Anne Marie Berry

Base editors can correct disease-causing genetic variants. After a neonate had received a diagnosis of severe carbamoyl-phosphate synthetase 1 deficiency, a disease with an estimated 50% mortality in early infancy, we immediately began to develop a customized lipid nanoparticle-delivered base-editing therapy. After regulatory approval had been obtained for the therapy, the patient received two infusions at approximately 7 and 8 months of age. In the 7 weeks after the initial infusion, the patient was able to receive an increased amount of dietary protein and a reduced dose of a nitrogen-scavenger medication to half the starting dose, without unacceptable adverse events and despite viral illnesses. No serious adverse events occurred. Longer follow-up is warranted to assess safety and efficacy. (Funded by the National Institutes of Health and others.).

Whole genome sequencing has been an effective tool in the discovery of variants that cause rare diseases. In this study, we determined the suitability of a novel avidity sequencing approach for rare disease applications. We built a sample to results workflow, combining this sequencing technology with standard library preparation kits, analysis workflows, and interpretation tools. We applied the workflow to ten pedigrees with inherited retinal degeneration (IRD) phenotype. Candidate variants of interest identified through whole genome sequencing were further evaluated using segregation analysis in the additional family members. Potentially causal variants in known IRD genes were detected in five of the ten cases. These high confidence variants were found in ABCA4, CERKL, MAK, PEX6 and RDH12 genes associated with retinal degeneration, that could be sufficient to cause pathology. Pending confirmatory clinical evaluation, we observed a 50% diagnostic yield, consistent with previously reported outcomes of IRD patient analysis. The study confirms that avidity sequencing is effective in detection of causal variants when used for whole genome sequencing in rare disease applications.

Abstract Whole genome sequencing has been an effective tool in the discovery of variants that cause rare disease. In this study, we determined the suitability of a novel avidity sequencing approach for rare disease applications. We built a sample to results workflow, combining the novel sequencing technology with standard library preparation kits, analysis workflows, and interpretation tools. We applied the workflow to ten pedigrees with inherited retinal degeneration (IRD) phenotype. Candidate variants of interest identified through whole genome sequencing were further evaluated using segregation analysis. Mutations in known IRD genes were detected in five of the ten cases. Genes with identified high confidence variants associated with retinal degeneration included PEX6, ABCA4, CERKL, MAK , and RDH12 . Pending confirmatory clinical sequencing, we observed a 50% diagnostic yield, consistent with previously reported outcomes of IRD patient analysis. The study confirms that avidity sequencing is effective in detection of causal mutations when used for whole genome sequencing in rare disease applications.

The high-temperature requirement A1 (HTRA1), a serine protease, has been demonstrated to play a pivotal role in the extracellular matrix (ECM) and has been reported to be associated with the pathogenesis of age-related macular degeneration (AMD). To delineate its role in the retina, the phenotype of homozygous Htra1-KO (Htra1-/-) mice was characterized to examine the effect of Htra1 loss on the retina and retinal pigment epithelium (RPE) with age. The ablation of Htra1 led to a significant reduction in rod and cone photoreceptor function, primary cone abnormalities followed by rods, and atrophy in the RPE compared with WT mice. Ultrastructural analysis of Htra1-/- mice revealed RPE and Bruch's membrane (BM) abnormalities, including the presence of sub-RPE deposits at 5 months (m) that progressed with age accompanied by increased severity of pathology. Htra1-/- mice also displayed alterations in key markers for inflammation, autophagy, and lipid metabolism in the retina. These results highlight the crucial role of HTRA1 in the retina and RPE. Furthermore, this study allows for the Htra1-/- mouse model to be utilized for deciphering mechanisms that lead to sub-RPE deposit phenotypes including AMD.

We previously identified a homozygous G178R mutation in human ASRGL1 (hASRGL1) through whole-exome analysis responsible for early onset retinal degeneration (RD) in patients with cone–rod dystrophy. The mutant G178R ASRGL1 expressed in Cos-7 cells showed altered localization, while the mutant ASRGL1 in E. coli lacked the autocatalytic activity needed to generate the active protein. To evaluate the effect of impaired ASRGL1 function on the retina in vivo, we generated a mouse model with c.578_579insAGAAA (NM_001083926.2) mutation (Asrgl1mut/mut) through the CRISPR/Cas9 methodology. The expression of ASGRL1 and its asparaginase activity were undetectable in the retina of Asrgl1mut/mut mice. The ophthalmic evaluation of Asrgl1mut/mut mice showed a significant and progressive decrease in scotopic electroretinographic (ERG) response observed at an early age of 3 months followed by a decrease in photopic response around 5 months compared with age-matched wildtype mice. Immunostaining and RT-PCR analyses with rod and cone cell markers revealed a loss of cone outer segments and a significant decrease in the expression of Rhodopsin, Opn1sw, and Opn1mw at 3 months in Asrgl1mut/mut mice compared with age-matched wildtype mice. Importantly, the retinal phenotype of Asrgl1mut/mut mice is consistent with the phenotype observed in patients harboring the G178R mutation in ASRGL1 confirming a critical role of ASRGL1 in the retina and the contribution of ASRGL1 mutations in retinal degeneration.