Faculty Bibliography

Glycogen storage disease type II (GSDII) is an autosomal recessive disorder resulting from inherited deficiency of the enzyme lysosomal acid alpha-glucosidase. Over 40 different mutations have been described but no large deletions have been previously identified. We now describe a homozygous large (9-kb) deletion extending from IVS 15 to 4 kb downstream of the terminal exon (exon 20), detected by polymerase chain reaction (PCR)-based methods. The deletion was initially suspected because of failure to amplify a contiguous group of exons by PCR. We hypothesized an Alu/Alu recombination, based on our prior demonstration by Southern blotting of Alu elements in the regions potentially flanking the deletion. Additional sequence analysis of genomic fragments confirmed the presence of Alu elements and allowed the design of flanking primers for PCR amplification. Amplification resulted in a smaller than normal fragment (0.7 vs. 10 kb) in homozygosity in the proband and in heterozygosity in her parents. Cloning and sequencing of the smaller than normal 0.7-kb deletion fragment revealed an Alu/Alu deletion junction. In heterozygosity this deletion would not be detected by currently standard PCR mutation detection methods. Based on other Alu-mediated deletions, this deletion is likely to be recurrent and should be screened for in all non-consanguineous GSDII patients, particularly when only one mutation has been identified and none of the 12 single-nucleotide polymorphisms in the deleted region are heterozygous. These observations also suggest that initial characterization of genes at disease-causing loci should include a search for Alu and other repetitive elements to facilitate subsequent PCR-based mutation analysis

Some recent publications indicate that inherited disorders can ameliorate or possibly disappear if mutations responsible for the disease revert to normal. This review tries to summarize our current knowledge about reverse mutations as this information may be of special interest for attempts at somatic gene therapy.

Glycogen storage disease type II (GSDII), an autosomal recessive myopathic disorder, results from deficiency of lysosomal acid alpha-glucosidase. We searched for mutations in an evolutionarily conserved region in 54 patients of differing phenotype. Four novel mutations (D645N, G448S, R672W, and R672Q) and a previously described mutation (C647W) were identified in five patients and their deleterious effect on enzyme expression demonstrated in vitro. Two novel frame-shifting insertions/deletions (delta nt766-785/insC and +insG@nt2243) were identified in two patients with exon 14 mutations. The remaining three patients were either homozygous for their mutations (D645N/D645 and C647W/C647W) or carried a previously described leaky splice site mutation (IVS1-13T-->G). For all patients 'in vivo' enzyme activity was consistent with clinical phenotype. Agreement of genotype with phenotype and in vitro versus in vivo enzyme was seen in three patients (two infantile patients carrying C647W/C647W and D645N/+insG@nt2243 and an adult patient heteroallelic for G648S/IVS1-13T-->G). Relative discordance was found in a juvenile patient homozygous for the non-expressing R672Q and an adult patient heterozygous for the minimally expressing R672W and delta nt766-785/+insC. Possible explanations include differences in in vitro assays vs in vivo enzyme activity, tissue specific expression with diminished enzyme expression/stability in fibroblasts vs muscle, somatic mosaicism, and modifying genes

Genetic deficiency of the purine salvage enzyme adenosine deaminase (ADA) results in varying degrees of immunodeficiency, ranging from neonatal onset Severe Combined Immunodeficiency (SCID) to an adult onset immunodeficiency disorder. Multiple different mutations have now been identified in these immunodeficient patients. Additional mutations, initially identified in healthy individuals, abolish ADA in erythrocytes but retain 10-80% of activity in non-erythroid cells ('partial deficiency mutations'). In general, severity of disease correlates inversely with the amount of residual ADA expressed by the mutant enzymes and directly with the accumulation of the toxic metabolites deoxyATP and deoxyadenosine. We report two newly identified mutations (Y97C and L106V), both carried on the same allele of an immunodeficient patient who was diagnosed prenatally and successfully transplanted with haploidentical bone marrow. Based on the ability of mutant cDNAs to express ADA in vitro , the L106V mutation resulted in activity similar to 'partial' mutations (30% of normal) while the Y97C mutation resulted in detectable but markedly reduced activity (1.5% of normal). However, the presence of both mutations on the same allele virtually abolished detectable enzyme activity. Analysis of the crystallographic structure of ADA to understand the marked deleterious effect of the Y97C mutation suggested a previously unappreciated role of salt bridges in the catalytic mechanism of ADA. The patient was also heteroallelic for a previously described deletion of the promoter and exon 1. Testing of additional patients in whom we had not identified a mutation on the second allele revealed presence of this deletion in three of four patients tested. This deletion is therefore relatively common, accounting for 10% of almost 100 chromosomes studied by this and other laboratories, but is easily missed by currently used methods of mutation detection. Lastly, the finding of two mutations on the same allele that interact to reduce residual enzyme function emphasizes hazards in evaluating potential genotype-phenotype correlations in individuals analyzed only for the presence of single specific mutations

The complete healing of wounds is the final step in a highly regulated response to injury. Although many of the molecular mediators and cellular events of healing are known, their manipulation for the enhancement and acceleration of wound closure has not proven practical as yet. We and others have established that adenosine is a potent regulator of the inflammatory response, which is a component of wound healing. We now report that ligation of the G alpha s-linked adenosine receptors on the cells of an artificial wound dramatically alters the kinetics of wound closure. Excisional wound closure in normal, healthy mice was significantly accelerated by topical application of the specific A2A receptor agonist CGS-21680 (50% closure by day 2 in A2 receptor antagonists. In rats rendered diabetic (streptozotocin-induced diabetes mellitus) wound healing was impaired as compared to nondiabetic rats; CGS-21680 significantly increased the rate of wound healing in both nondiabetic and diabetic rats. Indeed, the rate of wound healing in the CGS-21680-treated diabetic rats was greater than or equal to that observed in untreated normal rats. These results appear to constitute the first evidence that a small molecule, such as an adenosine receptor agonist, accelerates wound healing in both normal animals and in animals with impaired wound healing