The case for early use of rapid whole-genome sequencing in management of critically ill infants: late diagnosis of Coffin-Siris syndrome in an infant with left congenital diaphragmatic hernia, congenital heart disease, and recurrent infections.

Congenital diaphragmatic hernia (CDH) results from incomplete formation of the diaphragm leading to herniation of abdominal organs into the thoracic cavity. CDH is associated with pulmonary hypoplasia, congenital heart disease, and pulmonary hypertension. Genetically, it is associated with aneuploidies, chromosomal copy-number variants, and single gene mutations. CDH is the most expensive noncardiac congenital defect. Management frequently requires implementation of extracorporeal membrane oxygenation (ECMO), which increases management expenditures 2.4-3.5-fold. The cost of management of CDH has been estimated to exceed $250 million per year. Despite in-hospital survival of 80%-90%, current management is imperfect, as a great proportion of surviving children have long-term functional deficits. We report the case of a premature infant prenatally diagnosed with CDH and congenital heart disease, who had a protracted and complicated course in the intensive care unit with multiple surgical interventions, including postcardiac surgery ECMO, gastrostomy tube placement with Nissen fundoplication, tracheostomy for respiratory failure, recurrent infections, and developmental delay. Rapid whole-genome sequencing (rWGS) identified a de novo, likely pathogenic, c.3096_ 3100delCAAAG (p.Lys1033Argfs*32) variant in ARID1B, providing a diagnosis of Coffin-Siris syndrome. Her parents elected palliative care and she died later that day

Biallelic mutations in valyl-tRNA synthetase gene VARS are associated with a progressive neurodevelopmental epileptic encephalopathy.

Aminoacyl-tRNA synthetases (ARSs) function to transfer amino acids to cognate tRNA molecules, which are required for protein translation. To date, biallelic mutations in 31 ARS genes are known to cause recessive, early-onset severe multi-organ diseases. VARS encodes the only known valine cytoplasmic-localized aminoacyl-tRNA synthetase. Here, we report seven patients from five unrelated families with five different biallelic missense variants in VARS. Subjects present with a range of global developmental delay, epileptic encephalopathy and primary or progressive microcephaly. Longitudinal assessment demonstrates progressive cortical atrophy and white matter volume loss. Variants map to the VARS tRNA binding domain and adjacent to the anticodon domain, and disrupt highly conserved residues. Patient primary cells show intact VARS protein but reduced enzymatic activity, suggesting partial loss of function. The implication of VARS in pediatric neurodegeneration broadens the spectrum of human diseases due to mutations in tRNA synthetase genes

Nonaminoglycoside compounds induce readthrough of nonsense mutations

Large numbers of genetic disorders are caused by nonsense mutations for which compound-induced readthrough of premature termination codons (PTCs) might be exploited as a potential treatment strategy. We have successfully developed a sensitive and quantitative high-throughput screening (HTS) assay, protein transcription/translation (PTT)–enzyme-linked immunosorbent assay (ELISA), for identifying novel PTC-readthrough compounds using ataxia-telangiectasia (A-T) as a genetic disease model. This HTS PTT-ELISA assay is based on a coupled PTT that uses plasmid templates containing prototypic A-T mutated (ATM) mutations for HTS. The assay is luciferase independent. We screened ∼34,000 compounds and identified 12 low-molecular-mass nonaminoglycosides with potential PTC-readthrough activity. From these, two leading compounds consistently induced functional ATM protein in ATM-deficient cells containing disease-causing nonsense mutations, as demonstrated by direct measurement of ATM protein, restored ATM kinase activity, and colony survival assays for cellular radiosensitivity. The two compounds also demonstrated readthrough activity in mdx mouse myotube cells carrying a nonsense mutation and induced significant amounts of dystrophin protein

ATM-dependent MiR-335 targets CtIP and modulates the DNA damage response.

ATM plays a critical role in cellular responses to DNA double-strand breaks (DSBs). We describe a new ATM-mediated DSB-induced DNA damage response pathway involving microRNA (miRNA): irradiation (IR)-induced DSBs activate ATM, which leads to the downregulation of miR-335, a miRNA that targets CtIP, which is an important trigger of DNA end resection in homologous recombination repair (HRR). We demonstrate that CREB is responsible for a large portion of miR-335 expression by binding to the promoter region of miR-335. CREB binding is greatly reduced after IR, corroborating with previous studies that IR-activated ATM phosphorylates CREB to reduce its transcription activity. Overexpression of miR-335 in HeLa cells resulted in reduced CtIP levels and post-IR colony survival and BRCA1 foci formation. Further, in two patient-derived lymphoblastoid cell lines with decreased post-IR colony survival, a "radiosensitive" phenotype, we demonstrated elevated miR-335 expression, reduced CtIP levels, and reduced BRCA1 foci formation. Colony survival, BRCA1 foci, and CtIP levels were partially rescued by miRNA antisense AMO-miR-335 treatment. Taken together, these findings strongly suggest that an ATM-dependent CREB-miR-335-CtIP axis influences the selection of HRR for repair of certain DSB lesions

A new series of small molecular weight compounds induce read through of all three types of nonsense mutations in the ATM gene.

Chemical-induced read through of premature stop codons might be exploited as a potential treatment strategy for genetic disorders caused by nonsense mutations. Despite the promise of this approach, only a few read-through compounds (RTCs) have been discovered to date. These include aminoglycosides (e.g., gentamicin and G418) and nonaminoglycosides (e.g., PTC124 and RTC13). The therapeutic benefits of these RTCs remain to be determined. In an effort to find new RTCs, we screened an additional ~36,000 small molecular weight compounds using a high-throughput screening (HTS) assay that we had previously developed and identified two novel RTCs, GJ071, and GJ072. The activity of these two compounds was confirmed in cells derived from ataxia telangiectasia (A-T) patients with three different types of nonsense mutation in the ATM gene. Both compounds showed activity comparable to stop codons (TGA, TAG, and TAA) PTC124 and RTC13. Early structure-activity relationship studies generated eight active analogs of GJ072. Most of those analogs were effective on all three stop codons. GJ071 and GJ072, and some of the GJ072 analogs, appeared to be well tolerated by A-T cells. We also identified another two active RTCs in the primary screen, RTC204 and RTC219, which share a key structural feature with GJ072 and its analogs

A new series of small molecular weight compounds induce read through of all three types of nonsense mutations in the ATM gene.

Chemical-induced read through of premature stop codons might be exploited as a potential treatment strategy for genetic disorders caused by nonsense mutations. Despite the promise of this approach, only a few read-through compounds (RTCs) have been discovered to date. These include aminoglycosides (e.g., gentamicin and G418) and nonaminoglycosides (e.g., PTC124 and RTC13). The therapeutic benefits of these RTCs remain to be determined. In an effort to find new RTCs, we screened an additional ~36,000 small molecular weight compounds using a high-throughput screening (HTS) assay that we had previously developed and identified two novel RTCs, GJ071, and GJ072. The activity of these two compounds was confirmed in cells derived from ataxia telangiectasia (A-T) patients with three different types of nonsense mutation in the ATM gene. Both compounds showed activity comparable to stop codons (TGA, TAG, and TAA) PTC124 and RTC13. Early structure-activity relationship studies generated eight active analogs of GJ072. Most of those analogs were effective on all three stop codons. GJ071 and GJ072, and some of the GJ072 analogs, appeared to be well tolerated by A-T cells. We also identified another two active RTCs in the primary screen, RTC204 and RTC219, which share a key structural feature with GJ072 and its analogs

Rapid whole-genome sequencing identifies a novel AIRE variant associated with autoimmune polyendocrine syndrome type 1

Autoimmune polyendocrine syndrome type 1 (APS-1; OMIM #240300), also referred to as autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), is a rare monogenic autoimmune disorder caused by mutations in the autoimmune regulator (AIRE) gene. APS-1 is classically characterized by a triad of chronic mucocutaneous candidiasis, autoimmune hypoparathyroidism, and autoimmune adrenocortical insufficiency. We report a 5-yr-old female who presented with symptoms of tetany due to hypocalcemia and was subsequently found to be secondary to hypoparathyroidism. Rapid trio whole-genome sequencing revealed compound heterozygous variants in AIRE in the proband, with a paternally inherited, pathogenic, frameshift variant (c.1265delC; p.Pro422LeufsTer58) and a novel, likely pathogenic, maternally inherited missense variant (c.268T>C; p.Tyr90His)