Induced pluripotent stem cell (iPSC) modeling validates reduced GBE1 enzyme activity due to a novel variant, p.Ile694Asn, found in a patient with suspected glycogen storage disease IV

Chie Naito; Karis Kosar; Eriko Kishimoto; Loren Pena; Yilun Huang; Kaili Hao; Anas Bernieh; Jennifer Kasten; Chet Villa; Priya Kishnani; Bali Deeksha; Mingxia Gu; Akihiro Asai

doi:10.1016/j.ymgmr.2024.101069

Induced pluripotent stem cell (iPSC) modeling validates reduced GBE1 enzyme activity due to a novel variant, p.Ile694Asn, found in a patient with suspected glycogen storage disease IV

Mol Genet Metab Rep. 2024 Mar 14:39:101069. doi: 10.1016/j.ymgmr.2024.101069. eCollection 2024 Jun.

Authors

Chie Naito¹, Karis Kosar¹, Eriko Kishimoto¹, Loren Pena^{1

2}, Yilun Huang¹, Kaili Hao¹, Anas Bernieh^{1

2}, Jennifer Kasten^{1

2}, Chet Villa^{1

2}, Priya Kishnani³, Bali Deeksha³, Mingxia Gu^{1

2}, Akihiro Asai^{1

2}

Affiliations

¹ Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.
² Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
³ Department of Pediatrics, Division of Medical Genetics, Duke Health, Durham, NC, USA.

Abstract

Background: Glycogen Storage disease type 4 (GSD4), a rare disease caused by glycogen branching enzyme 1 (GBE1) deficiency, affects multiple organ systems including the muscles, liver, heart, and central nervous system. Here we report a GSD4 patient, who presented with severe hepatosplenomegaly and cardiac ventricular hypertrophy. GBE1 sequencing identified two variants: a known pathogenic missense variant, c.1544G>A (p.Arg515His), and a missense variant of unknown significance (VUS), c.2081T>A (p. Ile694Asn). As a liver transplant alone can exacerbate heart dysfunction in GSD4 patients, a precise diagnosis is essential for liver transplant indication. To characterize the disease-causing variant, we modeled patient-specific GBE1 deficiency using CRISPR/Cas9 genome-edited induced pluripotent stem cells (iPSCs).

Methods: iPSCs from a healthy donor (iPSC-WT) were genome-edited by CRISPR/Cas9 to induce homozygous p.Ile694Asn in GBE1 (iPSC-GBE1-I694N) and differentiated into hepatocytes (iHep) or cardiomyocytes (iCM). GBE1 enzyme activity was measured, and PAS-D staining was performed to analyze polyglucosan deposition in these cells.

Results: iPSC^GBE1-I694N differentiated into iHep and iCM exhibited reduced GBE1 protein level and enzyme activity in both cell types compared to iPSC^wt. Both iHep^GBE1-I694N and iCM^GBE1-I694N showed polyglucosan deposits correlating to the histologic patterns of the patient's biopsies.

Conclusions: iPSC-based disease modeling supported a loss of function effect of p.Ile694Asn in GBE1. The modeling of GBE1 enzyme deficiency in iHep and iCM cell lines had multi-organ findings, demonstrating iPSC-based modeling usefulness in elucidating the effects of novel VUS in ultra-rare diseases.

Keywords: Cardiomyocyte differentiation; GBE1 enzyme activity; Glycogen branching enzyme 1 (GBE1); Glycogen storage disease IV (GSD4); Hepatocyte differentiation; iPSCs (induced pluripotent stem cells).