Abnormalities in pharyngeal arch-derived structures in SATB2-associated syndrome.

SATB2-associated syndrome (SAS, glass syndrome, OMIM#612313) is a neurodevelopmental autosomal dominant disorder with frequent craniofacial abnormalities including palatal and dental anomalies. To assess the role of Satb2 in craniofacial development, we analyzed mutant mice at different stages of development. Here, we show that Satb2 is broadly expressed in early embryonic mouse development including the mesenchyme of the second and third arches. Satb2-/- mutant mice exhibit microglossia, a shortened lower jaw, smaller trigeminal ganglia, and larger thyroids. We correlate these findings with the detailed clinical phenotype of four individuals with SAS and remarkable craniofacial phenotypes with one requiring mandibular distraction in childhood. We conclude that the mouse and patient data presented support less well-described phenotypic aspects of SAS including mandibular morphology and thyroid anatomical/functional issues.

NR3C2 microdeletions-an underrecognized cause of pseudohypoaldosteronism type 1A: a case report and literature review.

OBJECTIVES: Pseudohypoaldosteronism type 1A (PHA1A) is caused by haploinsufficiency of the mineralocorticoid receptor (MR). Heterozygous small insertions/deletions, transitions, and/or transversions within NR3C2 comprise the majority (85%-90%) of pathogenic copy number variants. Structural chromosomal abnormalities, contiguous gene deletion syndromes, and microdeletions are infrequent. We describe a neonate with PHA1A due to a novel NR3C2 microdeletion involving exons 1-2. METHODS: Literature review identified 39 individuals with PHA1A due to NR3C2 microdeletions. Transmission modality, variant description(s), testing method(s), exon(s) deleted, and affected functional domain(s) were characterized. RESULTS: In total, 40 individuals with NR3C2 microdeletions were described: 19 involved contiguous exons encoding a single MR domain; 21 involved contiguous exons encoding multiple MR domains. Transmission modality frequency was familial (65%), de novo (20%), or unknown (15%). Sequencing (Sanger or short-read next-generation) failed to detect microdeletions in 100% of tested individuals (n = 38). All were detected using deletion/duplication testing modalities. In 2 individuals, only microarray-based testing was performed; microdeletions were detected in both cases. CONCLUSION: Initial testing for PHA1A should rely on sequencing to detect the most common genetic alterations. Deletion/duplication analysis should be performed when initial testing is nondiagnostic. Most NR3C2 microdeletions are parentally transmitted, thus highlighting the importance of familial genetic testing and counseling.

Bone health in SATB2-associated syndrome: Results from a large prospective cohort and recommendations for surveillance.

Alterations in SATB2 result in SATB2-associated syndrome (SAS; Glass syndrome, OMIM 612313), an autosomal dominant multisystemic disorder predominantly characterized by developmental delay, craniofacial anomalies, and growth retardation. The bone phenotype of SAS has been less explored until recently and includes a variety of skeletal deformities, increased risk of low bone mineral density (BMD) with a propensity to fractures, and other biochemical abnormalities that suggest elevated bone turnover. We present the results of ongoing surveillance of bone health from 32 individuals (47% females, 3-18 years) with molecularly-confirmed SAS evaluated at a multidisciplinary clinic. Five individuals (5/32, 16%) were documented to have BMD Z-scores by DXA scans of -2.0 SD or lower and 7 more (7/32, 22%) had Z-scores between -1 and - 2 SD at the lumbar spine or the total hip. Alkaline phosphatase levels were found to be elevated in 19 individuals (19/30, 63%) and determined to correspond to bone-specific alkaline phosphatase elevations when measured (11/11, 100%). C-telopeptide levels were found to be elevated when adjusted by age and gender in 6 individuals (6/14, 43%). Additionally, the two individuals who underwent bone cross-sectional geometry evaluation by peripheral quantitative computed tomography were documented to have low cortical bone density for age and sex despite concurrent DXA scans that did not have this level of decreased density. While we could not identify particular biochemical abnormalities that predicted low BMD, the frequent elevations in markers of bone formation and resorption further confirmed the increased bone turnover in SAS. Based on our results and other recently published studies, we propose surveillance guidelines for the skeletal phenotype of SAS.

Once-weekly TransCon CNP (navepegritide) in children with achondroplasia (ACcomplisH): a phase 2, multicentre, randomised, double-blind, placebo-controlled, dose-escalation trial.

Background: TransCon CNP (navepegritide) is an investigational prodrug of C-type natriuretic peptide (CNP) designed to allow for continuous CNP exposure with once-weekly dosing. This 52-week phase 2 (ACcomplisH) trial assessed the safety and efficacy of TransCon CNP in children with achondroplasia. Methods: ACcomplisH is a global, randomised, double-blind, placebo-controlled, dose-escalation trial. Study participants were recruited between June 10, 2020, and September 24, 2021. Eligible participants were prepubertal, aged 2-10 years, with genetically confirmed achondroplasia, and randomised 3:1 to once-weekly subcutaneous injections of TransCon CNP (6, 20, 50, or 100 µg CNP/kg/week) or placebo for 52 weeks. Primary objectives were safety and annualised growth velocity (AGV). ACcomplisH is registered with ClinicalTrials.gov (NCT04085523) and Eudra (CT 2019-002754-22). Findings: Forty-two participants received TransCon CNP at doses of 6 µg (n = 10; 7 female), 20 µg (n = 11; 3 female), 50 µg (n = 10; 3 female), or 100 µg (n = 11; 6 female) CNP/kg/week, with 15 receiving placebo (5 female). Treatment-emergent adverse events (TEAEs) were mild or moderate with no grade 3/4 events reported. There were 2 serious TEAEs that were assessed as not related to TransCon CNP. Eleven injection site reactions occurred in 8 participants receiving TransCon CNP and no symptomatic hypotension occurred. TransCon CNP demonstrated a dose-dependent improvement in AGV. At 52 weeks, TransCon CNP 100 µg CNP/kg/week significantly improved AGV vs placebo (least squares mean [95% CI] 5.42 [4.74-6.11] vs 4.35 [3.75-4.94] cm/year; p = 0.0218), and improved achondroplasia-specific height SDS from baseline (least squares mean [95% CI] 0.22 [0.02-0·41] vs -0·08 [-0.25 to 0.10]; p = 0.0283). All participants completed the randomised period and continued in the ongoing open-label extension period receiving TransCon CNP 100 µg CNP/kg/week. Interpretation: This phase 2 trial suggests that TransCon CNP is effective, safe, with low injection site reaction frequency, and may provide a novel, once-weekly treatment option for children with achondroplasia. These results support TransCon CNP at 100 µg CNP/kg/week in the ongoing pivotal trial. Funding: Ascendis Pharma, A/S.

Bi-allelic variants in INTS11 are associated with a complex neurological disorder.

The Integrator complex is a multi-subunit protein complex that regulates the processing of nascent RNAs transcribed by RNA polymerase II (RNAPII), including small nuclear RNAs, enhancer RNAs, telomeric RNAs, viral RNAs, and protein-coding mRNAs. Integrator subunit 11 (INTS11) is the catalytic subunit that cleaves nascent RNAs, but, to date, mutations in this subunit have not been linked to human disease. Here, we describe 15 individuals from 10 unrelated families with bi-allelic variants in INTS11 who present with global developmental and language delay, intellectual disability, impaired motor development, and brain atrophy. Consistent with human observations, we find that the fly ortholog of INTS11, dIntS11, is essential and expressed in the central nervous systems in a subset of neurons and most glia in larval and adult stages. Using Drosophila as a model, we investigated the effect of seven variants. We found that two (p.Arg17Leu and p.His414Tyr) fail to rescue the lethality of null mutants, indicating that they are strong loss-of-function variants. Furthermore, we found that five variants (p.Gly55Ser, p.Leu138Phe, p.Lys396Glu, p.Val517Met, and p.Ile553Glu) rescue lethality but cause a shortened lifespan and bang sensitivity and affect locomotor activity, indicating that they are partial loss-of-function variants. Altogether, our results provide compelling evidence that integrity of the Integrator RNA endonuclease is critical for brain development.

Bi-allelic variants in NAE1 cause intellectual disability, ischiopubic hypoplasia, stress-mediated lymphopenia and neurodegeneration.

Neddylation has been implicated in various cellular pathways and in the pathophysiology of numerous diseases. We identified four individuals with bi-allelic variants in NAE1, which encodes the neddylation E1 enzyme. Pathogenicity was supported by decreased NAE1 abundance and overlapping clinical and cellular phenotypes. To delineate how cellular consequences of NAE1 deficiency would lead to the clinical phenotype, we focused primarily on the rarest phenotypic features, based on the assumption that these would best reflect the pathophysiology at stake. Two of the rarest features, neuronal loss and lymphopenia worsening during infections, suggest that NAE1 is required during cellular stress caused by infections to protect against cell death. In support, we found that stressing the proteasome system with MG132-requiring upregulation of neddylation to restore proteasomal function and proteasomal stress-led to increased cell death in fibroblasts of individuals with NAE1 genetic variants. Additionally, we found decreased lymphocyte counts after CD3/CD28 stimulation and decreased NF-κB translocation in individuals with NAE1 variants. The rarest phenotypic feature-delayed closure of the ischiopubic rami-correlated with significant downregulation of RUN2X and SOX9 expression in transcriptomic data of fibroblasts. Both genes are involved in the pathophysiology of ischiopubic hypoplasia. Thus, we show that NAE1 plays a major role in (skeletal) development and cellular homeostasis during stress. Our approach suggests that a focus on rare phenotypic features is able to provide significant pathophysiological insights in diseases caused by mutations in genes with pleiotropic effects.

Loss-of-function variants in MYCBP2 cause neurobehavioural phenotypes and corpus callosum defects.

The corpus callosum is a bundle of axon fibres that connects the two hemispheres of the brain. Neurodevelopmental disorders that feature dysgenesis of the corpus callosum as a core phenotype offer a valuable window into pathology derived from abnormal axon development. Here, we describe a cohort of eight patients with a neurodevelopmental disorder characterized by a range of deficits including corpus callosum abnormalities, developmental delay, intellectual disability, epilepsy and autistic features. Each patient harboured a distinct de novo variant in MYCBP2, a gene encoding an atypical really interesting new gene (RING) ubiquitin ligase and signalling hub with evolutionarily conserved functions in axon development. We used CRISPR/Cas9 gene editing to introduce disease-associated variants into conserved residues in the Caenorhabditis elegans MYCBP2 orthologue, RPM-1, and evaluated functional outcomes in vivo. Consistent with variable phenotypes in patients with MYCBP2 variants, C. elegans carrying the corresponding human mutations in rpm-1 displayed axonal and behavioural abnormalities including altered habituation. Furthermore, abnormal axonal accumulation of the autophagy marker LGG-1/LC3 occurred in variants that affect RPM-1 ubiquitin ligase activity. Functional genetic outcomes from anatomical, cell biological and behavioural readouts indicate that MYCBP2 variants are likely to result in loss of function. Collectively, our results from multiple human patients and CRISPR gene editing with an in vivo animal model support a direct link between MYCBP2 and a human neurodevelopmental spectrum disorder that we term, MYCBP2-related developmental delay with corpus callosum defects (MDCD).

Growth in individuals with SATB2-associated syndrome.

SATB2-associated syndrome (SAS) is an autosomal dominant multisystemic disorder caused by alterations in the SATB2 gene. In addition to a predominant neurodevelopmental phenotype, individuals with SAS often present with feeding difficulties and growth retardation that persist past infancy. In this study, we present growth and measurement data from 211 individuals (53.6% male, 46.4% female) with SAS due to different molecular mechanisms. To delineate growth in this population, we constructed SAS-specific growth charts by sex from birth to 10 years of age. Smoothed SAS percentiles were superimposed with normative percentiles from WHO (birth to <24 months) and CDC (24 months to 10 years) growth charts. Individuals with SAS tend to display slower postnatal growth with 22.2% (32/144), 19.0% (26/137), and 21.6% having at least one weight, height, or weight-for-length /body mass index (BMI) measurement below -2 standard deviations, respectively. The SAS 50th centile BMI was consistently below the normative data 50th centile and negative mean Z-scores were seen across almost all age groups analyzed for both genders. Individuals with chromosomal abnormalities displayed significantly lower weight for age Z-score, height for age Z-scores, occipitofrontal head circumference for age Z-scores, and BMI for age Z-scores compared to either missense or null variants.

The landscape of submicroscopic structural variants at the OPN1LW/OPN1MW gene cluster on Xq28 underlying blue cone monochromacy.

Blue cone monochromacy (BCM) is an X-linked retinal disorder characterized by low vision, photoaversion, and poor color discrimination. BCM is due to the lack of long-wavelength-sensitive and middle-wavelength-sensitive cone photoreceptor function and caused by mutations in the OPN1LW/OPN1MW gene cluster on Xq28. Here, we investigated the prevalence and the landscape of submicroscopic structural variants (SVs) at single-base resolution in BCM patients. We found that about one-third (n = 73) of the 213 molecularly confirmed BCM families carry an SV, most commonly deletions restricted to the OPN1LW/OPN1MW gene cluster. The structure and precise breakpoints of the SVs were resolved in all but one of the 73 families. Twenty-two families-all from the United States-showed the same SV, and we confirmed a common ancestry of this mutation. In total, 42 distinct SVs were identified, including 40 previously unreported SVs, thereby quadrupling the number of precisely mapped SVs underlying BCM. Notably, there was no "region of overlap" among these SVs. However, 90% of SVs encompass the upstream locus control region, an essential enhancer element. Its minimal functional extent based on deletion mapping in patients was refined to 358 bp. Breakpoint analyses suggest diverse mechanisms underlying SV formation as well as in one case the gene conversion-based exchange of a 142-bp deletion between opsin genes. Using parsimonious assumptions, we reconstructed the composition and copy number of the OPN1LW/OPN1MW gene cluster prior to the mutation event and found evidence that large gene arrays may be predisposed to the occurrence of SVs at this locus.

Prevalence and Outcomes of Primary Left Ventricular Dysfunction in Marfan Syndrome.

Even in the absence of significant valvular disease, patients with Marfan syndrome (MFS) have evidence of impaired left ventricular (LV) performance, suggestive of a primary cardiomyopathy. However, the true prevalence and long-term outcomes of this disease process remain largely unknown. We performed a retrospective analysis of all adult patients with confirmed MFS followed at Stanford Health Care. Those with significant valvular regurgitation, coronary artery disease, or previous cardiac surgery were excluded. LV systolic dysfunction was defined as a LV ejection fraction (LVEF) <55% on transthoracic echocardiography. A total of 753 patients with confirmed MFS were followed up over a median duration of 8 years (interquartile range 4 to 13). Of those, 241 patients (53% women, 71% White) met inclusion criteria and comprised the study cohort. LV systolic dysfunction was present in 30 patients (12%), with a median age of onset of 25 years (interquartile range 19 to 37), median EF of 52% (interquartile range 48 to 54), and evidence of clinical heart failure (New York Heart Association functional class ≥II) in 10% of patients. LV systolic dysfunction was more common in patients with larger aortic root diameters (≥4.0 cm: Odds ratio = 4.5, 95% confidence interval = 1.2 to 17.1) but was not associated with other cardiovascular manifestations of MFS or traditional atherosclerotic risk factors. In conclusion, apart from significant valvular pathology, LV systolic dysfunction was prevalent in MFS from a young age, suggestive of a primary cardiomyopathy. LV dysfunction was typically mild and subclinical and occurred more commonly in patients with more pronounced aortopathies.

A clinical scoring system for early onset (neonatal) Marfan syndrome.

PURPOSE: This study aimed to develop objective diagnostic criteria for early onset Marfan syndrome (eoMFS) to facilitate early diagnosis and timely interventions. METHODS: On the basis of an extensive literature review and the responses from a survey distributed among providers with expertise in the diagnosis and management of eoMFS, we developed an age-based, diagnostic scoring system encompassing 10 features common to eoMFS (9 clinical + 1 laboratory) and divided them into cardiac, systemic, and FBN1 (on the basis of the location of the pathogenic FBN1 variant) scores. RESULTS: In total, 77 individuals with eoMFS (13 newly reported) and 49 individuals diagnosed with classical Marfan syndrome during early childhood were used to validate the criteria. Median cardiac (8 vs 0, P < .001), systemic (11 vs 3, P < .001), FBN1 (5 vs 0, P < .001), and total (23 vs 4, P < .001) scores were significantly higher in individuals with eoMFS than in those without. A proposed clinical score (cardiac + systemic) cutoff of ≥14 points showed excellent sensitivity (100%), specificity (92%), and reliability (correctly classified = 94%). CONCLUSION: Distinct from classical Marfan syndrome in phenotype and morbidity, eoMFS can be diagnosed clinically using an objective scoring system encompassing the typical physical features and cardiac disease manifestations. Although genetic testing can be suggestive of eoMFS, genetic testing alone is insufficient for diagnosis.

Genetic and phenotypic heterogeneity in KIAA0753-related ciliopathies.

Primary ciliopathies are heterogenous disorders resulting from perturbations in primary cilia form and/or function. Primary cilia are cellular organelles which mediate key signaling pathways during development, such as the sonic hedgehog (SHH) pathway which is required for neuroepithelium and central nervous system development. Joubert syndrome is a primary ciliopathy characterized by cerebellar/brain stem malformation, hypotonia, and developmental delays. At least 35 genes are associated with Joubert syndrome, including the gene KIAA0753, which is part of a complex required for primary ciliogenesis. The phenotypic spectrum associated with biallelic pathogenic variants in KIAA0753 is broad and not well-characterized. We describe four individuals with biallelic pathogenic KIAA0753 variants, including five novel variants. We report in vitro results assessing the function of each variant indicating that mutant proteins are not fully competent to promote primary ciliogenesis. Ablation of KIAA0753 in vitro blocks primary ciliogenesis and SHH pathway activity. Correspondingly, KIAA0753 patient fibroblasts have a deficit in primary ciliation and improper SHH and WNT signaling, with a particularly blunted response to SHH pathway stimulation. Our work expands the phenotypic spectrum of KIAA0753 ciliopathies and demonstrates the utility of patient-focused functional assays for proving causality of genetic variants.

Craniosynostosis is a feature of Costello syndrome.

Costello syndrome (CS) is an autosomal dominant disorder caused by pathogenic variants in HRAS. Craniosynostosis is a known feature of other RASopathies (Noonan and cardiofaciocutaneous syndromes) but not CS. We describe four individuals with CS and craniosynostosis and present a summary of all previously reported individuals with craniosynostosis and RASopathy.

Case Report: SATB2-Associated Syndrome Overlapping With Clinical Mitochondrial Disease Presentation: Report of Two Cases.

SATB2-associated syndrome (SAS) is an autosomal dominant neurogenetic multisystemic disorder. We describe two individuals with global developmental delay and hypotonia who underwent an extensive evaluation to rule out an underlying mitochondrial disorder before their eventual diagnosis of SAS. Although the strict application of the clinical mitochondrial disease score only led to the designation of "possible" mitochondrial disorder for these two individuals, other documented abnormalities included nonspecific neuroimaging findings on magnetic resonance imaging and magnetic resonance spectroscopy, decreased complex I activity on muscle biopsy for patient 2, and variation in the size and relative proportion of types of muscle fibers in the muscle biopsies that were aligned with mitochondrial diseases. SAS should be in the differential diagnoses of mitochondrial disorders, and broad-spectrum diagnostic tests such as exome sequencing need to be considered early in the evaluation process of undiagnosed neurodevelopmental disorders.

Variants in the degron of AFF3 are associated with intellectual disability, mesomelic dysplasia, horseshoe kidney, and epileptic encephalopathy.

The ALF transcription factor paralogs, AFF1, AFF2, AFF3, and AFF4, are components of the transcriptional super elongation complex that regulates expression of genes involved in neurogenesis and development. We describe an autosomal dominant disorder associated with de novo missense variants in the degron of AFF3, a nine amino acid sequence important for its binding to ubiquitin ligase, or with de novo deletions of this region. The sixteen affected individuals we identified, along with two previously reported individuals, present with a recognizable pattern of anomalies, which we named KINSSHIP syndrome (KI for horseshoe kidney, NS for Nievergelt/Savarirayan type of mesomelic dysplasia, S for seizures, H for hypertrichosis, I for intellectual disability, and P for pulmonary involvement), partially overlapping the AFF4-associated CHOPS syndrome. Whereas homozygous Aff3 knockout mice display skeletal anomalies, kidney defects, brain malformations, and neurological anomalies, knockin animals modeling one of the microdeletions and the most common of the missense variants identified in affected individuals presented with lower mesomelic limb deformities like KINSSHIP-affected individuals and early lethality, respectively. Overexpression of AFF3 in zebrafish resulted in body axis anomalies, providing some support for the pathological effect of increased amount of AFF3. The only partial phenotypic overlap of AFF3- and AFF4-associated syndromes and the previously published transcriptome analyses of ALF transcription factors suggest that these factors are not redundant and each contributes uniquely to proper development.

SATB2-associated syndrome in adolescents and adults.

The goal of this study was to investigate the medical, communication, activities of daily living (ADLs), and mental health concerns affecting adolescents and adults with SATB2-associated syndrome (SAS). A comprehensive questionnaire was administered to the caregivers of 49 individuals 12 years or older with SAS (mean age was 19.4 years, range 12-37 years). For all individuals, medical records, including laboratory results, were reviewed. Most individuals required some degree of assistance for ADLs and none of the adults were able to live independently. Health status was qualified as excellent or very good in 61% of individuals. The most common medical problems were dental anomalies, with a significantly higher frequency of hypotonia and gastroesophageal reflux in younger individuals. Medical and surgical interventions were often required. Sixty-nine percent (n = 33) of individuals spoke 10 or fewer words. Autism (41%), anxiety (37%), and attention deficit disorder (37%) were common with one third of individuals receiving medical treatments for these diagnoses. While medical and developmental problems in individuals with SAS were similar to those previously reported, many of these are persistent into adolescence and adulthood. This study provides better guidance for the challenges facing adults with SAS and their families.

Individuals with SATB2-associated syndrome with and without autism have a recognizable metabolic profile and distinctive cellular energy metabolism alterations.

SATB2-associated syndrome (SAS) is a multisystemic disorder characterized by developmental delay often with concurrent autistic tendencies. This study aimed to characterize cellular metabolic pathways and energy metabolism from cells derived from individuals with SAS. The cellular production of NADH (nicotinamide adenine dinucleotide, reduced form) as determined by the Phenotype Mammalian MicroArrays was measured in lymphoblastoid cell lines derived from 11 subjects with a molecularly confirmed diagnosis of SAS and compared to a control population of 50 age-matched typically developing individuals. All patients were evaluated clinically by a multidisciplinary team. Eleven individuals (five in a screening cohort and six in the validation cohort, mean age 6.1 years) were recruited to the study. All individuals had developmental delay and the diagnosis of autism was previously established in five of them. Key metabolic findings included reduced NADH production in the presence of phosphorylated carbohydrates (with corresponding increased production in the presence of alternative carbon-based energy sources), increased response to certain hormones (ß-estradiol in particular), and significantly reduced levels of NADH in wells containing tryptophan. The individual analysis revealed no particular differences among the SAS subjects based on molecular findings or phenotypic features. In conclusion, individuals with SAS have a common and recognizable metabolic profile. A lower capacity to utilize glucose as an energy substrate could be contributing to the neurodevelopment phenotype of SAS. The identified abnormalities offer previously unexplored insight into the potential pathophysiology of common SAS phenotypic features.

A dyadic approach to the delineation of diagnostic entities in clinical genomics.

The delineation of disease entities is complex, yet recent advances in the molecular characterization of diseases provide opportunities to designate diseases in a biologically valid manner. Here, we have formalized an approach to the delineation of Mendelian genetic disorders that encompasses two distinct but inter-related concepts: (1) the gene that is mutated and (2) the phenotypic descriptor, preferably a recognizably distinct phenotype. We assert that only by a combinatorial or dyadic approach taking both of these attributes into account can a unitary, distinct genetic disorder be designated. We propose that all Mendelian disorders should be designated as "GENE-related phenotype descriptor" (e.g., "CFTR-related cystic fibrosis"). This approach to delineating and naming disorders reconciles the complexity of gene-to-phenotype relationships in a simple and clear manner yet communicates the complexity and nuance of these relationships.