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Extensive genetic heterogeneity in patients with acid alpha glucosidase deficiency as detected by abnormalities of DNA and mRNA

Martiniuk F; Mehler M; Tzall S; Meredith G; Hirschhorn R
Acid maltase, or acid alpha glucosidase (GAA), is a lysosomal enzyme that hydrolyzes glycogen to glucose and is deficient in glycogen storage disease type II. We have previously isolated a partial cDNA (1.9 kb) for human GAA and detected abnormalities of mRNA in two infantile-onset and one adult-onset patient. We have now extended this study and examined mRNA and DNA from cell lines of eight additional infantile and three adult-onset patients. While five of the 10 infantile-onset patients expressed normal amounts and sizes of mRNA, the remaining five did not express detectable GAA mRNA. Two adult-onset patients had normal amounts and sizes of mRNA, while two adult-onset patients had mRNA of smaller size. Thus, half of the larger series of GAA-deficient patients also exhibited quantitative and/or qualitative abnormalities of mRNA. Of the five infantile-onset patients with normal mRNA, two exhibited an abnormal SacI fragment not found in DNA from 60 normals. To further characterize these patients, we determined GAA activity in several of the cell lines by using either the artificial substrate, 4-methylumbelliferyl-alpha-D-glucoside, or the natural substrate glycogen. Two adult-onset patients who both had normal size mRNA differed as to enzyme activity, with one patient exhibiting enzyme activity similar to that in infantile-onset patients. By combining these data with those for previously reported presence or absence of GAA-mutant protein cross-reacting to antibody, we provide evidence for a minimum of six different mutations in these 14 GAA-deficient cell lines
PMID: 2112341
ISSN: 0002-9297
CID: 15203

Sequence of the cDNA and 5'-flanking region for human acid alpha-glucosidase, detection of an intron in the 5' untranslated leader sequence, definition of 18-bp polymorphisms, and differences with previous cDNA and amino acid sequences

Martiniuk F; Mehler M; Tzall S; Meredith G; Hirschhorn R
Acid maltase or acid alpha-glucosidase (GAA) is a lysosomal enzyme that hydrolyzes glycogen to glucose and is deficient in glycogen storage disease type II. Previously, we isolated a partial cDNA (1.9 kb) for human GAA; we have now used this cDNA to isolate and determine sequence in longer cDNAs from four additional independent cDNA libraries. Primer extension studies indicated that the mRNA extended approximately 200 bp 5' of the cDNA sequence obtained. Therefore, we isolated a genomic fragment containing 5' cDNA sequences that overlapped the previous cDNA sequence and extended an additional 24 bp to an initiation codon within a Kozak consensus sequence. The sequence of the genomic clone revealed an intron-exon junction 32 bp 5' to the ATG, indicating that the 5' leader sequence was interrupted by an intron. The remaining 186 bp of 5' untranslated sequence was identified approximately 3 kb upstream. The promoter region upstream from the start site of transcription was GC rich and contained areas of homology to Sp1 binding sites but no identifiable CAAT or TATA box. The combined data gave a nucleotide sequence of 2,856 bp for the coding region from the ATG to a stop codon, predicting a protein of 952 amino acids. The 3' untranslated region contained 555 bp with a polyadenylation signal at 3,385 bp followed by 16 bp prior to a poly(A) tail. This sequence of the GAA coding region differs from that reported by Hoefsloot et al. (1988) in three areas that change a total of 42 amino acids. Direct determination of the amino acid sequence in one of these areas confirmed the nucleotide sequence reported here but also disagreed with the directly determined amino acid sequence reported by Hoefsloot et al. (1988). At two other areas, changes in base pairs predicted new restriction sites that were identified in cDNAs from several independent libraries. The amino acid changes in all three ares increased the homology to rabbit-human isomaltase. Therefore, we believe that our nucleotide sequence for GAA is more precise. We have also identified single base-pair polymorphisms at 18 sites for human GAA, some of which are not silent
PMID: 2111708
ISSN: 1044-5498
CID: 15206


Meredith, GS; Mitnick, HJ; Burstin, HE; Zimmerman, SS
ISSN: 0028-7628
CID: 31549

Polyarteritis nodosa presenting with migratory soft tissue swelling [Case Report]

Meredith GS; Mitnick HJ; Burstin HE; Zimmerman SS
PMID: 2888071
ISSN: 0028-7628
CID: 63008


Martiniuk, F; Meredith, G; Mehler, M; Pellicer, A; Hirschhorn, R
ISSN: 0009-9279
CID: 31204

Comparison and possible homology of isozymes of adenosine deaminase in Aves and humans

Ratech H; Thorbecke GJ; Meredith G; Hirschhorn R
Two kinetically distinct adenosine deaminase (ADA) isozymes with different molecular weights (35,000 and 100,000 daltons) are found in chicken liver in approximately equal amounts. The 100,000-dalton ADA has a markedly higher Km for adenosine and a markedly lower deaminating activity for deoxyadenosine relative to adenosine than does the 35,000-dalton ADA. A 100,000-dalton ADA isozyme has only recently been detected in mammalian tissues, where, in contrast to the chicken, it is only a trace component of total ADA activity. The human 100,000-dalton ADA isozyme, compared to the human 35,000-dalton ADA isozyme, has been reported to have a higher Km, a lower pH optimum, and a greater resistance to inhibition by erythro-9-(2-hydroxy-2-nonyl) adenine (EHNA). The similarity in KmS of the 100,000-dalton ADA isozyme in man and aves led us to hypothesize that these isozymes might be descended from a common ancestor and therefore also be similar as to other kinetic parameters. We now report that the chicken 100,000-dalton ADA, like the human 100,000-dalton isozyme, has a lower pH optimum and a greater resistance to inhibition by EHNA than does the avian or human 35,000-dalton isozyme. In addition, the avian 100,000-dalton isozyme is relatively resistant to inhibition by deoxycoformycin and has a cathodal rather than an anodal electrophoretic mobility at pH 6.5. Conversely, we report that the human 100,000-dalton ADA isozyme, similar to the avian 100,000-dalton ADA, has markedly lower relative deaminating activity for deoxyadenosine than does the 35,000-dalton ADA human isozyme. Thus, despite the marked difference in the relative amount of the 100,000- and 35,000-dalton ADA isozymes in man as compared to aves, the 100,000-dalton ADA isozymes from both species exhibit several similar kinetic properties, all of which are different from those of the 35,000-dalton ADA isozymes. We also report using a new sensitive assay, relative rates of degradation by the two chicken isozymes of several naturally occurring modified adenine nucleosides which are inhibitory to in vitro human lymphocyte proliferation.
PMID: 7238474
ISSN: 0013-9432
CID: 8878