Dried Blood Spots, an Affordable Tool to Collect, Ship, and Sequence gDNA from Patients with an X-Linked Agammaglobulinemia Phenotype Residing in a Developing Country.

Background: New sequencing techniques have revolutionized the identification of the molecular basis of primary immunodeficiency disorders (PID) not only by establishing a gene-based diagnosis but also by facilitating defect-specific treatment strategies, improving quality of life and survival, and allowing factual genetic counseling. Because these techniques are generally not available for physicians and their patients residing in developing countries, collaboration with overseas laboratories has been explored as a possible, albeit cumbersome, strategy. To reduce the cost of time and temperature-sensitive shipping, we selected Guthrie cards, developed for newborn screening, to collect dried blood spots (DBS), as a source of DNA that can be shipped by regular mail at minimal cost. Method: Blood was collected and blotted onto the filter paper of Guthrie cards by completely filling three circles. We enrolled 20 male patients with presumptive X-linked agammaglobulinemia (XLA) cared for at the Vietnam National Children's Hospital, their mothers, and several sisters for carrier analysis. DBS were stored at room temperature until ready to be shipped together, using an appropriately sized envelope, to a CLIA-certified laboratory in the US for sequencing. The protocol for Sanger sequencing was modified to account for the reduced quantity of gDNA extracted from DBS. Result: High-quality gDNA could be extracted from every specimen. Bruton tyrosine kinase (BTK) mutations were identified in 17 of 20 patients studied, confirming the diagnosis of XLA in 85% of the study cohort. Type and location of the mutations were similar to those reported in previous reviews. The mean age when XLA was suspected clinically was 4.6 years, similar to that reported by Western countries. Two of 15 mothers, each with an affected boy, had a normal BTK sequence, suggesting gonadal mosaicism. Conclusion: DBS collected on Guthrie cards can be shipped inexpensively by airmail across continents, providing sufficient high-quality gDNA for Sanger sequencing overseas. By using this method of collecting gDNA, we were able to confirm the diagnosis of XLA in 17 of 20 Vietnamese patients with the clinical diagnosis of agammaglobulinemia.

Ethanol tolerance and withdrawal responses in GABA(A) receptor alpha 6 subunit null allele mice and in inbred C57BL/6J and strain 129/SvJ mice.

We have been using a genetic strategy to define the contribution of specific candidate genes, such as those encoding subunits of the gamma-aminobutyric acid type A receptor, to various ethanol sensitive responses. We have used the gene knockout approach in mouse embryonic stem cells to create mice in which the gene encoding the alpha6 subunit of the gamma-aminobutyric acid type A receptor is rendered nonfunctional. In the present report, we provide a detailed characterization of several behavioral responses to ethanol in these null allele mice. In a separate series of experiments, behavioral response to ethanol was compared between two inbred strains of mice that are commonly used as background stock in knockout experiments, namely C57BL/6J and Strain 129/SvJ. Wild type (alpha6+/+) and homozygous null allele (alpha6-/-) mice did not differ to the ataxic effects of ethanol on acute functional tolerance (95.8 +/- 8.7 vs. 98.8 +/- 5.7 mg/dl +/- SEM, respectively). Withdrawal hyperexcitability was assessed following chronic exposure to ethanol vapor (EtOH) or air (CONT) in inhalation chambers in a multiple withdrawal treatment paradigm. At the end of the last treatment cycle, mice were scored for handling induced convulsions (HIC). After adjusting for differences in blood ethanol concentration between genotypes at the end of the final treatment cycle, we observed a greater area under the 24-hr HIC curves in mice treated with ethanol (p < 0.0001) but did not detect an effect of genotype (alpha6+/+/CONT 3.1 +/- 2.0; alpha6-/-/CONT 5.5 +/- 2.5; alpha6+/+/EtOH 30.1 +/- 6.2; alpha6-/-/EtOH 33.0 +/- 5.8 mean units +/- SEM). We also examined these mice for differences in protracted tolerance; at approximately 26 hr into the final withdrawal cycle, each mouse was injected with ethanol (3.5 mg/g body weight) and sleep time was measured. We detected a significant effect of treatment (p < 0.001) with ethanol-treated mice demonstrating signs of tolerance as reflected by a reduction in duration of sleep time. However, effect of genotype was not significant (alpha6+/+/CONT 57.4 +/- 7.6; alpha6-/-/CONT 59.0 +/- 7.6; alpha6+/ +/EtOH 34.8 +/- 7.4; alpha6-/-/EtOH 30.8 +/- 5.6 min +/- SEM). From these data we conclude that the alpha6 subunit of the GABA(A)-R exerts little if any influence on acute functional tolerance, withdrawal hyperexcitability, or protracted tolerance. Strain 129/SvJ and C57BL/6J mice were also compared for acute functional tolerance and were found not to differ (96.3 +/- 4.4 vs. 94.8 +/- 11.3 mg/dl +/- SEM, respectively). Withdrawal hyperexcitability was assessed by comparing the area under the 24 hr HIC curves. Strain 129/SvJ mice displayed a much greater basal HIC response compared to C57BL/6J mice (19.8 +/- 4.3 vs. 0.2 +/- 0.2 mean units +/- SEM, respectively); after adjusting for differences in blood ethanol concentration between strains at the end of the final ethanol treatment cycle, the HIC response was markedly enhanced by ethanol treatment in Strain 129/SvJ mice but not in C57BL/6J mice (50.4 +/- 3.1 vs. 9.5 +/- 5.4 mean units +/- SEM, respectively). The effects of treatment (p < 0.0001), strain (p < 0.0001), and the interaction of strain with treatment (p < 0.01) were significant. Since many gene knockout mice are maintained on a mixed genetic background of Strain 129/SvJ and C57BL/6J, we conclude that significant differences in tests of withdrawal hyperexcitability may be confounded by the influence of genes that cosegregate with the gene targeted allele.