Molecular features of 23 patients with glycogen storage disease type III in Turkey: a novel mutation p.R1147G associated with isolated glucosidase deficiency, along with 9 AGL mutations.

Glycogen storage disease type III (GSD III) is an autosomal recessive disorder caused by deficiency in the glycogen debranching enzyme (gene symbol: AGL) with two enzyme activities: transferase and glucosidase. A missense mutation causing isolated glucosidase deficiency has never been reported. In this study, we examined 23 patients of Turkish ancestry and identified a novel missense mutation p.R1147G with isolated glucosidase deficiency, along with nine AGL mutations: six nonsense mutations (p.W373X, p.R595X, p.Q667X, p.Q1205X, p.W1327X and p.Q1376X), one deletion (c.1019delA) and two splicing mutation (c.293+2T>G and c.958+1G>A). As p.R1147G impaired glucosidase activity, but maintained transferase activity in vitro, a 12-year-old girl homozygous for p.R1147G was diagnosed with having isolated glucosidase deficiency. Of nine other mutations, p.W1327X and c.1019delA were recurrent, whereas seven mutations were novel. Six patients with p.W1327X were all from two nearby cities on the East Black Sea and shared the same AGL haplotype, indicating a founder effect in Turkish patients. Patients with the same mutations had identical haplotypes. Our results provide the first comprehensive overview of clinical and molecular features of Turkish GSD III patients and the first description of the missense mutation associated with isolated glucosidase deficiency.

Egyptian glycogen storage disease type III - identification of six novel AGL mutations, including a large 1.5 kb deletion and a missense mutation p.L620P with subtype IIId.

BACKGROUND: Glycogen storage disease type III (GSD III) is caused by mutations in AGL which encodes for a single protein with two enzyme activities: oligo-1, 4-1, 4-glucantransferase (transferase) and amylo-1, 6-glucosidase. Activity of both enzymes is lost in most patients with GSD III, but in the very rare subtype IIId, transferase activity is deficient. Since the spectrum of AGL mutations is dependent on the ethnic group, we investigated the clinical and molecular characteristics in Egyptian patients with GSD III. METHODS: Clinical features were examined in five Egyptian patients. AGL was sequenced and AGL haplotypes were determined. RESULTS: Six novel AGL mutations were identified: a large deletion (c.3481-3588+1417del1525 bp), two insertions (c.1389insG and c.2368insA), two small deletions (c.2223-2224delGT and c.4041delT), and a missense mutation (p.L620P). p.L620P was found in a patient with IIId. Each mutation was located on a different AGL haplotype. CONCLUSIONS: Our results suggest that there is allelic and phenotypic heterogeneity of GSD III in Egypt. This is the second description of a large deletion in AGL. p.L620P is the second mutation found in GSD IIId.

Impact of elevated serum lipoprotein (a) concentrations on the risk of coronary heart disease in patients with type 2 diabetes mellitus.

Type 2 diabetes mellitus is associated with a marked increase of coronary heart disease (CHD). We aimed to assess the impact of elevated serum lipoprotein (a) (Lp[a]) concentrations on the risk of CHD in patients with type 2 diabetes mellitus. A consecutive series of 352 outpatients was investigated. We determined the serum lipid profile and checked the patients for a history of CHD and of its traditional risk factors. Furthermore, the patients were divided into 3 groups according to the degree of elevation of the serum Lp(a) concentration: serum Lp(a) concentrations greater than 50 mg/dL, between 30 and 50 mg/dL, and less than 30 mg/dL, a presumed high normal value; and the prevalence of CHD was compared among the 3 groups. The serum Lp(a) concentrations in the subjects varied widely from 0.4 to 163.6 mg/dL. Patients with CHD had significantly higher serum Lp(a) concentrations than those without CHD (P = .0045). Logistic regression analysis to identify factors associated with the presence of CHD revealed that elevated serum Lp(a) is a significant risk factor (P = .0246). The prevalence of CHD increased with increasing serum Lp(a) concentrations (P = .048). Patients with serum Lp(a) concentrations greater than 50 mg/dL had a significantly higher prevalence of CHD than those with serum Lp(a) concentrations less than 30 mg/dL: the odds ratio of an elevated serum Lp(a) concentration was 3.346 (P = .039). In conclusion, elevated serum Lp(a) is a significant risk factor; and the risk of CHD appears to increase with increasing serum Lp(a) concentrations. Serum Lp(a) concentration of 50 mg/dL might represent a threshold level in relation to the risk of CHD in patients with type 2 diabetes mellitus.

A 60-y-old chylomicronemia patient homozygous for missense mutation (G188E) in the lipoprotein lipase gene showed no accelerated atherosclerosis.

BACKGROUND: Familial lipoprotein lipase (LPL) deficiency is a rare autosomal recessive disorder caused by mutations in the LPL gene. Patients with LPL deficiency have chylomicronemia; however, whether they develop accelerated atherosclerosis remains unclear. METHODS: We investigated clinical and mutational characteristics of a 60-y-old Japanese patient with chylomicronemia. RESULTS: The patient's fasting plasma triglyceride levels were >9.0 mmol/l. In postheparin plasma, one fifth of the normal LPL protein mass was present; however, LPL activity was undetectable. Molecular analysis of the LPL gene showed the patient to be a homozygote of missense mutation replacing glycine with glutamine at codon 188 (G188E), which had been known to produce mutant LPL protein lacking lipolytic activity. Ultrasonographic examination of the patient's carotid and femoral arteries showed no accelerated atherosclerosis. Moreover, 64-slice mechanical multidetector-row computer tomography (MDCT) angiography did not detect any accelerated atherosclerotic lesions in the patient's coronary arteries. The patient had none of the risk factors such as smoking, hypertension, and diabetes. CONCLUSIONS: Our case suggests that accelerated atherosclerosis may not develop in patients with LPL deficiency, when they have no risk factors.

Severe hypertriglyceridemia in Japan: Differences in causes and therapeutic responses.

BACKGROUND: Severe hypertriglyceridemia (>1000 mg/dL) has a variety of causes and frequently leads to life-threating acute pancreatitis. However, the origins of this disorder are unclear for many patients. OBJECTIVE: We aimed to characterize the causes of and responses to therapy in rare cases of severe hypertriglyceridemia in a group of Japanese patients. METHODS: We enrolled 121 patients from a series of case studies that spanned 30 years. Subjects were divided into 3 groups: (1) primary (genetic causes); (2) secondary (acquired); and (3) disorders of uncertain causes. In the last group, we focused on 3 possible risks factors for hypertriglyceridemia: obesity, diabetes mellitus, and heavy alcohol intake. RESULTS: Group A (n = 20) included 13 patients with familial lipoprotein lipase deficiency, 3 patients with apolipoprotein CII deficiency, and other genetic disorders in the rest of the group. Group B patients (n = 15) had various metabolic and endocrine diseases. In Group C (uncertain causes; n = 86), there was conspicuous gender imbalance (79 males, 3 females) and most male subjects were heavy alcohol drinkers. In addition, 18 of 105 adult patients (17%) had histories of acute pancreatitis. CONCLUSION: The cause of severe hypertriglyceridemia is uncertain in many patients. In primary genetic forms of severe hypertriglyceridemia, genetic diversity between populations is unknown. In the acquired forms, we found fewer cases of estrogen-induced hypertriglyceridemia than in Western countries. In our clinical experience, the cause of most hypertriglyceridemia is uncertain. Our work suggests that genetic factors for plasma triglyceride sensitivity to alcohol should be explored.

Apolipoprotein C-II Tuzla: a novel large deletion in APOC2 caused by Alu-Alu homologous recombination in an infant with apolipoprotein C-II deficiency.

BACKGROUNDS: Familial apolipoprotein (apo) C-II deficiency is a very rare inherited disorder characterized by chylomicronemia. Since the discovery in 1978, reports on apo C-II deficient patients have been limited and only 13 different mutations in APOC2, a gene encoding apo C-II protein, were identified. OBJECTIVES: The objective is to investigate the biochemical and genetic features of a 3-month-old Bosniak girl with chylomicronemia whose apo C-II protein was undetectable in her plasma. METHODS: APOC2, LPL, APOA5, and GPIHBP1 were sequenced. Isoelectrofocusing and immunoblotting of chylomicrons and VLDL fraction from the patient were performed. RESULTS: Sequence analysis demonstrated a large deletion of 2978 base pairs in APOC2, which encompassed exons 2, 3, and 4. The patient was homozygous for the deletion. The 5' part of the breakpoint was located in an Alu Sx repetitive element in intron 1 of APOC2, whereas the 3' part of the breakpoint was in another Alu Sx between APOC2 and CLPTM1, a gene flanking APOC2. We speculate that the deletion was caused by a homologous recombination between two Alu Sx elements. No mutations were detected in LPL, APOA5, and GPIHBP1. Isoelectrofocusing and immunoblotting confirmed the absence of apo C-II protein. CONCLUSIONS: We diagnosed the patient as having apo C-II deficiency and designated the novel large deletion as apo C-II Tuzla. This is the first description of apo C-II deficiency caused by Alu-Alu recombination in APOC2.

Lipoprotein lipase activity in heterozygotes for lipoprotein lipase gene mutations reveals a gender bias.

BACKGROUND: Familial lipoprotein lipase (LPL) deficiency is a very rare autosomal recessive disorder characterized by marked elevation of plasma triglyceride concentrations. Since 1989, a variety of mutations have been reported in affected patients. Studies on subjects with heterozygous LPL deficiency, on the other hand, have been limited. METHODS: We examined post-heparin plasma LPL activity in 15 subjects with heterozygous LPL deficiency. RESULTS: The heterozygotes exhibited normal or slightly elevated plasma triglyceride concentrations. The mean LPL activity was reduced by 25% in the heterozygotes relative to controls. Interestingly, LPL activity was reduced specifically in female heterozygotes. CONCLUSION: LPL activity is decreased in female, but not in male, subjects heterozygous for a number of different LPL gene mutations.

Hypertriglyceridemia and pancreatitis in a patient with apolipoprotein E7 (p.[E244K; E245K])/E4.

BACKGROUND: The etiology of hypertriglyceridemia is complex and one of the common variants in affecting plasma lipid levels is apolipoprotein (apo) E isoform. Scores of apo E variants have been reported, including apo E7. However, a clinical lipid phenotype of apo E7 has not been fully elucidated. METHODS: A 48-year-old Japanese male had hypertriglyceridemia and a history of repeated episodes of acute pancreatitis. The measurement of serum apolipoproteins and apo E phenotyping, and the sequencing analyses of several genes regulating triglyceride metabolism were performed in the patient. RESULTS: The apo E phenotype of the patient was E7/E4. Apo E7 had the same point mutations p.[E244K; E245K] in APOE as reported previously. In addition, he had APOA5 haplotypes associated with hypertriglyceridemia. Laboratory examinations excluded deficiency of apolipoproteins, lipoprotein lipase, and GPI-HBP1 in this patient. CONCLUSIONS: This is, to our knowledge, the first report of severe hypertriglyceridemia and acute pancreatitis in a patient with apo E7.

Hepatic lipase activity is decreased in Japanese patients with type III hyperlipoproteinemia.

BACKGROUND: Type III hyperlipoproteinemia (HLP), a disorder associated with a high incidence of premature cardiovascular diseases, is characterized by the accumulation of remnant lipoproteins in the plasma. The primary genetic defect in patients with type III HLP is the presence of apolipoprotein E2 (apoE2), an isoform of apoE, and accumulation of remnant lipoproteins in the plasma has been thought to be attributable to the presence of apoE2, which bind poorly to low density lipoprotein receptors, resulting in defective remnant lipoprotein clearance. On the other hand, the activity of hepatic lipase (HL), the enzyme that plays a pivotal role in the removal of remnant lipoproteins, in type III HLP has not been investigated. METHODS: We examined post-heparin plasma lipoprotein lipase (LPL) and HL activities in 7 patients with type III HLP. The activities of HL and LPL in post-heparin plasma were measured separately by an immunochemical method using antiserum specifically directed against HL. RESULTS: The post-heparin plasma HL activity was significantly reduced, while the LPL activity was normal. CONCLUSIONS: Reduced HL activity may account, at least in part, for the accumulation of remnant lipoproteins in the plasma, a characteristic feature of type III HLP.

Serum aspirin esterase is strongly associated with glucose and lipids in healthy subjects: different association patterns in subjects with type 2 diabetes mellitus.

BACKGROUND: Aspirin esterase (AE) activity can account for part of aspirin pharmacokinetics in the circulation, possibly being associated with the impairment of aspirin effectiveness as an inhibitor of platelet aggregation. AIMS: The study was aimed at investigating the correlations of serum AE activity with cholinesterase (ChE) and metabolic variables in healthy subjects in comparison to subjects with type 2 diabetes mellitus (T2DM). METHODS: In cardiovascular disease-free T2DM subjects and healthy controls, the AE activity levels and/or the correlation patterns between AE and the other variables were analyzed. RESULTS: Neither AE nor ChE activities were higher in the subjects with T2DM. Serum AE activity strongly correlated with ChE as well as glucose/lipids variables such as total cholesterol and triglyceride in healthy subjects, while the correlations between AE and glucose/lipids variables were not present in T2DM subjects. CONCLUSIONS: These data may reflect the pathophysiological changes between healthy and T2DM subjects. Our data may thus provide the basis for future studies to unravel the mechanisms.

A novel complex deletion-insertion mutation mediated by Alu repetitive elements leads to lipoprotein lipase deficiency.

Lipoprotein lipase (LPL) deficiency is a rare autosomal recessive inherited disorder, characterized by marked hypertriglyceridemia, eruptive xanthoma, hepatosplenomegaly, recurrent attacks of pancreatitis, and markedly low or absent LPL activity in postheparin plasma. A majority of LPL deficient patients have been reported to have point mutations in the LPL gene; however, we find a complex deletion-insertion mutation by Alu elements, mobile retrotransposons, in a patient with LPL deficiency. This patient suffered from acute pancreatitis, showed chylomicronemia and lacked detectable LPL activity or mass in her postheparin plasma. Southern blot analysis and long-range PCR of the patient's DNA demonstrated a 2.2-kb deletion encompassing exon 2. Sequence analysis revealed (1) a 2.3-kb deletion between an AT-rich region adjacent to an Alu element in intron 1 and another Alu element in intron 2; (2) an insertion of approximately 150bp 5'-truncated Alu sequence with a poly (A) tail at the deletion point. The inserted sequence belongs to Alu Yb9, the youngest subfamily of Alu elements. The deletion occurred at the consensus cleavage site (3'-A|TTTT-5') without target site duplication. These findings indicated that Alu retrotransposition caused the complex deletion-insertion. The patient was homozygous for this complex mutation, which eliminates exon 2 and leads to LPL deficiency. To our knowledge, the patient is the first case with LPL deficiency due to a complex deletion-insertion mediated by Alu repetitive elements.

A Japanese patient with cardiomyopathy caused by a novel mutation R285X in the AGL gene.

Left ventricular hypertrophy (LVH) is primarily or secondarily caused by a cardiovascular or systemic disease. The pattern of LVH is distinctive in hypertrophic or metabolic cardiomyopathy and differs from that seen in LVH caused by hypertension or aortic stenosis. A 42-year-old Japanese man had LVH similar to that with hypertrophic cardiomyopathy. The patient was diagnosed with glycogen storage disease type IIIa (GSD-IIIa). Echocardiography showed that he had severe LVH, and concomitant hepatomegaly and hypoglycemia, which led to measurement of glycogen debranching enzyme (GDE) activity; it was undetectable. Sequence analysis of the AGL gene encoding GDE showed a novel nonsense mutation: a C-to-T transition at codon 285 in exon 8, resulting in substitution of the arginine codon by the stop codon (R285X). The patient was homozygous for the mutation. Cardiomyopathy in this patient was caused by a nonsense mutation in the AGL gene. Five other Japanese GSD-IIIa patients over 30 years of age have all presented with cardiomyopathy, as well as hepatomegaly and hypoglycemia. Patients with LVH associated with hepatomegaly and hypoglycemia should undergo biochemical and genetic analyses for GSD-IIIa.

Molecular analysis of the AGL gene: heterogeneity of mutations in patients with glycogen storage disease type III from Germany, Canada, Afghanistan, Iran, and Turkey.

Glycogen storage disease type III (GSD III) is an autosomal recessive disorder characterized by excessive accumulation of abnormal glycogen in the liver and/or muscles and caused by deficiency in the glycogen debranching enzyme (AGL). Previous studies have revealed that the spectrum of AGL mutations in GSD III patients depends on ethnic grouping. We investigated nine GSD III patients from Germany, Canada, Afghanistan, Iran, and Turkey and identified six novel AGL mutations: one nonsense (W255X), three deletions (1019delA, 3202-3203delTA, and 1859-1869del11-bp), and two splicing mutations (IVS7 + 5G > A and IVS21 + 5insA), together with three previously reported ones (R864X, W1327X, and IVS21 + 1G > A). All mutations are predicted to lead to premature termination, which abolishes enzyme activity. Our molecular study on GSD III patients of different ethnic ancestry showed allelic heterogeneity of AGL mutations. This is the first AGL mutation report for German, Canadian, Afghan, Iranian and Turkish populations.

Molecular characterization of Egyptian patients with glycogen storage disease type IIIa.

Glycogen storage disease type IIIa (GSD IIIa) is an autosomal recessive disorder characterized by excessive accumulation of abnormal glycogen in the liver and muscles and caused by a deficiency in the glycogen debranching enzyme. The spectrum of AGL mutations in GSD IIIa patients depends on ethnic group-prevalent mutations have been reported in the North African Jewish population and in an isolate such as the Faroe islands, because of the founder effect, whereas heterogeneous mutations are responsible for the pathogenesis in Japanese patients. To shed light on molecular characteristics in Egypt, where high rate of consanguinity and large family size increase the frequency of recessive genetic diseases, we have examined three unrelated patients from the same area in Egypt. We identified three different individual AGL mutations; of these, two are novel deletions [4-bp deletion (750-753delAGAC) and 1-bp deletion (2673delT)] and one the nonsense mutation (W1327X) previously reported. All are predicted to lead to premature termination, which completely abolishes enzyme activity. Three consanguineous patients are homozygotes for their individual mutations. Haplotype analysis of mutant AGL alleles showed that each mutation was located on a different haplotype. Our results indicate the allelic heterogeneity of the AGL mutation in Egypt. This is the first report of AGL mutations in the Egyptian population.

Lipoprotein lipase mRNA in white adipose tissue but not in skeletal muscle is increased by pioglitazone through PPAR-gamma.

Lipoprotein lipase (LPL), a key enzyme for triglyceride hydrolysis, is an insulin-dependent enzyme and mainly synthesized in white adipose tissue (WAT) and skeletal muscles (SM). To explore how pioglitazone, an enhancer of insulin action, affects LPL synthesis, we examined the effect of pioglitazone on LPL mRNA levels in WAT or SM of brown adipose tissue (BAT)-deficient mice, which develop insulin resistance and hypertriglyceridemia. Both LPL mRNA of WAT and SM were halved in BAT-deficient mice. Pioglitazone increased LPL mRNA in WAT by 8-fold, which was substantially associated with a 4-fold increase of peroxisome proliferator activated receptor (PPAR)-gamma mRNA (r=0.97, p<0.0001), whereas pioglitazone did not affect LPL mRNA in SM. These results suggest that pioglitazone exclusively increases LPL production in WAT via stimulation of PPAR-gamma synthesis. Since pioglitazone does not affect LPL production in SM, this would contribute to prevent the development of insulin resistance due to lipotoxicity.