95% CI 0.18-0.75) and higher odds of isolated septal defects (OR=4.68, 95% CI 1.71-12.83), although precision was limited by the smaller A-B group. PC2 was associated with lower odds of pulmonary stenosis/atresia with additional lesions (OR=0.73, 95% CI 0.61-0.87), and PC4 with higher odds of abnormal origin of the subclavian arteries (OR=2.59, 95% CI 1.37-4.89). ADMIXTURE-derived continental ancestry proportions did not show independent associations with these two PC-associated outcomes. These lesion-specific findings suggest the hypothesis that deletion interval and broader genetic background may contribute to CHD variability in 22q11.2DS, pending future replication. KEY MESSAGES: What is already known on this topic: Chromosome 22q11.2DS is a rare genetic disorder associated with serious medical challenges. Most affected individuals have congenital heart disease but with variable phenotypic expression. One of the main questions still an open topic in the field is why individuals with 22q11.2DS show extensive phenotypic heterogeneity. What this study adds: Analysis of retrospective data on 3,016 individuals with 22q11.2DS and 1,868 with sequence data suggest that deletion type and genetic variation influence phenotypic expression of congenital heart disease. How this study might affect research, practice or policy: This work informs the clinician as to the importance of testing for deletion type and obtaining accurate cardiac phenotypes to diagnose 22q11.2DS that will improve the prognosis and disease progression. It also implicates the complexity of sex and ancestry as confounding factors that will help guide future research studies to identify genetic modifiers of congenital heart disease.

Poly(rC)-binding protein 1 (PCBP1), a splicing factor and key member of the hnRNP E family, was initially characterized for its tumor suppressive properties. More recently, its role in regulating gene expression in the brain and nervous system has attracted growing interest. Through an international multicenter collaboration, we identified 13 de novo pathogenic variants in PCBP1 across 13 unrelated families. All affected individuals exhibited intellectual disability (ID), with autism spectrum disorder (ASD) as a prominent feature. Functional analysis in primary hippocampal mouse neuron cultures demonstrated that PCBP1 variants impair dendritic arborization, underscoring their deleterious effects. Transcriptomic profiling by RNA sequencing of subjects-derived T cells showed a distinctive signature characterized by significantly increased exon skipping. These results highlight the essential role of PCBP1 in neurogenesis and neuritogenesis, revealing the impact of loss-of-function variants in cells harboring PCBP1 pathogenic variants, thereby confirming the link between splicing defects and neurodevelopmental disorders. Collectively, our findings demonstrate the critical role of PCBP1 in neurodevelopment, reaffirming the importance of splicing regulation in mammalian neurodevelopment.

Variants in RNU4-2 , encoding the small nuclear RNA (snRNA) U4, were recently identified as a major cause of neurodevelopmental disorders (ReNU syndrome). Here, we investigated de novo variants in 50 snRNAs in a French cohort of 23,649 individuals with rare disorders and collected data of additional patients through an international collaboration. Altogether, we identified 133 probands with pathogenic or likely pathogenic variants in RNU4-2 and 15 individuals with de novo and/or recurrent variants in constrained regions of RNU5B-1 , one of five genes encoding U5. These variants cluster in evolutionarily conserved regions of U4 and U5 essential for splicing. RNU4-2 variants affecting stem III are associated with milder phenotypes than those in the T-loop (quasi-pseudoknot). Phaseable variants associated with severe phenotypes occurred on the maternal allele. Individuals with RNU4-2 variants show specific defects in alternative 5’ splice site usage, correlating with variant location and clinical severity. Additionally, we report an episignature associated with severe ReNU syndrome. This study further highlights the importance of de novo variants in snRNAs and establishes RNU5B-1 as a new neurodevelopmental disorder gene.

Pre-mRNA splicing is a highly coordinated process. While its dysregulation has been linked to neurological deficits, our understanding of the underlying molecular and cellular mechanisms remains limited. We implicated pathogenic variants in U2AF2 and PRPF19, encoding spliceosome subunits in neurodevelopmental disorders (NDDs), by identifying 46 unrelated individuals with 23 de novo U2AF2 missense variants (including 7 recurrent variants in 30 individuals) and 6 individuals with de novo PRPF19 variants. Eight U2AF2 variants dysregulated splicing of a model substrate. Neuritogenesis was reduced in human neurons differentiated from human pluripotent stem cells carrying two U2AF2 hyper-recurrent variants. Neural loss of function (LoF) of the Drosophila orthologs U2af50 and Prp19 led to lethality, abnormal mushroom body (MB) patterning, and social deficits, which were differentially rescued by wild-type and mutant U2AF2 or PRPF19. Transcriptome profiling revealed splicing substrates or effectors (including Rbfox1, a third splicing factor), which rescued MB defects in U2af50-deficient flies. Upon reanalysis of negative clinical exomes followed by data sharing, we further identified 6 patients with NDD who carried RBFOX1 missense variants which, by in vitro testing, showed LoF. Our study implicates 3 splicing factors as NDD-causative genes and establishes a genetic network with hierarchy underlying human brain development and function.

Cat Eye Syndrome (CES) is a rare genetic disease caused by the presence of a small supernumerary marker chromosome derived from chromosome 22, which results in a partial tetrasomy of 22p‐22q11.21. CES is classically defined by association of iris coloboma, anal atresia, and preauricular tags or pits, with high clinical and genetic heterogeneity. We conducted an international retrospective study of patients carrying genomic gain in the 22q11.21 chromosomal region upstream from LCR22‐A identified using FISH, MLPA, and/or array‐CGH. We report a cohort of 43 CES cases. We highlight that the clinical triad represents no more than 50% of cases. However, only 16% of CES patients presented with the three signs of the triad and 9% not present any of these three signs. We also highlight the importance of other impairments: cardiac anomalies are one of the major signs of CES (51% of cases), and high frequency of intellectual disability (47%). Ocular motility defects (45%), abdominal malformations (44%), ophthalmologic malformations (35%), and genitourinary tract defects (32%) are other frequent clinical features. We observed that sSMC is the most frequent chromosomal anomaly (91%) and we highlight the high prevalence of mosaic cases (40%) and the unexpectedly high prevalence of parental transmission of sSMC (23%). Most often, the transmitting parent has mild or absent features and carries the mosaic marker at a very low rate (<10%). These data allow us to better delineate the clinical phenotype associated with CES, which must be taken into account in the cytogenetic testing for this syndrome. These findings draw attention to the need for genetic counseling and the risk of recurrence.

Objectives: Aortic dissection in patients with Marfan and related syndromes (HTAD) is a serious pathology whose treatment by thoracic endovascular repair (TEVAR) is still under debate. The aim of this study was to assess the results of the TEVAR for aortic dissection in patients with HTAD as compared to a young population without HTAD. Methods: The study received the proper ethical oversight. We performed an observational exposed (confirmed HTAD) vs. non-exposed (p = 0.005. An increase in the whole aortic volume > 10% was observed in ten cases (58.8%) in the HTAD group and in five cases (22.7%) in the non-HTAD group (p = 0.022). FL thrombosis was achieved in nine cases (52.9%) in the HTAD group vs. twenty (90.9%) cases in the non-HTAD group (p < 0.01). The risk factors for unfavorable anatomical evolution were male gender and the STABILISE technique. With a linear model, we observed a significantly different aortic volume evolution between the two groups (p < 0.01) with the STABILISE technique; this statistical difference was not found in the TEVAR subgroup. In the HTAD patients, there was a significant difference in the total aortic volume evolution progression between the patients treated with the STABILISE technique and the patients treated with TEVAR (+160.1 ± 52.3% vs. +47 ± 22.5%, p < 0.01 and +189.5 ± 92.5% vs. +58.6 ± 34.8%, p < 0.01 at 1 year and at the end of follow-up, respectively). Conclusions: TEVAR in the HTAD patients seemed to be associated with poorer anatomical outcomes at 1 year. This result was strongly related to the STABILISE technique which should be considered with care in these specific patients.

(1) Background: The vascular type of Ehlers–Danlos syndrome (vEDS) is a rare genetic connective tissue disorder caused by pathogenic variants in the COL3A1 gene that result in arterial and organ fragility and premature death. We present five cases of vEDS that highlight the diagnosis and treatment challenges encountered by clinicians with these patients. (2) Case presentations: we present the cases of five patients with vascular complications of vEDS who were successfully managed using endovascular interventions or hybrid techniques at our institution from 2005 to 2022. (3) Conclusions: These data emphasize that a multidisciplinary approach is needed for vEDS patients and that when endovascular or hybrid treatment is performed in a timely manner by a skilled team of interventional radiologists, good results can be achieved. Our report also demonstrates that the prognosis of vEDS patients has improved over the past 20 years with a new prevention program including celiprolol therapy, physical activity adaptation and limitation, and scheduled monitoring by expert clinicians.

Exon skipping is a promising therapeutic approach. One important condition for this approach is that the exon-skipped form of the gene can at least partially perform the required function and lead to improvement of the phenotype. It is therefore critical to identify the exons that can be skipped without a significant deleterious effect on the protein function. Pathogenic variants in the DMD gene are responsible for Duchenne muscular dystrophy (DMD). We report for the first time a deletion of the in-frame exon 49 associated with a strikingly normal muscular phenotype. Based on this observation, and on previously known therapeutic approaches using exon skipping in DMD for other single exons, we aimed to extend the clinical use of exon skipping for patients carrying truncating mutations in exon 49. We first determined the precise genomic position of the exon 49 deletion in our patients. We then demonstrated the feasibility of skipping exon 49 using an in vitro AON (antisense oligonucleotide) approach in human myotubes carrying a truncating pathogenic variant as well as in healthy ones. This work is a proof of concept aiming to expand exon-skipping approaches for DMD exon 49.

Purpose Despite exome (ES) or genome sequencing (GS) availability, chromosomal microarray (CMA) remains the first-line diagnostic tests in most rare disorders diagnostic work-up, looking for Copy-number variations (CNV), with a diagnostic yield of 10-20%. The question of the equivalence of CMA and ES in CNV calling is an organisational and economic question, especially when ordering a GS after a negative CMA and/or ES. Methods This work measures the equivalence between CMA and GATK4 exome sequencing depth of coverage method in detecting coding CNV on a retrospective cohort of 615 unrelated individuals. A prospective detection of ES CNV on a cohort of 1803 unrelated individuals was performed. Results On the retrospective validation cohort every CNV was accurately detected (64/64 events). In the prospective cohort, 32 diagnostics were performed among the 1803 individuals with CNVs ranging from 704bp to aneuploidy. An incidental finding was reported. The overall increase in diagnostic yield was of 1.7%, varying from 1.2% in individuals with multiple congenital anomalies to 1.9% in individuals with chronic kidney failure. Conclusions Combining SNV and CNV detection increases the suitability of exome sequencing as a first-tier diagnostic test for suspected rare mendelian disorders. Before considering the prescription of a GS after a negative ES, a careful reanalysis with updated CNV calling and SNV annotation should be considered.