Biomedical subjects
Publications and source records attributed to M E Hodes.
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A C-to-T transition in exon 4 of the PLP gene was found in 2 affected males and two obligate carriers in a German family with Pelizaeus-Merzbacher disease. The mutation, which causes loss of an HphI site and changes amino acid 155 from threonine to isoleucine, was absent from 108 normal chromosomes. There are 5 concordances and 1 discrepancy between these results and those obtained by magnetic resonance imaging in this family.
There are extremely complicated and numerous gene expressions in mammalian brains. Each region of the brain contains a set of mRNA expressed only in that region and these mRNA would be related to presumed region of specific function. Genes preferentially expressed in cerebellum (CB) are therefore the reasonable candioate genes for site of genetic lesions in CB neurodegenerative disorders. In this study, the subtractive hybridization was introduced into screening and isolating CB specific mRNA. Testing CB and front cortex (CTX) cDNA libraries were built, which covered most low abundantly expressed mRNA. By applying over 100 fold excessive CTX and liver single strand cDNA to the CB double strand cDNA, subtractive hybridization was carried out by phenol-emulsion-reassociation-technique (PERT), in which the common expressed housekeep genes would be eluted by restrict site ligation, and CB specific cDNA flanking with EcoRI site in both ds cDNA ends could be cloned into lamda gill phage. PERT subtraction reduced CB cDNA libraries from initial 5.15 x 10(6) to remaining 2.5 x 10(3) recombinants, which were most likely CB specific expressed cDNA. Random Twenty recombinants (over 2 kb inserts) were amplified randomly by polymerase chain reaction (PCR) and were hybridized with CB cDNA and liver cDNA probes in dot blot. 14 inserts positively hybridized with CB cDNA probe and only 3 inserts showed signals with liver cDNA probe. Colony blot presented similar results. 10 cDNA clones which affinited to CB cDNA probe were selected for the Northern blot. In five tissures, 2 cDNA clones binded only in CB mRNA channel, 6 were proved preferential expression in CB and low abundant expression in 4 other tissues, 1 clone was a housekeep gene and 1 could not be detected in all tissues. The partially sequencing of clone PC7 and PD8 were introduced into Computer Gene Bank Data Base. No homologous complements were found between these two clones and over ten thousands genes that have been sequenced. The two clones were newly reported. According to our experiment, we believe that: 1) CB specifically expressed mRNAs are much less than expected comparing with other brain regions; 2) most of CB mRNA expressions are in a preferential way other than unique expression.
We have developed a highly sensitive and rapid coupled reverse transcription-polymerase chain reaction (RT-PCR) technique for detection of alpha-amylase-encoding gene transcripts and for distinguishing between the human salivary (AMY1) and pancreatic (AMY2) gene transcripts. The two genes are 93-94% homologous. However, the AMY1 gene has an additional exon known as exon S, and an extra 32 bp in exon 1. Genotyping of the different AMYs by RT-PCR was based on this unique feature of the AMY1 mRNA sequence. Detection of AMY gene (AMY1 and AMY2) transcripts in cellular RNA was achieved with a set of primers common to both human AMY1 and AMY2 genes and derived from the exon 3-4 regions. In contrast, AMY1 gene transcripts were distinguished from the pancreatic AMY2 gene transcripts by use of primers specific to the exon S-1 regions of the AMY1 gene. To distinguish AMY1 transcripts from a mixture of AMY1 and AMY2, use was made of the differences in the ethidium bromide-stained agarose gel patterns obtained after digestion of the amplified exon 3-4 fragments with TaqI. AMY gene transcripts were detectable by autoradiography in RT-PCR amplified DNA obtained from as little as 5 pg of human pancreatic or parotid total RNA. A comparison of sensitivity of Northern blotting vs. RT-PCR suggested that the RT-PCR method is about 3-6 x 10(3)-fold more sensitive than Northern blotting in detecting AMY gene transcripts in human pancreatic total RNA.
We studied the distribution of alpha-amylase mRNA in normal dog tissues by northern blotting (NB) and reverse transcription-polymerase chain reaction (RT-PCR) with human pancreatic (AMY2) and salivary (AMY1) alpha-amylase cDNA-specific primers. Analysis of poly(A+) RNA from various normal tissues by NB indicated the presence of detectable levels of alpha-amylase mRNA transcripts only in pancreas. Dot-blot analysis of DNA amplified with primers common to both (human) isoamylase mRNAs showed presence of alpha-amylase gene transcripts not only in pancreas but also in liver, small intestine, large intestine and fallopian tube. Traces of amylase gene transcripts were also observed in ovary, uterus and lung. Interestingly, amylase transcripts were not detectable in the parotid gland by NB or RT-PCR. We have also localized alpha-amylase mRNA transcripts to dog pancreas by in situ transcription and in situ hybridization. Our results suggest that there is high degree of homology between the alpha-amylase mRNA sequences in dog and human at least in the exon 3-4 regions of the human gene.
Pelizaeus-Merzbacher disease (PMD) is a human X chromosome-linked dysmyelination disorder of the central nervous system for which the genetic defect has not yet been established. The jimpy mutation jp of the mouse is an X chromosome-linked disorder of myelin formation. The mutation is at an intron/exon splice site in the mouse gene for proteolipid protein (PLP). With the jimpy mouse mutation as a precedent, we focused our attention on the human PLP gene, which is found at Xq22. The polymerase chain reaction was used to amplify the exons of the PLP gene of an affected male from a large Indiana PMD kindred. DNA sequencing showed a C----T transition at nucleotide 40 of the second exon. An affected third cousin also showed this sequence variation, while two unaffected male relatives (sons of an obligate carrier female) had the normal cytidine nucleotide. Allele-specific oligonucleotides were used to generate data for linkage studies on the above mentioned PMD kindred. Our results show tight linkage (theta = 0) of PMD to PLP with a lod (logarithm of odds) score of 4.62. In six other unrelated PMD kindreds, only the normal-sequence oligonucleotide hybridized, which indicates genetic heterogeneity. The radical nature of the predicted amino acid change (proline to leucine), suggests that the PMD-causing defect may have been delineated in one kindred.
X-linked nephrogenic diabetes insipidus (NDI) was segregating in a large Indiana family. It was tested for linkage of the NDI gene to X-chromosome molecular markers. Maximum lod scores of 3.15 and 3.01 (theta = 0) obtained for the molecular markers F8A (F8C) and DXS15 (DX13) respectively, indicate that the NDI gene is located in Xq28. A lod score of 3.61 (theta = 0) was obtained with multipoint linkage analysis of F8A and DXS15.
Salivary alpha-amylase (EC 3.2.1.1) is the major protein component of human parotid gland secretion. We studied amylase gene structure and expression in tissue from a series of normal and neoplastic parotid glands by Southern blot analysis, in situ hybridization, and immunohistochemistry. Thirty-two tumors were examined. Southern blot analysis of DNA extracted from a Warthin tumor, an adenoid cystic carcinoma, and a mucoepidermoid carcinoma showed no evidence of structural rearrangement of amylase genes. Eleven parotid Warthin tumors were negative for amylase protein and mRNA by immunocytochemistry and in situ hybridization. One pleomorphic adenoma in the group of 10 examined showed focal staining for amylase protein, although amylase mRNA could not be demonstrated in the same population of cells by in situ hybridization in serial tissue sections. Five mucoepidermoid carcinomas and three acinar cell carcinomas were devoid of amylase protein and mRNA. Normal parotid tissue obtained from all patients studied revealed abundant acinar cell amylase mRNA and protein. In situ hybridization, in conjunction with immunocytochemistry, allows precise cellular localization of mRNA and protein, thereby establishing the site of production of specific transcripts. We conclude that the interruption in amylase gene expression in parotid gland neoplasms occurs at the transcriptional level.
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We encountered six female infants with a specific pattern of developmental abnormalities of the urogenital and lower intestinal tracts. The anomalies included ambiguous genitalia, lack of perineal openings, and müllerian and urinary tract anomalies. Each patient had normal female chromosomes and normal adrenal gland function. We believe that this combination of anomalies represents a recognizable and specific sequence that is due to a failure of migration to and/or fusion of the urorectal septum with the cloacal membrane. This, in turn, we postulate, leads to persistence of the cloaca and cloacal membrane and failure of normal differentiation of the external genitalia. Persistence of the cloacal membrane results in absence of the urethral and vaginal openings and an imperforate anus. We propose calling this entity the urorectal septum malformation sequence.
Two sibs who had hypesthetic corneas, absence of the peripapillary choriocapillaris and retinal pigment epithelium, sensorineural hearing loss bilaterally, persistent ductus arteriosus, moderate mental retardation, and unusual facial appearance are described. Their mother had mild to moderate sensorineural hearing loss, retinal changes and similar facial features. The differential diagnosis is discussed. We believe this is a clinically distinct syndrome with autosomal dominant inheritance.
Long-term storage of DNA is required for a number of genetic studies; prior to extraction, blood samples may be subject to elevated temperatures for variable intervals. We have studied the effect of temperatures ranging from -70 degrees C to +65 degrees C on human blood and on DNA extracted from it. DNA in solution stored at ambient temperatures up to 37 degrees C for 6 months was digestible by three different restriction endonucleases, whereas storage at 45 degrees C is deleterious after 6-7 weeks. DNA can be extracted from blood samples stored at -70 degrees C for at least 2 months or at 23 degrees C for a week or more, but blood stored at these temperatures may yield less high-molecular-weight DNA. Cell pellets from which plasma has been removed also can serve as a source of DNA. Isolated DNA stored dry for years (up to 30) is difficult to dissolve and may appear degraded, but a sample stored dry for 13 years and then in solution at -20 degrees C for 7 years appeared to be intact.
Roentgencephalometric findings are presented for a family with an unusual facial morphology. Twenty measurements defining the size and shape of major anatomic areas of the head and face were taken. The measurements were transformed into Z-scores. Using the sigma z (i.e., standard deviation of the Z-scores) value, an estimate of craniofacial pattern deviation from the norms was made for each family member. All members of this family have highly aberrant, i.e., dysmorphic, craniofacial pattern profiles. Familial as well as syndromic craniofacial similarities vs. dissimilarities between Z-score values of pairs of family members were assessed by the Pearson's correlation coefficient rz. Results of correlation coefficients demonstrate a high level (far in excess of the expected value of rz 0.50) of craniofacial pattern profile (CFPP) similarity between sibs and between sibs and their mother. This strongly suggests that the two sibs and their mothers are the carrier of the same genetic syndrome.
An Indiana family segregating a syndrome of X-linked mental retardation and skeletal anomalies was tested for linkage of the mutant gene to X-chromosome molecular markers. Lod scores of 3.27 and 3.06 (theta = 0) for the molecular probes St14-1 (DXS52) and Dx13 (DXS15), respectively, indicate that the disease gene is located in the terminal portion of Xq.
The distribution of human salivary amylase mRNA was studied by in situ hybridization to a [32P]-labeled amylase cDNA probe. Amylase mRNA was localized to the apical portion of acinar cells in frozen sections of human parotid salivary gland. No hybridization was noted in ductal cells, skeletal muscle, or in connective tissue. These results were consistent with immunohistochemical localization of amylase. The technique of in situ hybridization was modified to permit localization of amylase mRNA in variously fixed, paraffin-embedded parotid glands. Although the hybridization signal decreased with all fixatives, the pattern of localization paralleled that obtained with frozen sections. No advantage was noted in fixation with ethanol-acetic acid or Bouin solution over routine fixation with formalin. These results have important implications for researchers interested in studies of gene expression. We have demonstrated that routinely fixed paraffin blocks of human tissue can be used for cellular localization of specific mRNA. In coordination with immunocytochemistry, in situ hybridization offers a powerful tool for studies of mRNA and protein expression in individual cells.
Phosphodiesterase I (PDE I) is an exonuclease capable of hydrolyzing a variety of phosphate ester and pyrophosphate bonds. Cell fractionation and histochemical studies in animal tissues have localized PDE I in the plasma membrane of various epithelia. This suggests a role for the enzyme in active transport. Distribution of PDE I in human tissues has not previously been studied. We have produced a polyclonal antiserum to bovine intestinal PDE I and have demonstrated crossreactivity with the human intestinal enzyme. This polyclonal antiserum was used in PAP immunocytochemistry to localize immunoreactive PDE I in a variety of human tissues. Localization was prominent in the gastrointestinal tract, including the cytoplasm of gastric mucosa parietal cells, cytoplasm of surface epithelium and isolated crypt cells in small intestine, and the colonic epithelial cytoplasm and brush border. Parotid gland acinar cells and scattered ductal cells showed positive cytoplasmic staining. Acinar and scattered pancreatic islet cells contained immunoreactive PDE I, as did Kupffer cells of the liver sinusoids. Immunoreactive PDE I was found in all vascular endothelia. The epithelium of the urinary tract showed extensive immunoreactivity. This included the distal convoluted and collecting tubules of the kidney, and ureteral and bladder urothelium. In previous histochemical studies of animal tissues, no evidence of PDE I activity was noted in male or female reproductive tract. In this study, immunoreactive PDE I was localized to human Sertoli cells and to basal epithelium of the epididymis and prostate acini. Fallopian tube epithelium of female reproductive tract also demonstrated immunoreactive PDI I, as did several cell types in term placenta. Our immunocytochemical results with human tissues differ significantly from previous histochemical studies in animal tissues, principally in the genitourinary system. This may be due in part to the different detection systems employed as well as the higher sensitivity of the immunoperoxidase technique. This underscores the importance of adjunct techniques in tissue surveys. The widespread epithelial distribution of immunoreactive PDE I detected by this polyclonal antibody implies an integral role in cell function, probably in active transport.
Three generations of a family exhibit a unique syndrome of X-linked ataxia, pyramidal tract signs, and adult-onset dementia. Initial signs, manifested by 2 to 3 years of age, are delayed walking and tremor. During their teens, the patients develop mild but progressive ataxia and pyramidal tract signs. Memory problems in the third decade initiate a progressive dementia, leading to death in the sixth decade. Laboratory investigations failed to disclose a biochemical basis for the syndrome. Preliminary molecular linkage studies have been conducted, and although the specific position of the responsible gene on the X chromosome has not yet been determined, the q26-qter region and much of the p arm are unlikely sites for this gene. The linkage studies are continuing.