POLR1A variants underlie phenotypic heterogeneity in craniofacial, neural, and cardiac anomalies.

Heterozygous pathogenic variants in POLR1A, which encodes the largest subunit of RNA Polymerase I, were previously identified as the cause of acrofacial dysostosis, Cincinnati-type. The predominant phenotypes observed in the cohort of 3 individuals were craniofacial anomalies reminiscent of Treacher Collins syndrome. We subsequently identified 17 additional individuals with 12 unique heterozygous variants in POLR1A and observed numerous additional phenotypes including neurodevelopmental abnormalities and structural cardiac defects, in combination with highly prevalent craniofacial anomalies and variable limb defects. To understand the pathogenesis of this pleiotropy, we modeled an allelic series of POLR1A variants in vitro and in vivo. In vitro assessments demonstrate variable effects of individual pathogenic variants on ribosomal RNA synthesis and nucleolar morphology, which supports the possibility of variant-specific phenotypic effects in affected individuals. To further explore variant-specific effects in vivo, we used CRISPR-Cas9 gene editing to recapitulate two human variants in mice. Additionally, spatiotemporal requirements for Polr1a in developmental lineages contributing to congenital anomalies in affected individuals were examined via conditional mutagenesis in neural crest cells (face and heart), the second heart field (cardiac outflow tract and right ventricle), and forebrain precursors in mice. Consistent with its ubiquitous role in the essential function of ribosome biogenesis, we observed that loss of Polr1a in any of these lineages causes cell-autonomous apoptosis resulting in embryonic malformations. Altogether, our work greatly expands the phenotype of human POLR1A-related disorders and demonstrates variant-specific effects that provide insights into the underlying pathogenesis of ribosomopathies.

Stx4 is required to regulate cardiomyocyte Ca2+ handling during vertebrate cardiac development.

Requirements for vesicle fusion within the heart remain poorly understood, despite the multitude of processes that necessitate proper intracellular trafficking within cardiomyocytes. Here, we show that Syntaxin 4 (STX4), a target-Soluble N-ethylmaleimide sensitive factor attachment receptor (t-SNARE) protein, is required for normal vertebrate cardiac conduction and vesicular transport. Two patients were identified with damaging variants in STX4. A patient with a homozygous R240W missense variant displayed biventricular dilated cardiomyopathy, ectopy, and runs of non-sustained ventricular tachycardia, sensorineural hearing loss, global developmental delay, and hypotonia, while a second patient displayed severe pleiotropic abnormalities and perinatal lethality. CRISPR/Cas9-generated stx4 mutant zebrafish exhibited defects reminiscent of these patients' clinical presentations, including linearized hearts, bradycardia, otic vesicle dysgenesis, neuronal atrophy, and touch insensitivity by 3 days post fertilization. Imaging of Vamp2+ vesicles within stx4 mutant zebrafish hearts showed reduced docking to the cardiomyocyte sarcolemma. Optical mapping of the embryonic hearts coupled with pharmacological modulation of Ca2+ handling together support that zebrafish stx4 mutants have a reduction in L-type Ca2+ channel modulation. Transgenic overexpression of zebrafish Stx4R241W, analogous to the first patient's STX4R240W variant, indicated that the variant is hypomorphic. Thus, these data show an in vivo requirement for SNAREs in regulating normal embryonic cardiac function and that variants in STX4 are associated with pleiotropic human disease, including cardiomyopathy.

Mandibulofacial dysostosis with microcephaly: An expansion of the phenotype via parental survey.

Mandibulofacial dysostosis with microcephaly (MFDM) is due to haploinsufficiency of spliceosomal GTPase EFTUD2. Features include microcephaly, craniofacial dysmorphology, developmental disability, and other anomalies. We surveyed parents of individuals with MFDM to expand knowledge about health, development, and parental concerns. Participants included attendees of the inaugural MFDM family conference in June 2019 and members of the MFDM online group. To explore MFDM variable expressivity, we offered targeted Sanger sequencing for untested parents. Forty-seven parents participated in the survey. 59% of individuals with MFDM were male, with mean age 6.4 years (range 8 months to 49 years). Similar to the literature (n = 123), common features include microcephaly, cleft palate, choanal stenosis, tracheoesophageal fistula, heart problems, and seizures. New information includes airway intervention details, age-based developmental outcomes, rate of vision refractive errors, and lower incidences of prematurity and IUGR. Family concerns focused on development, communication, and increased support. Targeted Sanger sequencing for families of seven individuals demonstrated de novo variants, for a total of 91.9% de novo EFTUD2 variants (n = 34/37). This study reports the largest single cohort of individuals with MFDM, expands phenotypic spectrum and inheritance patterns, improves understanding of developmental outcomes and care needs, and identifies development as the biggest concern for parents.

Prioritized Dietary Supplement Information Needs of 307 NCAA Division I Student Athletes.

OBJECTIVE: To evaluate dietary supplement information needs among collegiate athletes. METHODS: Three hundred seven (n = 154 male; n = 153 female) student athletes participating in a National Collegiate Athletic Association Division I team completed a dietary supplement survey. Qualitative coding addressed open-ended responses, and chi-square test of independence explored differences among athlete subgroups. RESULTS: Five themes representing athletes' information needs included quality/composition (53.5%; n = 77), general information (31.9%; n = 46), nutrition information (30.6%; n = 44), performance (18.8%; n = 27), and body composition (13.2%; n = 19). Athletes with "no" or "minimal" (n = 63), vs "moderate" or "strong" (n = 195), perceived knowledge of supplement safety were more likely to list a question about supplement quality or composition (34.9% [n = 22/63] vs 21.5% [n= 42/195]; P = .03; chi-square = 4.6). CONCLUSIONS AND IMPLICATIONS: Further research is needed to corroborate findings to inform educational efforts and promote safe and effective use of dietary supplements by student athletes.