Jianjun Shen

Mutations in exon 3 of the glycogen debranching enzyme gene are associated with glycogen storage disease type III that is differentially expressed in liver and muscle.

Glycogen storage disease type HI (GSD-III), an autosomal recessive disease, is caused by deficient glycogen debranching enzyme (GDE) activity. Most GSD-III patients are GDE deficient in both liver and muscle (type IIIa), and some GSD-III patients have GDE absent in liver but retained in muscle (type IIIb). The molecular basis for this enzymatic variability is largely unknown. In the present study, the analysis of the GDE gene in three GSD-IIIb patients by single-strand conformation polymorphism (SSCP), DNA sequencing, restriction analysis, and family studies, revealed each of them as being a compound heterozygote for two different mutations. The first mutant alleles in all three patients involved mutations in exon 3 at amino acid codon 6 of the GDE protein. Two had an AG deletion at nucleotides 17 and 18 of the GDE cDNA (17delAG) which resulted in change of subsequent amino acid sequence and a truncated protein (25X); the other had a C to T transition at nucleotide 16 of the cDNA which changed a Glutamine codon to a stop codon (Q6X). The 17delAG mutation was also found in 8 of the 10 additional GSD-IIIb patients. The Q6X mutation was found in one of the remaining two GSD-IIIb patients. These two mutations were not found in any of the 31 GSD-IIIa patients, 2 GSD-IIId patients, nor 28 unrelated normal controls. The second mutant alleles in each of the three GSD-IIIb patients were R864X, R1228X, and W68OX. The R864X and R1228X were not unique for GSD-IIIb as they were also found in GSD-IIIa patients (frequency of 10.3% and 5.2% in Caucasian patients, respectively). Our data demonstrated that both IIIa and IIIb had mutations in the same GDE gene and established for the first time the molecular basis of GSD-III that differentially expressed in liver and muscle. The striking and specific association of exon 3 mutations with GSD-IIIb may provide insight into mechanisms controlling tissue-specific expression of the GDE gene. The identification of exon 3 mutations has clinical significance as well because it distinguished GSD-IIIb from IIIa hence permitting diagnosis from a blood sample rather than a more invasive muscle biopsy.

Molecular Characterization of Glycogen Storage Disease Type III

Deficiency of the glycogen debranching enzyme (gene, AGL) causes glycogen storage disease type III (GSD-III), an autosomal recessive disease affecting glycogen metabolism. Most GSD-III patients have AGL deficiency in both the liver and muscle (type IIIa), but some have it in the liver but not muscle (type IIIb). Cloning of human AGL cDNAs and determination of the genomic structure and mRNA isoforms of AGL have allowed for the study of GSD-III at the molecular level. In turn, the resulting information has greatly facilitated our understanding of the molecular basis of this storage disease with remarkable clinical and enzymatic variability. In this review, we summarize all 31 GSD-III mutations in the literature and discuss their clinical and laboratory implications. Most of the mutations are nonsense mutations caused by a nucleotide substitution or small insertion or deletion; only one is caused by a missense amino acid change. Some important genotype-phenotype correlation have emerged, in particular, that exon 3 mutations (17delAG and Q6X) are specifically associated with GSD-IIIb. Three other mutations have appeared to have some phenotype correlation. Specifically, the splice mutation IVS32-12A>G was found in GSD-III patients having mild clinical symptoms, while the mutations 3965delT and 4529insA are associated with a severe phenotype and early onset of clinical manifestations. A molecular diagnostic scheme has been proposed to diagnose GSD-III noninvasively. The characterization of AGL mutations in GSD-III patients has also helped the structure-function analysis of this bifunctional enzyme important for glycogen metabolism.

Acylcarnitines in fibroblasts of patients with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency and other fatty acid oxidation disorders

Mitochondrial fatty acid oxidation disorders cause hypoglycaemia, hepatic dysfunction, myopathy, cardiomyopathy and encephalopathy. Despite their recognition for more than 15 years, diagnosis and treatment remain difficult. To help design rational diagnostic and therapeutic strategies, we studied the pathophysiology of accumulating metabolites in a whole-cell system. Acylcarnitines were quantified in cells and media of cultured fibroblasts after incubation with L-carnitine and fatty acids. Following incubation with palmitate, long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD)-deficient fibroblasts compared with controls showed elevation of hydroxypalmitoyl- and palmitoylcarnitine and reduction of C10- and shorter acylcarnitines, and following incubation with linoleate an increase in C14:2-, C18:2- and hydroxy-C18:2-acylcarnitines and reduction in C10:1-acylcarnitines. Hydroxyacylcarnitines remained more intracellular compared to corresponding saturated acylcarnitines. Incubation with decanoate and octanoate showed absence of hydroxylated acylcarnitines and correction of secondary metabolic disturbances, suggesting that optimal treatment should include medium-chain triglycerides of these chain lengths. Fibroblasts of patients with other fatty acid oxidation disorders showed distinct elevations of disease-specific acylcarnitines. This acylcarnitine analysis allows the diagnosis of LCHAD deficiency and its differentiation from other fatty acid oxidation disorders, which can pose difficulties in vivo. The strategy has allowed in-depth analysis with different substrates, providing suggestions for the rational design of treatment trials.

Clinical and laboratory findings in four patients with the non-progressive hepatic form of type IV glycogen storage disease

SummaryThe classic clinical presentation for type IV glycogen storage disease (branching enzyme deficiency, GSD IV) is hepatosplenomegaly with failure to thrive occurring in the first 18 months of life, followed by progressive liver failure and death by age 5 years. Although there have been two patients without apparent liver progression previously reported, no long-term follow-up clinical data have been available. We present here the clinical spectrum of the non-progressive liver form of GSD IV in four patients, and long-term follow-up of the oldest identified patients (ages 13 and 20 years). None has developed progressive liver cirrhosis, skeletal muscle, cardiac or neurological involvement, and none has been transplanted. Branching enzyme activity was also measured in cultured skin fibroblasts from patients with the classic liver progressive, the early neonatal fatal, and the non-progressive hepatic presentations of GSD IV. The residual branching enzyme activity in the patients without progression was not distinguishable from the other forms and could not be used to predict the clinical course. Our data indicate that GSD IV does not always necessitate hepatic transplantation and that caution should be used when counselling patients regarding the prognosis of GSD IV. Patients should be carefully monitored for evidence of progression before recommending liver transplantation.

Acylcarnitines in plasma and blood spots of patients with long-chain 3-hydroxyacyl-coenzyme A dehydrogenase defiency

The acylcarnitines in plasma and blood spots of 23 patients with proven deficiency of long-chain 3-hydroxyacylcoenzyme A dehydrogenase were reviewed. Long-chain 3-hydroxyacylcarnitines of C14:1, C14, C16 and C18:1 chain length, and long-chain acylcarnitines of C12, C14:1, C14, C16, C18:2 and C18:1 chain length were elevated. Acetylcarnitine was decreased. In plasma, elevation of hydroxy-C18:1 acylcarnitine over the 95th centile of controls, in combination with an elevation of two of the three acylcarnitines C14, C14:1 and hydroxy-C16, identified over 85% of patients with high specificity (less than 0.1% false positive rate). High endogenous levels of long-chain acylcarnitines in normal erythrocytes reduced the diagnostic specificity in blood spots compared with plasma samples. The results were also diagnostic in asymptomatic patients, and were not influenced by genotype. Treatment with diet low in fat and high in medium-chain triglyceride decreased all disease-specific acylcarnitines, often to normal, suggesting that this assay is useful in treatment monitoring.

A nonsense mutation due to a single base insertion in the 3'-coding region of glycogen debranching enzyme gene associated with a severe phenotype in a patient with glycogen storage disease type IIIa

Glycogen storage disease type III (GSD‐III) is an autosomal recessive disease resulting from deficient glycogen debranching enzyme (GDE) activity. A child with GDE deficient in both liver and muscle (GSD‐IIIa) had recurrent hypoglycemia, seizures, severe cardiomegaly, and hepatomegaly and died at 4 years of age. Analysis of the GDE gene in this child by single‐strand conformation polymorphism, followed by direct DNA sequencing and restriction analysis, revealed an insertion of a nucleotide A into position 4529 of the GDE cDNA (4529insA). This insertion resulted in substitution of a tyrosine to a stop codon at amino acid 1510 (Y1510X). The 4529insA mutation appeared to be homozygous in this patient and was not found in 20 unrelated controls or 18 other GSD‐III patients (14 GSD‐IIIa and 4 GSD‐IIIb). This is the first identification of a disease mutation in this gene, and the data suggest that homozygous 4529insA may be associated with a severe phenotype in GSD‐IIIa.

Novel donor splice site mutations of AGL gene in glycogen storage disease type IIIa.

Novel donor splice site mutations of AGL gene in glycogen storage disease type IIIa G. M. Hadjigeorgiou1,2, G. P. Comi1*, A. Bordoni1, J. Shen3, Y .-T . Chen3, S. Salani1, A. T oscano4, F. Fortunato1, S. L ucchiari1, N. Bresolin1, C. Rodolico4, M. G. Piscaglia5, L . Franceschina1, A. Papadimitriou2 and G. Scarlato1 1Centro Dino Ferrari, Istituto di Clinica Neurologica, Università degli Studi di Milano, IRCCS Ospedale Maggiore Policlinico, Milan, Italy ; 2Division of Neurology, Red Cross Hospital, Athens, Greece ; 3Division of Medical Genetics, Department of Pediatrics, Duke University Medical Center, Durham, North Carolina, USA; 4 Intituto di Clinica Neurologica II, Università di Messina, Messina ; 5 Servizio di Neurologia, Ospedale delgi Infermi, Rimini, Italy * Correspondence Instituto di Clinica Neurologica, Ospedale Maggiore Policlinico, Via Sforza 35, 20122 Milano, Italy. E-mail : gpcomi=imiucca.csi.unimi.it

Prenatal diagnosis of glycogen storage disease type IV using PCR-based DNA mutation analysis

Deficiency of glycogen branching enzyme activity causes glycogen storage disease type IV (GSD‐IV). Clinically, GSD‐IV has variable clinical presentations ranging from a fatal neonatal neuromuscular disease, to a progressive liver cirrhosis form, and to a milder liver disease without progression. Current methods for prenatal and postnatal diagnosis are based on an indirect method of measuring the enzyme activity, which has a limited sensitivity and cannot be used to distinguish patients with these variable clinical phenotypes. In this study, a GSD‐IV family with a non‐progressive hepatic form of the disease requested prenatal diagnosis. Determination of the branching enzyme activity in cultivated amniocytes showed 20 per cent residual activity overlapping with the level detected in the heterozygotes. Mutation analysis revealed that the fetus carried two mutant alleles, L224P and Y329S, the same as the proband of this family. The fetus was predicted to be affected and postnatally his clinical presentation is consistent with the diagnosis. We conclude that DNA mutation analysis should be used in the prenatal diagnosis of GSD‐IV, especially in the situation of high residual enzyme activity. Copyright

Prenatal diagnosis and carrier detection for glycogen storage disease type III using polymorphic DNA markers

Deficiency of glycogen debranching enzyme gene (AGL) causes glycogen storage disease type III (GSD‐III), an autosomal recessive disease. Prenatal diagnosis and carrier detection using enzymatic methods are technically difficult and have limited ability to distinguish a carrier from an affected patient. Mutations in the AGL gene can be used for these purposes. However, the mutations identified thus far account for less than half of the total mutant alleles, and no common mutations have been detected except in North African Jews and in a rare subtype of the disease (GSD‐IIIb). Our recent identification of three highly informative DNA polymorphic markers in the AGL gene allowed us to perform prenatal diagnosis and carrier detection in two GSD‐III families with unknown mutations, using the polymerase chain reaction (PCR) and restriction analysis. In one family, a fetus was diagnosed to be a GSD‐III carrier and his carrier status was confirmed postnatally. A newborn in the second family was postnatally diagnosed with the disease.

Two new mutations in the 3' coding region of the glycogen debranching enzyme in a glycogen storage disease type IIIa Ashkenazi Jewish patient.

Glycogen storage disease type III (GSD III) is an autosomal recessive disease caused by the deficiency of glycogen debranching enzyme (AGL). We report the finding of two new mutations in a GSD IIIa Ashkenazi Jewish patient. Both mutations are insertion of an adenine into a stretch of 8 adenines towards the 3′ end of the coding region: one at position 3904 (3904insA) in exon 30, the second at position 4214 (4214insA) in exon 32. The mutations cause frameshifts and premature terminations of the glycogen debranching enzyme, the first causing a frameshift at amino acid 1304, the second causing a frameshift at amino acid 1408 of the total of 1532. These mutations demonstrate the importance of the 125 amino acids at the carboxy-terminus of the debrancher enzyme for its activity and support the suggestion that the putative glycogen binding domain is located in the carboxy-terminus of the AGL. The mutations cause distinctive single-strand conformation polymorphism (SSCP) patterns enabling easy detection.