Characterization of the genetic lesions causing Types A and B Niemann-Pick Disease in the Ashkenazi Jewish population.
Item
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Title
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Characterization of the genetic lesions causing Types A and B Niemann-Pick Disease in the Ashkenazi Jewish population.
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Identifier
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AAI9304692
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identifier
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9304692
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Creator
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Lev-Ran, Orna.
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Contributor
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Adviser: Edward H. Schuchman
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Date
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1992
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Genetics | Health Sciences, Medicine and Surgery | Health Sciences, General
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Abstract
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Acid sphingomyelinase (ASM; sphingomyelin phosphodiesterase, E.C. 3.1.4.12) is the lysosomal enzyme responsible for the degradation of the phospholipid sphingomyelin. A deficiency of this enzyme results in Types A and B Niemann-Pick Disease (NPD). This thesis describes the isolation and characterization of the cDNA and genomic sequences encoding ASM and the identification of three genetic lesions causing NPD. Human urinary ASM was purified and two groups of cDNA clones were isolated using oligonucleotide mixtures based on tryptic peptide sequences. DNA sequencing studies revealed that group-2 cDNAs did not have an internal 172 bp sequence, but had instead an in-frame 40 bp sequence which was not present in group-1 cDNAs. These initial results suggested the occurrence of alternative RNA splicing, a finding that was confirmed when the ASM genomic region was isolated and sequenced. The ASM gene was small ({dollar}\sim{dollar}5 kb) and contained six exons. The alternatively spliced 172 bp sequence was encoded by exon 3, whereas the 40 bp sequence was located at the 5{dollar}\sp\prime{dollar} end of intron 2. An Alu 1 element in the reverse orientation was identified in intron 2. The regulatory region was GC rich and contained putative promoter elements including Sp1, TATA, CAAT, NF-1 and AP-1 binding sites.;In order to identify the molecular lesions causing Types A and B NPD, reverse-transcribed total RNA, and/or genomic DNA from Ashkenazi Jewish patients were amplified by the polymerase chain reaction (PCR) using ASM-specific primers. In Type A patients two mutations have been identified: a G to T transversion of nucleotide 3599, (designated R496L), and a T to C transition at nucleotide 1370 (designated L302P). In Type B patients, a three base in-frame deletion of nucleotides 3933-3935, (designated {dollar}\Delta{dollar}R608) was identified. Screening other NPD patients and normal individuals with allele-specific oligonucleotides (ASOs) excluded the possibility that these nucleotide changes were common polymorphisms and revealed that the R496L and the L302P mutations occurred in about 32% and 24% of the Ashkenazi Jewish NPD Type A alleles, respectively. The R496L mutation also occurred as one allele in an Ashkenazi Jewish Type B patient and the second allele of this patient was the {dollar}\Delta{dollar}R608 mutation. This small deletion was homoallelic in another unrelated Ashkenazi Jewish Type B patient, but was not found in Type A patients. To provide additional evidence for the authenticity of these mutations, the mutant cDNAs constructs were transiently expressed in COS-1 cells. As expected, they did not express catalytically active ASM. These findings should facilitate molecular carrier detection and prenatal diagnosis for NPD in the Ashkenazi Jewish population and provide initial genotype/phenotype correlations for this disease.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
