Asako Horinishi

No evidence of accelerated atherosclerosis in a 66-yr-old chylomicronemia patient homozygous for the nonsense mutation (Tyr61→Stop) in the lipoprotein lipase gene

Whether chylomicronemia is atherogenic or not has yet to be determined in humans. We investigated a 66-yr-old female with severe chylomicronemia resulting from a lipoprotein lipase (LPL) deficiency. The patients plasma triglyceride level was approximately 2000 mg/dl. Both LPL activity and the mass of postheparin plasma in this patient were virtually absent. A nonsense mutation in exon 3 (Tyr61-->Stop) was identified in the patients LPL gene, and a restriction fragment length polymorphism analysis established that the patient was homozygous for this mutation. The patient was neither a diabetic nor a smoker. Clinically, the patient had never experienced pancreatitis or angia pectoris. An examination of her carotid, femoral and coronary arteries by ultrasonogram and electrocardiogram after exercise-tolerance testing showed no accelerated atherosclerosis. This case suggests that atherosclerosis may not occur despite massive hyperlipidemia, when LPL bridging was not present due to the absence of LPL secretion and circulating mass.

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

AbstractGlycogen 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.

A novel point mutation in an acceptor splice site of intron 32 (IVS32 A-12-->G) but no exon 3 mutations in the glycogen debranching enzyme gene in a homozygous patient with glycogen storage disease type IIIb.

Abstract Genetic deficiency of the glycogen-debranching enzyme (debrancher) causes glycogen storage disease type III (GSD III), which is divided into two subtypes: IIIa and IIIb. In GSD IIIb, glycogen accumulates only in the liver, whereas both liver and muscles are involved in GSD IIIa. The molecular basis for the differences between the two subtypes has not been fully elucidated. Recently, mutations in exon 3 of the debrancher gene were reported to be specifically associated with GSD IIIb. However, we describe a homozygous GSD IIIb patient without mutations in exon 3. Analysis of the patient’s debrancher cDNA revealed an 11-bp insertion in the normal sequence. An A to G transition at position –12 upstream of the 3′ splice site of intron 32 (IVS 32 A–12→G) was identified in the patient’s debrancher gene. No mutations were found in exon 3. Mutational analysis of the family showed the patient to be homozygous for this novel mutation as well as three polymorphic markers. Furthermore, the mother was heterozygous and the parents were first cousins. The acceptor splice site mutation created a new 3′ splice site and resulted in insertion of an 11-bp intron sequence between exon 32 and exon 33 in the patient’s debrancher mRNA. The predicted mutant enzyme was truncated by 112 amino acids as a result of premature termination. These findings suggested that a novel IVS 32 A–12→G mutation caused GSD IIIb in this patient.

Mutational and haplotype analysis of AGL in patients with glycogen storage disease type III

AbstractGlycogen storage disease type III (GSD III) is a rare autosomal recessive inherited disorder caused by a deficiency of the glycogen-debranching enzyme (AGL). We investigated two GSD III patients and identified four different mutations. Nucleotide sequence analysis revealed patient 1 of Chinese descent to be a compound heterozygote for a novel nonsense mutation, R34X, and the splicing mutation (IVS32−12A > G) reported in a Japanese patient. Patient 2 of Japanese origin was found to be compound heterozygous for a novel nonsense mutation, Y1148X, and the splicing mutation (IVS14+1G > T) that we had described previously. To determine whether splicing mutations occurred independently, we performed intense AGL haplotype analysis using 21 intragenic polymorphic markers plus a novel polymorphism IVS32−97 A/G in the vicinity of the IVS32 splicing mutation. Patient 1 of Chinese origin and the Japanese patient homozygous for the IVS32−12A > G were found to have different haplotypes, indicating the IVS32−12A > G mutation to be a recurrent mutation. This is the first recurrent mutation established by intense haplotyping in the AGL gene.

Molecular characterization of Egyptian patients with glycogen storage disease type IIIa

AbstractGlycogen 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.

Compound heterozygous patient with glycogen storage disease type III: identification of two novel AGL mutations, a donor splice site mutation of Chinese origin and a 1-bp deletion of Japanese origin.

Glycogen storage disease type III (GSD III) is an autosomal recessive disorder caused by deficiency of glycogen-debranching enzyme (AGL). We studied a 2-year-old GSD III patient whose parents were from different ethnic groups. Nucleotide sequence analysis of the patient showed two novel mutations: a single cytosine deletion at nucleotide 2399 (2399delC) in exon 16, and a G-to-A transition at the +5 position at the donor splice site of intron 33 (IVS33+5G>A). Analysis of the mRNA produced by IVS33+5G>A showed aberrant splicing: skipping of exon 33 and activation of a cryptic splice site in exon 34. Mutational analysis of the family revealed that the 2399delC was inherited from her father, who is of Japanese origin, and the IVS33+5G>A from her mother, who is of Chinese descent, establishing that the patient was a compound heterozygote. To our knowledge, this is the first report of a mutation identified in a GSD III patient from the Chinese population.

Detection of a new compound heterozygote (del G916/G1401A) for lipoprotein lipase deficiency and a comparative haplotype analysis of the mutant lipoprotein lipase gene from Japanese patients

A patient with early-onset chylomicronemia from a non-consanguineous family to be a compound heterozygote for two different mutations in the lipoprotein lipase (LPL) gene has been demonstrated. Reports on compound heterozygotes for LPL deficiency have been limited. The patient, a 2-month-old Japanese boy, was admitted to Urayasu Hospital because of hyperlipidemia. He showed eruptive exanthomas on his face and buttocks, but had no signs of pancreatitis or hepatosplenomegaly. The patient’s plasma triglyceride (TG) and total cholesterol (TC) concentrations were 14 183 and 763 mg/dl, respectively (Table 1). The apolipoprotein (apo) CII concentration was a predictable value for plasma TG levels from a previous study [1]. LPL activity, assayed as described elsewhere [2], and mass, determined with a sandwich enzyme-linked immunosorbent assay (ELISA) kit (Dai-nippon Pharmaceutical, Japan) [3], in the patient’s postheparin plasma were virtually undetectable. The father’s plasma lipid levels were within the normal range, whereas the mother exhibited moderate hypertriglyceridemia shortly after the delivery. The LPL activities of the parents were nearly normal, but their LPL masses were approximately half the normal value. Informed consent for molecular analysis was obtained from the parents. Nucleotide sequence analysis of the patient’s LPL gene showed two separate point mutations: a single guanosine deletion at nucleotide 916 (del G) in exon 5, leading to premature termination at codon 224 by a frameshift; and a G-to-A transition at nucleotide 1401 (G1401A) in exon 8, resulting in a substitution of tryptophan (TGG) at codon 382 by a stop codon (TGA). Southern blot hybridization was performed first, as described previously [4], but showed no major rearrangements (data not shown). The polymerase chain reaction (PCR) was then carried out as previously described [4]. PCR products were directly inserted into a plasmid vector pCRII (Invitrogen, USA), and the nucleotide sequence of subcloned plasmids were determined in both directions by dideoxy termination with an AmpliTaq cycle sequencing kit (Perkin-Elmer, USA) * Corresponding author. Tel.: +81-3-3588-1111; fax: +81-3-35827068. E-mail address: QFG00550@nifty.ne.jp (M. Okubo) 1 Present address: Department of Medicine, Urayasu Hospital, Juntendo University, Chiba 279, Japan.

1176C Polymorphism in Japanese patients with glycogen storage disease type 1a

The authors stated that a G727T mutation in the glucose-6phosphatase gene that was first shown to be prevalent in Japan (Kajihara et al. 1995; Okubo et al. 1997), and later in China (Lam et al. 1998a), was linked to a 1176C polymorphism in Chinese patients with glycogen storage disease type 1a (GSD 1a). In order to determine whether this linkage is also present in Japan, we analyzed Japanese G727T alleles in 4 homozygous GSD 1a patients with the G727T mutation plus one heterozygote [3 patients and one healthy carrier (Okubo et al. 1997) and a newly found unrelated patient (K. Hamada et al. unpublished)]. Using the DraI restriction fragment length polymorphism as described by Lam et al. (1998b), we found that all G727T alleles had 1176C; we confirmed this finding by sequencing analysis. Our observation supports the notion that Japanese and Chinese GSD 1a patients share a common ancestor, providing interesting evidence of the anthropological origin of these two populations. References

Glycogen storage disease III subtypes and muscle weakness during childhood

Professor Chen has raised a question concerning our recent article in which we described a novel splicing mutation in a homozygous glycogen storage disease (GSD) type IIIb patient (Okubo et al. 1998). Professor Chen states that definitive classification of GSD III subtypes requires measurement of enzyme activities in muscle. We believe that our patient is properly classified as subtype IIIb, even though no muscle specimens to assay enzyme activity are available at the moment. Our patient, now 32 years old, showed clinical and pathological findings (Hashimoto et al. 1998) that were clearly different from those of patients with GSD subtype IIIa. She had no muscular symptoms and showed no evidence of muscle weakness, physically or electromyographically. Histologically, she exhibited massive accumulation of glycogen in the liver, resulting in liver cirrhosis, but had no deposition of glycogen in her muscles. In our experience, patients with GSD subtype IIIa usually complain of muscle weakness beginning in childhood, and muscle weakness, though minimal, is indeed usually present during childhood (reviewed in Chen and Burchell 1995). Coleman et al. described all their seven patients with subtype IIIa as manifesting muscle weakness clinically during childhood, whereas three IIIb patients did not (Coleman et al. 1992). Other researchers have also reported that Japanese GSD patients with muscle weakness develop the symptom in childhood (Fukuda et al. 1996). Muscle glycogen concentrations in subtype IIIb are usually lower than in subtype IIIa (reviewed in Hers et al. 1989). Professor Chen cited a report showing variations in muscle glycogen content in GSD IIIa patients (Brown 1985). Brown described 10 patients as lacking the debranching enzyme and having normal muscle glycogen content (Brown and Brown 1983), but all of them showed some clinical findings related to muscle involvement. Because the role of glycogen in muscle is to provide a substrate for generation of the ATP needed for muscle contraction (Chen and Burchell 1995), it is not surprising for IIIa patients to complain of muscle weakness before excessive glycogen accumulation is detectable in biopsy specimens. At the age of 32 years, our patient does not show such muscle weakness. We believe this finding suggests that her muscular debranching enzyme activity is intact. If our patient’s debrancher activity in muscle were deficient, a kind of compensatory mechanism preventing glycogen accumulation would have to be assumed. We think that is an intriguing issue. REPLY