Clinical, pathological and molecular spectrum of patients with glycogen storage diseases in Pakistan

Objectives Evaluation of clinical, biochemical and molecular analysis of Pakistani patients with hepatic GSDs. Methods Medical charts, biochemical, histopathological and molecular results of patients with hepatic GSD were reviewed. Results Out of 55 GSD patients, 41 (74.5%) were males and 14 (25.5%) were females with consanguinity in 50 (91%) patients. The median age of initial symptoms, clinic diagnosis and molecular diagnosis were 450 (IQR: 270–960), 1,095 (IQR: 510–1,825) and 1717 (IQR: 796–3,011) days, respectively. Molecular analysis and enzyme activity was available for 33 (60%) and two patients, respectively. GSD III (n=9) was most prevalent followed by GSD Ib (n=7), GSD IXc (n=6), GSD VI (n=4), GSD Ia (n=3), GSD XI (n=3), GSD IXb (n=2) and GSD IXa (n=1). In patients (n=33) who underwent molecular analysis; 19 different variants in eight genes associated with GSD were identified. We also report five novel variants, two in SLC37A4, one in AGL and two in PYGL contributing to the diagnosis of GSD Ib, GSD III and GSD VI, respectively. Conclusions Fifty-five patients of GSDs in 26 families from a single care provider indicate a relatively high frequency of GSD in Pakistan, with multiple unrelated families harboring identical disease-causing variants, on molecular analysis, including two known pathogenic variants in SLC37A4 and PHKG2, and a novel variant in AGL.

Evaluation of Glycogen Storage Patients: Report of Twelve Novel Variants and New Clinical Findings in a Turkish Population

Glycogen storage diseases (GSDs) are clinically and genetically heterogeneous disorders that disturb glycogen synthesis or utilization. Although it is one of the oldest inherited metabolic disorders, new genetic methods and long-time patient follow-ups provide us with unique insight into the genotype–phenotype correlations. The aim of this study was to share the phenotypic features and molecular diagnostic results that include new pathogenic variants in our GSD cases. Twenty-six GSD patients were evaluated retrospectively. Demographic data, initial laboratory and imaging features, and current findings of the patients were recorded. Molecular analysis results were classified as novel or previously defined variants. Novel variants were analyzed with pathogenicity prediction tools according to American College of Medical Genetics and Genomics (ACGM) criteria. Twelve novel and rare variants in six different genes were associated with the disease. Hearing impairment in two patients with GSD I, early peripheral neuropathy after liver transplantation in one patient with GSD IV, epilepsy and neuromotor retardation in three patients with GSD IXA were determined. We characterized a heterogeneous group of all diagnosed GSDs over a 5-year period in our institution, and identified novel variants and new clinical findings. It is still difficult to establish a genotype–phenotype correlation in GSDs.

Mechanism of glycogen synthase inactivation and interaction with glycogenin

The macromolecule glycogen is the major glucose reserve in eukaryotes and defects of glycogen metabolism and structure lead to glycogen storage diseases and neurodegeneration. Glycogenesis begins with self-glucosylation of glycogenin (GN), which recruits glycogen synthase (GS). GS is activated by glucose-6-phosphate (G6P) and inactivated by phosphorylation, but how these opposing processes are coupled is unclear. We provide the first structure of phosphorylated human GS-GN complex revealing an autoinhibited GS tetramer flanked by two GN dimers. Phosphorylated N- and C-terminal tails from two GS protomers converge to form dynamic "spike" regions, which are buttressed against GS regulatory helices. This keeps GS in a constrained "tense" conformation that is inactive and more resistant to G6P activation. Mutagenesis that weaken the interaction between the regulatory helix and phosphorylated tails leads to a moderate increase in basal/unstimulated GS activity, supporting the idea that phosphorylation contributes to GS inactivation by constraining GS inter-subunit movement. We propose that multivalent phosphorylation supports GS autoinhibition through interactions from a dynamic "spike" region, thus allowing a "tuneable rheostat" for regulating GS activity. Our structures of human GS-GN provide new insights into the regulation of glycogen synthesis, facilitating future studies of glycogen storage diseases.

Molecular basis for the regulation of human glycogen synthase by phosphorylation and glucose-6-phosphate

Glycogen synthase (GYS1), in complex with glycogenin (GYG1), is the central enzyme of muscle glycogen biosynthesis, and its inhibition has been proposed as a therapeutic avenue for various glycogen storage diseases (GSDs). GYS1 activity is inhibited by phosphorylation of its N- and C- termini, which can be relieved by allosteric activation of glucose-6-phosphate. However, the structural basis of GYS1 regulation is unclear. Here, we present the first cryo-EM structures of phosphorylated human GYS1 complexed with a minimal interacting region of GYG1 in the inhibited, activated, and catalytically competent states at resolutions of 3.0-4.0 Å. These structures reveal how phosphorylations of specific N- and C- terminal residues are sensed by different arginine clusters that lock the GYS1 tetramer complex in an inhibited state via inter-subunit interactions. The allosteric activator, glucose-6-phopshate, promotes a conformational change by disrupting these interactions and increases flexibility of GYS1 allowing for a catalytically competent state to occur when bound to the sugar donor UDP-glucose. We also identify an inhibited-like conformation that has not transitioned into the activated state, whereby the locking interaction of phosphorylation with the arginine cluster impedes the subsequent conformational changes due to glucose-6-phosphate binding. Finally, we show that the PP1 phosphatase regulatory subunit PPP1R3C (PTG) is recruited to the GYS1:GYG1 complex through direct interaction with glycogen. Our data provide the first mechanistic insights into human glycogen synthase regulation.

Identification of a novel mutation in the PHKA2 gene in a child with liver cirrhosis

Objectives Glycogen storage diseases (GSDs) are heterogeneous disorders caused by various enzyme deficiencies. GSD type IX α2, the most common subtype of GSD IX, is due to a deficiency of hepatic phosphorylase kinase. Herein we will report a novel mutation in this disease with an unusual presentation. Case presentation we describe a 3-year-old boy who suffered from hepatomegaly, fatty liver disease, and liver cirrhosis. The cause of cirrhosis at a young age was unknown based on the laboratory data and liver biopsy, so we performed a targeted-gene sequencing (TGS) covering 450 genes involved in inborn metabolic diseases consisting of glycogen storage disorders genes with hepatic involvement. He was found out to have a rare novel pathogenic variant in the PHKA2 gene. Conclusions This novel variant c.2226+2T > C expands the mutational spectrum of the PHKA2 gene. Also, severe liver damage (cirrhosis) in patients with GSD- IX α2 has rarely been reported, which needs further discussion. We hypothesize that unidentified PHKA2 variants may be a rare cause of childhood liver cirrhosis.

A Patient with Recurrent Severe Hypoglycemic Attacks and Mitochondrial Complex III Deficiency, Nuclear Type 3: a Novel UQCRB Variant

Common causes of hypoglycemia include hyperinsulinism, hormonal deficiencies, fatty acid oxidation disorders, and glycogen storage diseases; however, rare causes should also be considered for the condition. Mitochondrial complex III deficiency shows an autosomal recessive or a mitochondrial inheritance pattern. To date, mitochondrial complex III deficiency, nuclear type 3 attributable to a pathogenic variant of the <i>UQCRB</i> gene (MIM 615158) has been identified in only 2 pediatric patients; both presented with hypoglycemia and lactic acidosis. In this paper, we present a patient with mitochondrial complex III deficiency, nuclear type 3, <i>UQCRB</i> variant associated with acute hypoglycemia and lactic acidosis episodes. The male patient was admitted on the first day of life with tachypnea, metabolic acidosis, and hypoglycemia. Up to 10 years of age, he was admitted 7 times with abdominal pain, vomiting, and fever. His blood tests revealed hypoglycemia, metabolic acidosis, and hyperlactatemia. At 10 years of age, a whole-exome sequencing (WES) analysis was performed identifying a homozygous c.309_313delAGAAA (p.Glu104ArgfsTer10) pathogenic variant of the <i>UQCRB</i> gene. Once the common causes of hypoglycemia are excluded, it is essential to perform a WES analysis for other rare causes. Thus, rare disorders such as mitochondrial complex III deficiency can be diagnosed.