Pompe disease is a genetic disorder resulting from a deficiency of lysosomal acid alpha-glucosidase (GAA) that manifests as a clinical spectrum with regard to symptom severity and rate of progression. In this study, we used microarrays to examine gene expression from the muscle of two cohorts of infantile-onset Pompe patients to identify transcriptional differences that may contribute to the disease phenotype. We found strong similarities among the gene expression profiles generated from biceps and quadriceps, and identified a number of signaling pathways altered in both cohorts. We also found that infantile-onset Pompe patient muscle had a gene expression pattern characteristic of immature or regenerating muscle, and exhibited many transcriptional markers of inflammation, despite having few overt signs of inflammatory infiltrate. Further, we identified genes exhibiting correlation between expression at baseline and response to therapy. This combined dataset can serve as a foundation for biological discovery and biomarker development to improve the treatment of Pompe disease.

Pompe disease is caused by deficiency of acid alpha-glucosidase (GAA), a lysosomal enzyme responsible for the degradation of glycogen. The resulting accumulation of lysosomal glycogen leads to vacuolation and weakness in muscle. The course of the disease is heterogeneous. Enzyme replacement therapy has been shown to reduce glycogen levels, improve morphology, and restore function and muscle strength; the response to therapy is variable but early treatment may lead to better clinical outcomes.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in the PKD1 or PKD2 gene, but cellular mechanisms of cystogenesis remain unclear. In an attempt to display the array of cyst-specific molecules and to elucidate the disease pathway, we have performed comprehensive high-throughput expression analysis of normal and ADPKD epithelia in a two-step fashion. First, we generated expression profiles of normal and cystic epithelia derived from kidney and liver using serial analysis of gene expression (SAGE). We found 472 and 499 differentially expressed genes with fivefold difference in liver and kidney libraries, respectively. These genes encode growth factors, transcription factors, proteases, apoptotic factors, molecules involved in cell-extracellular matrix interactions, and ion channels. As a second step, we constructed a custom cDNA microarray using a subset of the differentially regulated genes identified by SAGE and interrogated ADPKD patient samples. Subsequently, a set of differentially expressed genes was refined to 26 up-regulated and 48 down-regulated genes with ap value of <0.01. This study may provide valuable insights into the pathophysiology of ADPKD and suggest potential therapeutic targets.

The Hutterite population is a genetic isolate with an increased incidence of cystic fibrosis (CF). Previously we identified three CF haplotypes defined by polymorphisms flanking the CF transmembrane conductance regulator (CFTR) gene. DELTAF508 was present on one of the haplotypes in only 35% of CF chromosomes. We hypothesized that the other two CF haplotypes, one of which was the most common and the other of which is rare, each harbored different non-DELTAF508 mutations. Single-strand conformation polymorphism analysis detected a missense mutation, M1101K, in both chromosomes of a Hutterite patient carrying the two non-DELTAF508 haplotypes. M1101K appears to have originated on an uncommon CFTR allele and to be infrequent outside the Hutterite population. The presence of M1101K on two haplotypes is likely the result of a CFTR intragenic recombination which occurred since the founding, 10-12 generations ago, of the Hutterite population. The crossover was located between exons 14a and 17b, an interval of approximately 15 kbp. DELTAF508 and M1101K accounted for all of the CF mutations in patients from 16 CF families representing the three subdivisions of the Hutterite population.

Traditionally, DNA used for PCR-based diagnostic analysis has originated from white cells fractionated from whole blood. Although this method yields substantial quantities of DNA, there are some drawbacks to the procedure, including the inconvenience of drawing blood, risk of exposure to blood-borne pathogens, liquid sample handling, and the somewhat involved extraction procedure. Alternatively, DNA for genetic diagnosis has been derived from finger stick blood samples, hair roots, cheek scrapings, and urine samples. Oral saline rinses have also been used extensively as a means of collecting buccal epithelial cells as a DNA source. However, this method still requires liquid sample handling. Herein, we present our results involving the rapid extraction of DNA from buccal cells collected on cytology brushes and swabs for use in PCR reactions, specifically the multiplex amplification of 5 exons within the CFTR gene. The quality of DNA isolated from buccal cells, collected in this manner, has been sufficient to reproducibly support multiplex amplification. Cheek cell samples and the DNA prepared from them as described here are highly stable. The success rate of PCR amplification on DNA prepared from buccal cells is 99%. In a blind study comparing the analysis of 12 mutations responsible for cystic fibrosis in multiplex products amplified with DNA from both blood and buccal cell samples from 464 individuals, there was 100% correlation of results for blood and cheek cell DNA, validating the use of DNA extracted from cheek cells collected on cytology brushes for use in genetic testing.

Dinuclrotide repeat polymorphism closely linked to the cystic fibrosis (CFTR) gene Get access B. Riachards, B. Riachards Department of Genetics Disease Research, Integrated GeneticsFramingham, MA 01701, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar C. Reeves, C. Reeves Department of Genetics Disease Research, Integrated GeneticsFramingham, MA 01701, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar G.T. Horn G.T. Horn Department of Genetics Disease Research, Integrated GeneticsFramingham, MA 01701, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 19, Issue 20, 25 October 1991, Page 5798, https://doi.org/10.1093/nar/19.20.5798 Published: 25 October 1991

Journal Article Detection by PCR of the VNTR polymorphism at D4S95 Get access Bernice A. Allitto, Bernice A. Allitto Search for other works by this author on: Oxford Academic PubMed Google Scholar Glenn T. Horn, Glenn T. Horn 1Department of Human Genetics, Integrated GeneticsOne Mountain Road, Framingham, MA 01752 Search for other works by this author on: Oxford Academic PubMed Google Scholar Michael R. Altherr, Michael R. Altherr 2Department of Biological Chemistry, University of CaliforniaIrvine, CA 92717, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Brenda Richards, Brenda Richards 1Department of Human Genetics, Integrated GeneticsOne Mountain Road, Framingham, MA 01752 Search for other works by this author on: Oxford Academic PubMed Google Scholar Andrea I. McClatchey, Andrea I. McClatchey Search for other works by this author on: Oxford Academic PubMed Google Scholar John J. Wasmuth, John J. Wasmuth 2Department of Biological Chemistry, University of CaliforniaIrvine, CA 92717, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar James F. Gusella James F. Gusella * *To whom correspondence should be addressed Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 19, Issue 14, 25 July 1991, Page 4015, https://doi.org/10.1093/nar/19.14.4015-a Published: 25 July 1991

Introduction: Some segments of the human genome exhibit polymorphism due to a variable number of tandem repeats (VNTR). The enzymatic amplification of VNTR loci can allow for rapid analysis and discrimination of closely sized alleles. We report a PCR-based method for analysis of the highly polymorphic Stl4 VNTR. Source and Description: The S t l4 -1 probe is a 3 kb EcoRI fragment cloned from human DNA which detects several polymorphisms at the DXS52 loci, including a highly polymorphic VNTR RFLP revealed by Taql. Analysis of this VNTR by Southern transfer has revealed ten alleles ranging in size from 3.4—6.6 kb, with alleles as large as 15 kb in Black individuals (1, 3). The region responsible for the Taql polymorphism was sequenced from a cloned allele, and was found to be an almost perfectly duplicated 60 bp repeat (2). To assay the VNTR by PCR, amplification primers were designed on either side of the repeat. However, due to additional repetitive sequence flanking the hypervariable region, the PCR primers were not placed directly adjacent to the VNTR. As a result, amplified products include about 650 bp of flanking sequence. PCR Amplification: We used 1 /xM in each of the primers 5'GGCATGTCATCACTTCTCTCATGTT-3' and 5'-CACCACTGCCCTCACGTCACTT-3', 'standard' PCR buffer and dNTP concentrations (4), 1 /tg genomic DNA, and 24 cycles of PCR consisting of 20 sec at 94°C, 30 sec at 55°C, and 20 sec at 74°C, with a final incubation for 5 min at 74°C. Less favorable results were obtained with additional cycles. Size differences between the amplified products were found to correspond with those seen on Southern blots; Mendelian inheritance of the allelic products was also verified (data not shown). Frequency: 50 unrelated Caucasian males were analyzed using the above primers. The amplified allele sizes and frequencies observed are: 3000 bp (2%), 2900 (8%), 2400 (12%), 1690 (36%), 1630(2%), 1570(14%), 1390(10%), 1300(2%), 1220 (2%), and 700 (12%). We have also seen other rare allelic products (880, 1750, 1810 and 2100 bp) that were not present in these 50 samples. All of these products (except for die 880 bp product) are shown below. The size of the smallest product (700 bp) suggests that it represents an allele with just one 60 bp repeat; and the most frequent product of 1690 bp appears to contain 17 tandem repeats. Chromosomal Location: The DXS52 loci map to Xq26-28, and are linked to several disease genes including hemophilias A and B, fragile X syndrome, and adrenoleukodystrophy (2). The Stl4 VNTR specifically maps to Xq28 and is about 2 cM from the hemophilia A-coagulation factor VDI locus, yet its large number of alleles makes it very useful in the diagnosis of hemophilia A.

Journal Article Detection by PCR of a VNTR polymorphism at D4S43 Get access Glenn T. Horn, Glenn T. Horn Search for other works by this author on: Oxford Academic PubMed Google Scholar Andrea I. McClatchey, Andrea I. McClatchey 1Molecular Neurogenetics Laboratory, Massachusetts General HospitalChartlestown, MA 02129, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Brenda Richards, Brenda Richards Search for other works by this author on: Oxford Academic PubMed Google Scholar Marcy E. MacDonald, Marcy E. MacDonald 1Molecular Neurogenetics Laboratory, Massachusetts General HospitalChartlestown, MA 02129, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar James F. Gusella James F. Gusella 1Molecular Neurogenetics Laboratory, Massachusetts General HospitalChartlestown, MA 02129, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 19, Issue 17, 11 September 1991, Page 4772, https://doi.org/10.1093/nar/19.17.4772 Published: 11 September 1991 Article history Accepted: 21 June 1991 Published: 11 September 1991

The highly polymorphic VNTR locus pYNZ32 has been more extensively characterized, and its analysis converted to a rapid PCR-based format. DNA sequencing in the areas within and flanking the repeated segment allowed the design of specific amplification primers. The repeated region of pYNZ32 consists of an imperfectly duplicated 27-bp motif, 16 bases of which are more highly conserved. Allelic products from PCR amplification were resolved into nine different size classes ranging from approximately 1400 to 2200 bp. Additional polymorphism was revealed when the amplified products were analyzed by restriction enzyme digestion. Both the overall size variation and the internal sequence polymorphism were used to determine a heterozygosity value of 86% for YNZ32 in 50 unrelated individuals. The rapid analysis and improved resolution of amplified alleles on agarose gels, and the internal variability within YNZ32, increase its diagnostic utility as a VNTR and as a linkage marker for the nearby Huntington disease gene.

Six genetic polymorphisms, closely linked to the cystic fibrosis gene and useful in clinical linkage analysis, have been characterized and converted to a more rapid form of assay. Sequences flanking the metD (Ban I), metH (Msp I), XV-2c (Taq I), KM.19 (Pst I), MP6d-9 (Msp I), and J3.11 (Msp I) polymorphic restriction sites have been determined and used to design specific polymerase chain reaction (PCR) amplification primers and allele-specific oligonucleotide probes. All six of these polymorphisms were found to involve single-base alterations, and the XV-2c polymorphism was found to lie within an Alu repeat segment. These PCR-based tests, in conjunction with the CS.7 (Hha I) assay described elsewhere (Stanier P et al. Hum Genet 1988;80:309-10; Williams C et al. Lancet 1988;ii:102-3), provide a convenient, rapid, and reliable method of haplotype and linkage analysis, clinically useful in those situations where direct detection of mutations is not possible.