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Glycogen storage disease, type II (GSDII; Pompe disease; acid maltase deficiency) is an autosomal recessive disease caused by mutations of the GAA gene that lead to deficient acid alpha-glucosidase enzyme activity and accumulation of lysosomal glycogen. Although measurement of acid alpha-glucosidase enzyme activity in fibroblasts remains the gold standard for the diagnosis of GSDII, analysis of the GAA gene allows confirmation of clinical or biochemical diagnoses and permits predictive and prenatal testing of individuals at risk of developing GSDII. We have developed a clinical molecular test for the detection of GAA mutations based on cycle sequencing of the complete coding region. GAA exons 2-20 are amplified in six independent PCR using intronic primers. The resulting products were purified and sequenced. Preliminary studies using this protocol were conducted with DNA from 21 GSDII-affected individuals from five centers across Canada. In total, 41 of 42 mutations were detected (96.7% detection rate). Mutations spanned intron 1 through exon 19 and included nine novel mutations. Haplotype analysis of recurrent mutations further suggested that three of these mutations are likely to have occurred independently at least twice. Additionally, we report the identification of the c.-32-13T>G GAA mutation in an individual with infantile variant GSDII, despite reports of this mutation being associated almost exclusively with late-onset forms of the disease. The development of a clinical molecular test provides an important tool for the management and counseling of families and individuals with GSDII, and has provided useful information about the GAA mutation spectrum in Canada.

Recently, we reported on a family showing transmission of the FRAXA gene by three nonpenetrant, normally intelligent, full and half brothers to their affected grandsons [Kirkilionis et al., 1992]. We have reanalyzed this family for CGG repeat size by polymerase chain reaction (PCR) amplification/Southern blot and FMR1 methylation status using EcoRI/BssHII double digests with pE5-1 as the hybridization probe. The half brother was found to have a premutation allele size of 59 CGG repeats. MnlI digestion of PCR products showed the absence of intervening AGG sequences. All of his obligate carrier daughters had CGG alleles ranging from 65 to 90 repeats, with a final expansion of more than 200 repeats in his FRAXA-affected grandson and 131 repeats in his carrier granddaughter. Two full brothers were shown to have inherited a 47-CGG repeat premutation allele. Analysis of one brother showed that he stably transmitted the 47-repeat allele to his daughter. Analysis of the second brother, his daughter, and his granddaughter showed that this allele was meiotically unstable, with the allele size increasing from 47, to 48, to 49 from the father, to the daughter to the granddaughter, respectively. MnlI digestion and DNA sequencing of PCR products showed the absence of intervening AGG sequences. This is the first case in which the lack of AGG interspersions has been associated with instability of a gray zone allele resulting in a one-repeat increase in two successive generations.

Rationale and Objectives: We assessed the usefulness of the resistive index (RI) and renal length in predicting a significant renal artery stenosis (RAS) and evaluated the effect of captopril on the RI in kidneys with and without a significant RAS., Methods: The RIs and renal lengths of both kidneys were measured in 39 patients who were referred for captopril renography for suspected renovascular hypertension. The difference in RIs (delta RI), the smaller RI (SRI), the difference in lengths (delta L), and the shorter length (SL) of the patient's two kidneys were determined. The accuracy of each of these parameters was calculated using captopril renography (n = 39) and arteriography (n = 9) as the gold standards., Results: There was a significant difference in the delta RI (P < .05), SRI (p < .001), and delta L (p < .05) in patients with a positive captopril renogram for a significant RAS. Captopril increased delta RI (p = .052) in patients with a positive captopril renogram (n = 6). Use of an SRI threshold of less than .55 resulted in ultrasound being as accurate as captopril renography in predicting an angiographically documented stenosis of greater than or equal to 50%., Conclusion: The RI and renal length are useful in detecting a significant RAS. In this preliminary study, captopril was shown to increase delta RI in patients with a significant RAS, but larger prospective studies are necessary to further assess the value of captopril sonography in detecting a significant RAS.

Rationale and Objectives: We evaluated the changes in the down-stream Doppler waveforms caused by a proximal stenosis in the main renal artery of dogs., Methods: Renal parenchymal arterial waveforms downstream from mild (< 50%), moderate (50-75%), and severe (76-95%) stenoses were compared with nonstenotic baseline waveforms in five mongrel dogs. Waveform shapes were categorized as biphasic or monophasic. The percentage of biphasic and monophasic waveforms was determined for each stenosis. The acceleration index (AI) and the acceleration time (AT) were determined using traditional and modified calculations (AI' and AT'). Late systolic deceleration (DS), diastolic deceleration (DD), and the resistive index (RI) also were measured., Results: AT, AI', and AT' demonstrated significant differences between the severe stenoses and nonstenotic baselines (p < .05); however, there was no difference between the mild and moderate stenoses versus baselines. The percentage distribution of monophasic and biphasic waveforms was highly correlated with the degree of stenosis. Monophasic waveforms increased on average from 22.5% of baseline waveforms to 76.5% of waveforms in the severe stenoses. Biphasic waveforms decreased on average from 69.9% of baseline waveforms to 18.7% of waveforms in the severe stenoses., Conclusion: Quantitative evaluation of the downstream waveform parameters (AI, AT, AI', AT', DS, DD, and RD in the dog kidney is not sufficiently accurate, but calculation of the percentage of the monophasic and biphasic waveforms present may be useful to predict a hemodynamically significant renal artery stenosis (> or = 50%).

Nearly all cases of fragile X syndrome result from expansion of a CGG trinucleotide repeat found in the 5' untranslated portion of the FMR1 gene. Methylation of the expanded repeats correlates with down-regulation of transcription of FMR1; thus fragile X syndrome is postulated to be due to a loss of function of the FMR1 gene product, and this has been demonstrated at the protein level. However, the nature of the mutation offers the possibility of methylation spreading to adjacent genes with consequent loss of expression and contribution to the phenotype. Deletions of FMR1 and flanking sequence (some of substantial size) have been reported in patients with phenotypes consistent with a diagnosis of fragile X-syndrome, however, none is strictly intragenic. We report here the identification of two different intragenic loss of function mutations in FMR1: a single de novo nucleotide deletion in a young male patient (IJ) and an inherited two basepair change in an Adult male (SD), each with classical features of fragile X syndrome.

A physical map for 13 loci on chromosome 10 was developed by determining the dosage of the corresponding DNA sequences in cell lines with unbalanced chromosome 10 rearrangements. Nine of the sequences were assigned to a smaller segment of the chromosome than previously and four sublocalizations were confirmed. The physical map covers most of chromosome 10, from 10p13 to 10q23. The linear order of loci within the physical map agrees with existing linkage maps of chromosome 10. A comparison between the physical map and existing genetic maps indicate an uneven distribution of recombination for chromosome 10. There appear to be hot spots of recombination in the regions defined by q21.1 and q22-q23. In addition, there is a suppression of recombination in the pericentromeric region in males which is not evident in females.

Norepinephrine administration causes progressive hypertrophy of the mammalian heart as measured by myocardial mass. The purpose of this study was to determine the growth response of the myocardial tissue components as well as the myocardial cell itself to norepinephrine. Young, adult cats were given low doses of norepinephrine in dextrose or dextrose alone twice daily for 15 days. On day 16, there were no changes in the animals body weight, right ventricular systolic pressure, right ventricular end-diastolic pressure, heart rate, cardiac index, or blood pressure. However, the right ventricle/body weight, the left ventricle/body weight and the total heart weight/body weight were increased significantly in the norepinephrine treated animals. The increase was on the order of 40%. The cardiac muscle cell was also significantly increased in size and both the right and left ventricular cardiac muscle cells exhibited a dramatic increase in size as measured by cross sectional area. Upon stereological examination it was found that the amount of hypertrophy as seen in the cardiac muscle cells was paralleled by the hypertrophy seen in the other tissue components of the myocardium. The volume density of the muscle cells, the interstitial components, as well as the blood vessel compartment were identical in the control and in the norepinephrine-treated groups. In conclusion, this study demonstrates that the response of the myocardium to norepinephrine is similar to that seen in response to a volume overload rather than that seen in response to pressure overload.

Two new morphs (F and G) detected by the centromeric alpha satellite probe p alpha 10RP8 and D10Z1 in HinfI digests are linked to the PstI polymorphisms of D10Z1, confirming their chromosome 10 location. The F and G morphs were in strong linkage disequilibrium with each other but were in weak linkage disequilibrium with the A and B morphs defined in PstI digests. Data for haplotypes formed by using the A and F morphs improved the lod score for linkage between the disease locus for multiple endocrine neoplasia type 2A (MEN2A) and D10Z1 (Z = 14.06 at theta = 0) in the six large families studied by Wu et al. Furthermore, the locus that codes for a distinct phenotype, medullary thyroid carcinoma (MTC) with parathyroid tumors (PTs) and no pheochromocytomas (PHEOs) (referred to as MTC with PTs), in one of the families was closely linked to two markers, D10Z1 and RBP3, with lodscores of 2.86 and 3.54, respectively, at theta = 0. A possible allelic association was noted between disease phenotypes and centromeric haplotypes. The phenotype MTC and PHEOs with and without PTs was associated with the same relatively common centromeric haplotype (A + B-F-G-) in the four families in which all four morphs could be determined, while the phenotype MTC with PTs was associated with the rare centromeric haplotype (A-B-F-G+) in one family.

Multiple endocrine neoplasia type 2A (MEN2A) is a rare cancer syndrome that is inherited in an apparently autosomal dominant fashion. Previous linkage studies had assigned the MEN2A locus to chromosome 10 in the pericentromeric region. We recently have described several new easily scorable RFLPs for the chromosome 10-specific alpha satellite DNA (the D10Z1) locus that is known, on the basis of previous in situ hybridization experiments, to lie at the centromere. We report here tight linkage between MEN2A and D10Z1, as demonstrated by a maximum lod score of 12.02 at the recombination frequency of zero (1-lod-unit support interval 0-4 cM), indicating that the genetic defect in MEN2A lies in the immediate vicinity of the centromere. By means of a set of ordered polymorphic DNA markers from the pericentromeric region, multipoint as well as pairwise linkage analyses place the MEN2A locus at the middle of a small region (approximately 11 cM) bracketing the centromere with FNRB (at 10p11.2) and RBP3 (at 10q11.2) on either side, providing further support for the centromeric location of the MEN2A locus. Marked sex difference in recombination frequencies exists in this pericentromeric region: significantly (P less than .01) more female than male crossovers were observed across all of the adjacent intervals D10S24-FNRB, FNRB-D10Z1, and D10Z1-RBP3. However, a sex difference was not seen in the 7-cM interval from RBP3 to D10S5, suggesting that large variation in the sex difference in recombination can occur over small chromosomal regions.(ABSTRACT TRUNCATED AT 250 WORDS)

The beta subunit of the human fibronectin receptor (FNRB) is a transmembrane protein belonging to the VLA (very late antigens of activation) family. Using pGEM-32, a 2.5-kb partial cDNA clone corresponding to the 3' portion of the human FNRB locus, multiple restriction fragment length polymorphisms (RFLPs) were revealed on DNAs from unrelated Caucasians. RFLPs detected by five enzymes, BanII, HinfI, KpnI, BglII, and SacI, are of the simple two-allele form, and pairwise linkage analyses of these RFLPs with numerous known DNA markers from the chromosome-10 pericentromeric region not only confirmed the chromosome-10 assignment of the functional FNRB gene but also supported its localization at p11.2 suggested by in situ hybridization. An infrequent MspI RFLP was detected by pB/R2, a 4.6-kb genomic clone from the FNRB locus. Another type of DNA polymorphism was also revealed by the cDNA clone and it was visualized on the Southern blot analyses as the presence or absence of an extra band (or a set of extra bands). It seems to stem from a stretch of DNA sequence present in some individuals at one single locus but absent in others, and is of non-chromosome-10 origin based on linkage analyses with known chromosome 10 markers. This "presence/absence" type of polymorphism could be revealed by all of the 25 restriction enzymes tested and is similar in nature to that previously reported with one of the human dihydrofolate reductase pseudogenes, DHFRP1. Dissection of the pGEM-32 clone demonstrated that the region revealing the non-chromosome-10 sequences is within a fragment about 1.7 kb in length extending from about 600 nucleotides preceding the stop codon down to the end of the cloned FNRB 3' untranslated region. Due to its high polymorphism information content (PIC) value (0.71 for haplotypes of BanII, HinfI, and KpnI RFLPs) and proximity to the centromere. FNRB will prove to be a highly useful marker for genetic linkage studies of multiple endocrine neoplasia type 2A (MEN2A) as well as for chromosome-10 linkage studies in general.