Development of a clinical assay for detection of GAA mutations and characterization of the GAA mutation spectrum in a Canadian cohort of individuals with glycogen storage disease, type II
Introduction
Glycogen storage disease type II (GSDII; Pompe disease; acid α-glucosidase deficiency; acid maltase deficiency) is an inborn error of metabolism caused by accumulation of glycogen within the lysosomes of various tissues. Lysosomal storage of glycogen leads to cardiomegaly, hypotonia, macroglossia, and hepatomegaly in the infantile form of the disease, and to progressive muscle weakness involving both skeletal and diaphragmatic muscles in the late-onset forms. Although individuals with the infantile-onset forms frequently die during the first year of life, those with the later onset forms can survive well into adulthood depending on the age of onset and extent of cardiac and respiratory involvement. A subtype of GSDII, called the infantile variant form [1], has also been proposed in which affected individuals present during the first year of life with symptoms similar to the infantile-onset form, but with mild or absent cardiomegaly [2]. Individuals with this subtype often survive the first year of life, and may live for several years afterward. The frequency of the infantile-onset form of GSDII varies among different ethnic groups and ranges from 1/100,000 to 1/200,000 in outbred Caucasian populations to 1/14,000 among African-Americans. The prevalence of the adult-onset form of GSDII has been estimated at 1/60,000 in the Caucasian population [3].
GSDII is caused by a deficiency of the acid α-glucosidase enzyme (EC 3.2.1.20) encoded by the GAA gene on chromosome 17q25.3. The GAA gene is comprised of 20 exons spanning 18 kb of DNA. Its coding sequence, located in exons 2–20, is highly polymorphic. At least 20 polymorphic nucleotide residues have been described, many of which result in amino acid substitutions. In addition to these polymorphisms, at least 120 pathogenic mutations of the GAA gene have been reported in association with GSDII (http://www2.eur.nl/fgg/ch1/pompe/index.html). A common mutation, c.-32-13T > G, has been identified on at least one allele in over two-thirds of Caucasian patients with adult-onset GSDII [4], [5], [6]. Other common mutations include c.525del and exon 18 deletions in patients of Dutch ancestry [7], p.Arg854X mutation in patients of African-American ancestry [8], [9], p.Asp645Glu, c.1411_1414del and p.Gly615Arg mutations in patients of Chinese ancestry [10], [11], and p.Ser529Val, p.Arg600Cys, and p.Arg672Gln in patients of Japanese ancestry [12]. Despite the relative high prevalence of the above mutations in each population, most of the GAA mutations identified thus far are private.
Traditionally the diagnosis of GSDII has been based on the finding of deficient acid α-glucosidase activity, often in fibroblasts or muscle. These tests are invasive and time to reported results is generally at least 2–3 months. The enzyme assay can be performed on white blood cells but is complicated by the presence of overlapping activity towards the substrate by maltase-glucoamylase (renal isozyme) in granulocytes. This necessitates a preparation of pure lymphocytes or the use of either antibodies or inhibitors to eliminate renal isozyme activity [13], [14], [15], [16], [17], [18], [19]. Availability of a clinical molecular test for the detection of mutations in the GAA coding sequence is advantageous as such a test can provide a noninvasive method for confirmation of the clinical and/or biochemical diagnosis. It would also allow the detection of rare and/or novel mutations, facilitate genotype–phenotype correlations, and improve the accuracy of genetic counseling, of prenatal diagnosis, and potentially of pre-implantation genetic diagnosis. Importantly, the availability of a clinical molecular test for the diagnosis of GSDII would permit pre-symptomatic testing at birth of at-risk individuals, allowing optimal conditions for enzyme replacement therapy.
We report the development of a clinical molecular assay that allows sequence analysis of exons 2 through 20 as well as the 3′-untranslated region and the identification of 41 mutations on 42 alleles (sensitivity = 97.6%), including nine novel mutations. This represents a substantial proportion of the GSDII cases in Canada, and provides valuable information about the GAA mutation spectrum among affected individuals in this population. In addition, we report the identification of the c.-32-13T > G mutation in an individual with non-classical infantile-onset GSDII. This finding is significant and confirms a recent report indicating that this mutation can occur in infants with GSDII.
Section snippets
Patient samples
DNA from 5 controls and 23 unrelated individuals with clinical symptoms suggestive of GSDII were analyzed. All 23 individuals from the latter group were ascertained through metabolic clinics at one of five Canadian centers located in either Ottawa, Toronto, London, Calgary or Edmonton. Enzyme studies confirmed acid α-glucosidase deficiencies in 21 of these individuals. Technicians performing the molecular analysis were blinded with respect to clinical phenotype, and enzyme activity levels in
GAA mutational analysis
Mutations were not detected in exons 2 through 20 of the GAA gene in the five sequence controls nor two of the clinical samples. The two submitted clinical samples for which mutations were not identified corresponded to individuals with normal levels of acid α-glucosidase activity who are unaffected with GSDII (refer to Table 3). Failure to identify GAA mutations in these samples is consistent with the biochemical and clinical data. For the remaining 21 clinical samples with acid α-glucosidase
Discussion
We have developed a clinical molecular test for GSDII based on sequence analysis of the complete GAA coding region, flanking intronic sequences and 3′-untranslated region. GSDII is a rare disorder with no specific ethnic predisposition. It is characterized by a wide range of causative mutations that are largely private and there are no known genetic modifiers. Therefore, we feel that sequencing is the most appropriate method for mutation analysis of GAA. Sequence analysis of the complete coding
Acknowledgments
We thank the staff at the Children’s Hospital of Eastern Ontario, Molecular Diagnostic Laboratory and Tara Dzwiniel for their technical support during the sample collection and test development phases of this study. This research was funded in part by grants from Genzyme Corporation.
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