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Glycogen Storage Disease Type Ia Screening Using Dried Blood Spots on Filter Paper: Application of COP-PCR for Detection of the c.648G>T G6PC Gene Mutation.

ABSTRACT: Glycogen storage disease type Ia (GSDIa, OMIM #232200) is an autosomal recessive metabolic disease characterized by impaired glucose homeostasis and has a long-term complication of hepatocellular adenoma/carcinoma. GSDIa is caused by deleterious mutations in the glucose-6-phosphatase gene (G6PC). Recent studies have suggested that early treatment by gene replacement therapy may be a good solution to correct the glucose metabolism and prevent serious late complications. Early treatment of the disease needs an early disease detection system. Thus, we aimed to develop a screening system for GSDIa using dried blood spots (DBS) to detect the c.648G>T mutation in G6PC, which is a frequent mutation in the East Asian population. In this study, a total of 51 DBS samples (50 healthy controls and one patient with c.648G>T) were tested by modified competitive oligonucleotide priming PCR (mCOP-PCR). In control DBS samples, the c.648G allele was amplified at lower Cq (quantification cycle) values (<11), while the c.648T allele was amplified at higher Cq values (>14). In the patient DBS sample, the c.648T allele was amplified at a lower Cq value (<11), and the c.648G allele was amplified at a higher Cq value (>14). Based on these findings, we concluded that our mCOP-PCR system clearly differentiated the wild-type and mutant alleles, and may be applicable for screening for GSDIa with the c.648G>T mutation in G6PC.

SUBMITTER: Wijaya YOS

PROVIDER: S-EPMC8622217 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Real-time quantitative nucleic acid sequence-based amplification (QT-NASBA) is a sensitive method for detection of sub-microscopic gametocytaemia by measuring gametocyte-specific mRNA. Performing analysis on fresh whole blood samples is often not feasible in remote and resource-poor areas. Convenient methods for sample storage and transport are urgently needed. METHODS: Real-time QT-NASBA was performed on whole blood spiked with a dilution series of purified in-vitro cultivated gametocytes. The blood was either freshly processed or spotted on filter papers. Gametocyte detection sensitivity for QT-NASBA was determined and controlled by microscopy. Dried blood spot (DBS) samples were subjected to five different storage conditions and the loss of sensitivity over time was investigated. A formula to approximate the loss of Pfs25-mRNA due to different storage conditions and time was developed. RESULTS: Pfs25-mRNA was measured in time to positivity (TTP) and correlated well with the microscopic counts and the theoretical concentrations of the dilution series. TTP results constantly indicated higher amounts of RNA in filter paper samples extracted after 24 hours than in immediately extracted fresh blood. Among investigated storage conditions freezing at -20°C performed best with 98.7% of the Pfs25-mRNA still detectable at day 28 compared to fresh blood samples. After 92 days, the RNA detection rate was only slightly decreased to 92.9%. Samples stored at 37°C showed most decay with only 64.5% of Pfs25-mRNA detectable after one month. The calculated theoretical detection limit for 24 h-old DBS filter paper samples was 0.0095 (95% CI: 0.0025 to 0.0380) per ?l. CONCLUSIONS: The results suggest that the application of DBS filter papers for quantification of Plasmodium falciparum gametocytes with real-time QT-NASBA is practical and recommendable. This method proved sensitive enough for detection of sub-microscopic densities even after prolonged storage. Decay rates can be predicted for different storage conditions as well as durations.

Project description:BackgroundThe use of filter paper as a simple, inexpensive tool for storage and transportation of blood, 'Dried Blood Spots' or Guthrie cards, for diagnostic assays is well-established. In contrast, there are a paucity of diagnostic evaluations of dried cerebrospinal fluid (CSF) spots. These have potential applications in low-resource settings, such as Laos, where laboratory facilities for central nervous system (CNS) diagnostics are only available in Vientiane. In Laos, a major cause of CNS infection is Japanese encephalitis virus (JEV). We aimed to develop a dried CSF spot protocol and to evaluate its diagnostic performance using the World Health Organisation recommended anti-JEV IgM antibody capture enzyme-linked immunosorbent assay (JEV MAC-ELISA).Methodology and principal findingsSample volumes, spotting techniques and filter paper type were evaluated using a CSF-substitute of anti-JEV IgM positive serum diluted in Phosphate Buffer Solution (PBS) to end-limits of detection by JEV MAC-ELISA. A conventional protocol, involving eluting one paper punch in 200 μl PBS, did not detect the end-dilution, nor did multiple punches utilising diverse spotting techniques. However, pre-cut filter paper enabled saturation with five times the volume of CSF-substitute, sufficiently improving sensitivity to detect the end-dilution. The diagnostic accuracy of this optimised protocol was compared with routine, neat CSF in a pilot, retrospective study of JEV MAC-ELISA on consecutive CSF samples, collected 2009-15, from three Lao hospitals. In comparison to neat CSF, 132 CSF samples stored as dried CSF spots for one month at 25-30 °C showed 81.6% (65.7-92.3 95%CI) positive agreement, 96.8% (91.0-99.3 95%CI) negative agreement, with a kappa coefficient of 0.81 (0.70-0.92 95%CI).Conclusions/significanceThe novel design of pre-cut filter paper saturated with CSF could provide a useful tool for JEV diagnostics in settings with limited laboratory access. It has the potential to improve national JEV surveillance and inform vaccination policies. The saturation of filter paper has potential use in the wider context of pathogen detection, including dried spots for detecting other analytes in CSF, and other body fluids.

Project description:BackgroundGlycogen storage disease (GSD) Ia, caused by mutations in the glucose-6-phosphatase (G6PC) gene, is characterized by hepatomegaly, hypoglycemia, lactic acidosis, dyslipidemia, and hyperuricemia. This study aimed to investigate clinical and molecular features and late complications in Korean patients with GSD Ia.ResultsFifty-four Korean patients (33 males and 21 females) from 47 unrelated families, who were diagnosed with GSD Ia, based on genetic and biochemical data, between 1999 and 2017, were included in this study. The median age at diagnosis was 3.9 years (range: 5 months to 42 years), and the follow-up period was 8.0 ± 6.8 years. Most patients presented with hepatomegaly during infancy, but hypoglycemic symptoms were not predominant. Genetic analysis showed that all the patients had at least one c.648G > T allele. Homozygous c.648G > T mutations in the G6PC gene were identified in 34 families (72.3%), and compound heterozygotes with c.648G > T were found in the other families. The allele frequency of c.648G > T was 86.2% (81/94), and p.F51S, p.R83H, p.G122D, p.Y128*, p.G222R, and p.T255A were identified. Of 26 adult patients, 14 had multiple hepatic adenomas, and two were diagnosed with hepatocellular carcinoma. Thirteen patients showed renal complications, and seven patients presented gout, despite preventive allopurinol treatment. Twelve patients had osteoporosis, and two patients had pulmonary hypertension. The final heights were 157.9 cm (standard deviation score: - 3.1) in males and 157.8 cm (standard deviation score: - 0.6) in females.ConclusionIn our Korean patients with GSD Ia, the most common mutation in the G6PC gene was c.648G > T, suggesting a founder effect. Because of only mild hypoglycemia, the patients tended to be diagnosed late. Thus, adult patients with GSD Ia eventually developed diverse and serious complications, which indicates a need for careful monitoring and proper management of this disease.

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Glycogen Storage Disease Type Ia Screening Using Dried Blood Spots on Filter Paper: Application of COP-PCR for Detection of the c.648G>T G6PC Gene Mutation.

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