Insulin secretion deficits in a Prader-Willi syndrome ß-cell model are associated with a concerted downregulation of multiple endoplasmic reticulum chaperones.

Prader-Willi syndrome (PWS) is a multisystem disorder with neurobehavioral, metabolic, and hormonal phenotypes, caused by loss of expression of a paternally-expressed imprinted gene cluster. Prior evidence from a PWS mouse model identified abnormal pancreatic islet development with retention of aged insulin and deficient insulin secretion. To determine the collective roles of PWS genes in ß-cell biology, we used genome-editing to generate isogenic, clonal INS-1 insulinoma lines having 3.16 Mb deletions of the silent, maternal- (control) and active, paternal-allele (PWS). PWS ß-cells demonstrated a significant cell autonomous reduction in basal and glucose-stimulated insulin secretion. Further, proteomic analyses revealed reduced levels of cellular and secreted hormones, including all insulin peptides and amylin, concomitant with reduction of at least ten endoplasmic reticulum (ER) chaperones, including GRP78 and GRP94. Critically, differentially expressed genes identified by whole transcriptome studies included reductions in levels of mRNAs encoding these secreted peptides and the group of ER chaperones. In contrast to the dosage compensation previously seen for ER chaperones in Grp78 or Grp94 gene knockouts or knockdown, compensation is precluded by the stress-independent deficiency of ER chaperones in PWS ß-cells. Consistent with reduced ER chaperones levels, PWS INS-1 ß-cells are more sensitive to ER stress, leading to earlier activation of all three arms of the unfolded protein response. Combined, the findings suggest that a chronic shortage of ER chaperones in PWS ß-cells leads to a deficiency of protein folding and/or delay in ER transit of insulin and other cargo. In summary, our results illuminate the pathophysiological basis of pancreatic ß-cell hormone deficits in PWS, with evolutionary implications for the multigenic PWS-domain, and indicate that PWS-imprinted genes coordinate concerted regulation of ER chaperone biosynthesis and ß-cell secretory pathway function.

Characterization of exonic variants of uncertain significance in very long-chain acyl-CoA dehydrogenase identified through newborn screening.

Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is an autosomal recessive disease resulting from mutations in the ACADVL gene and is among the disorders tested for in newborn screening (NBS). Confirmatory sequencing following suspected VLCADD NBS results often identifies variants of uncertain significance (VUS) in the ACADVL gene, leading to uncertainty of diagnosis and providing effective treatment regimen. Currently, ACADVL has >300 VUSs in the ClinVar database that requiring characterization to determine potential pathogenicity. In this study, CRISPR/Cas9 genome editing was used to knock out ACADVL in HEK293T cells, and targeted deletion was confirmed by droplet digital polymerase chain reaction (PCR). No VLCAD protein was detected and an 84% decrease in enzyme activity using the electron transfer flavoprotein fluorescence reduction assay and C21-CoA as substrate was observed compared to control. Plasmids containing control or variant ACADVL coding sequence were transfected into the ACADVL null HEK293T. While transfection of control ACADVL restored VLCAD protein and enzyme activity, cells expressing the VLCAD Val283Ala mutant had 18% VLCAD enzyme activity and reduced protein compared to control. VLCAD Ile420Leu, Gly179Arg, and Gln406Pro produced protein comparable to control but 25%, 4%, and 5% VLCAD enzyme activity, respectively. Leu540Pro and Asp570_Ala572dup had reduced VLCAD protein and 10% and 3% VLCAD enzyme activity, respectively. VLCADD fibroblasts containing the same variations had decreased VLCAD protein and activity comparable to the transfection experiments. Generating ACADVL null HEK293T cell line allowed functional studies to determine pathogenicity of ACADVL exonic variants. This approach can be applied to multiple genes for other disorders identified through NBS.

Characterization of variants of uncertain significance in isovaleryl-CoA dehydrogenase identified through newborn screening: An approach for faster analysis.

INTRODUCTION: Clinical standard of care for newborn screening (NBS) is acylcarnitine metabolites quantitation by tandem mass spectrometry (MS/MS) from dried blood spots. Follow up sequencing often results in identification of one or more variants of uncertain significance (VUS). Isovaleric acidemia (IVA) is an autosomal recessive inborn error of metabolism caused by deficiency of isovaleryl-CoA dehydrogenase (IVDH) in the Leu catabolism pathway. Many IVD mutations are characterized as VUS complicating IVA clinical diagnoses and treatment. We present a testing platform approach to confirm the functional implication of VUS identified in newborns with IVA applicable to multiple inborn errors of metabolism identified by NBS. METHODS: An IVD null HEK293T cell culture model was generated by using a dual sgRNA CRISPR/Cas9 genome-editing strategy targeting IVD exons 2-3. Clonal cell lines were confirmed by a combination of genomic breakpoint sequencing and droplet digital PCR. The IVD null model had no IVDH antigen signal and 96% reduction in IVDH enzyme activity. The IVD null model was transfected with vectors containing control or variant IVD and functional assays were performed to determine variant pathogenicity. RESULTS: c.149G > C (p.Arg50Pro; precursor numbering), c.986T > C (p.Met329Thr), and c.1010G > A (p.Arg337Gln), c.1179del394 f. mutant proteins had reduced IVDH protein and activity. c.932C > T (p.Ala311Val), c.707C > T (p.Thr236Ile), and c.1232G > A (p.Arg411Gln) had stable IVDH protein, but no enzyme activity. c.521T > G (p.Val174Gly) had normal IVDH protein and activity. IVD variant transfection results confirmed results from IVA fibroblasts containing the same variants. CONCLUSIONS: We have developed an IVD null HEK293T cell line to rapidly allow determination of VUS pathogenicity following identification of novel alleles by clinical sequencing following positive NBS results for suspected IVA. We suggest similar models can be generated via genome-editing for high throughput assessment of VUS function for a multitude of inborn errors of metabolism and can ideally supplement NBS programs.

A porcine model of phenylketonuria generated by CRISPR/Cas9 genome editing.

Phenylalanine hydroxylase-deficient (PAH-deficient) phenylketonuria (PKU) results in systemic hyperphenylalaninemia, leading to neurotoxicity with severe developmental disabilities. Dietary phenylalanine (Phe) restriction prevents the most deleterious effects of hyperphenylalaninemia, but adherence to diet is poor in adult and adolescent patients, resulting in characteristic neurobehavioral phenotypes. Thus, an urgent need exists for new treatments. Additionally, rodent models of PKU do not adequately reflect neurocognitive phenotypes, and thus there is a need for improved animal models. To this end, we have developed PAH-null pigs. After selection of optimal CRISPR/Cas9 genome-editing reagents by using an in vitro cell model, zygote injection of 2 sgRNAs and Cas9 mRNA demonstrated deletions in preimplantation embryos, with embryo transfer to a surrogate leading to 2 founder animals. One pig was heterozygous for a PAH exon 6 deletion allele, while the other was compound heterozygous for deletions of exon 6 and of exons 6-7. The affected pig exhibited hyperphenylalaninemia (2000-5000 µM) that was treatable by dietary Phe restriction, consistent with classical PKU, along with juvenile growth retardation, hypopigmentation, ventriculomegaly, and decreased brain gray matter volume. In conclusion, we have established a large-animal preclinical model of PKU to investigate pathophysiology and to assess new therapeutic interventions.