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P I Patel

Publications and source records attributed to P I Patel.

At least 19 recordsLinked to original sources
PubMed2001

Identification of a positive regulatory element in the myelin-specific promoter of the PMP22 gene.

Over- and under expression of the 22 kDa peripheral myelin protein (PMP22) results in dysmyelinating peripheral neuropathies, such as Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy, with the liability to pressure palsies (HNPP). Expression of the PMP22 gene is driven by two alternative promoters, P1 and P2, with transcripts originating from P1 associated with peripheral nerve myelination by Schwann cells. Transient transfections of constructs containing P1 (3.5 kb) or P2 (2.5 kb) resulted in high levels of reporter gene expression in the RT4-D6P2T schwannoma cell line. Serial deletions of P1 revealed that region P1-A (-105 to -43), situated upstream of the minimal promoter, contained a positive regulatory element. The 62 bp P1-A region conferred in cis a sevenfold increase in expression of luciferase driven by a heterologous promoter in an orientation-dependent manner. Interspecies comparison of the P1-A region revealed a 98% degree of identity between the human, mouse, and rat sequences. A prominent sequence-dependent DNA-protein complex (C-I) was detected in electrophoretic mobility shift assays with P1-A using RT4-D6P2T nuclear extract and was localized to a minimal 21 bp region within P1-A. Site-directed mutagenesis of this region revealed nucleotides at positions -46 to -43 as being necessary for formation of C-I. Functional analysis of the mutated P1-A element indicated that positions -46 and -45 were essential for transactivation mediated by this element. Characterization of the transacting factor(s) interacting with this key regulatory element will shed light on its role in regulating peripheral nerve myelination.

Base Sequence↗
PubMed2001

Genomic organisation of the approximately 1.5 Mb Smith-Magenis syndrome critical interval: transcription map, genomic contig, and candidate gene analysis.

Smith-Magenis syndrome (SMS) is a multiple congenital anomalies/mental retardation syndrome associated with an interstitial deletion of chromosome 17 involving band p11.2. SMS is hypothesised to be a contiguous gene syndrome in which the phenotype arises from the haploinsufficiency of multiple, functionally-unrelated genes in close physical proximity, although the true molecular basis of SMS is not yet known. In this study, we have generated the first overlapping and contiguous transcription map of the SMS critical interval, linking the proximal 17p11.2 region near the SMS-REPM and the distal region near D17S740 in a minimum tiling path of 16 BACs and two PACs. Additional clones provide greater coverage throughout the critical region. Not including the repetitive sequences that flank the critical interval, the map is comprised of 13 known genes, 14 ESTs, and six genomic markers, and is a synthesis of Southern hybridisation and polymerase chain reaction data from gene and marker localisation to BACs and PACs and database sequence analysis from the human genome project high-throughput draft sequence. In order to identify possible candidate genes, we performed sequence analysis and determined the tissue expression pattern analysis of 10 novel ESTs that are deleted in all SMS patients. We also present a detailed review of six promising candidate genes that map to the SMS critical region.

Abnormalities, Multiple↗
PubMed2001

Competitive binding of triplex-forming oligonucleotides in the two alternate promoters of the PMP22 gene.

Overexpression of the 22-kDa peripheral myelin protein (PMP22) causes the inherited peripheral neuropathy, Charcot-Marie-Tooth disease type 1A (CMT1A). In an attempt to alter PMP22 gene expression as a possible therapeutic strategy for CMT1A, antiparallel triplex-forming oligonucleotides (TFO) were designed to bind to purine-rich target sequences in the two PMP22 gene promoters, P1 and P2. Target region I in P1 and region V in P2 were also shown to specifically bind proteins in mammalian nuclear extracts. Competition for binding of these targets by TFO vs. protein(s) was compared by exposing proteins to their target sequences after triplex formation (passive competition) or by allowing TFO and proteins to simultaneously compete for the same targets (active competition). In both formats, TFO were shown to competitively interfere with the binding of protein to region I. Oligonucleotides directed to region V competed for protein binding by a nontriplex-mediated mechanism, most likely via the formation of higher-order, manganese-destabilizable structures. Given that the activity of the P1 promoter is closely linked to peripheral nerve myelination, TFO identified here could serve as useful reagents in the investigation of promoter function, the role of PMP22 in myelination, and possibly as rationally designed drugs for the therapy of CMT1A. The nontriplex-mediated action of TFO directed at the P2 promoter may have wider implications for the use of such oligonucleotides in vivo.

Animals↗
PubMed2000

Human frataxin maintains mitochondrial iron homeostasis in Saccharomyces cerevisiae.

Frataxin is a nuclear-encoded mitochondrial protein widely conserved among eukaryotes. Human frataxin (fxn) is severely reduced in Friedreich ataxia (FRDA), a frequent autosomal recessive neuro- and cardio-degenerative disease. Whereas the function of fxn is unknown, the yeast frataxin homolog (Yfh1p) has been shown to be involved in mitochondrial iron homeostasis and protection from free radical toxicity. Evidence of iron accumulation and oxidative damage in cardiac tissue from FRDA patients suggests that fxn may have a similar function, but whether yeast and human frataxin actually have interchangeable roles in mitochondrial iron homeostasis is unknown. We show that a wild-type FRDA cDNA can complement Yfh1p-deficient yeast (yfh1 delta) by preventing the mitochondrial iron accumulation and oxidative damage associated with loss of Yfh1p. We analyze the functional effects of two FRDA point mutations, G130V and W173G, associated with a mild and a severe clinical presentation, respectively. The G130V mutation affects protein stability and results in low levels of mature (m) fxn, which are nevertheless sufficient to rescue yfh1 delta yeast. The W173G mutation affects protein processing and stability and results in severe m-fxn deficiency. Expression of the FRDA (W173G) cDNA in yfh1 delta yeast leads to increased levels of mitochondrial iron which are not as elevated as in Yfh1p-deficient cells but are above the threshold for oxidative damage of mitochondrial DNA and iron-sulfur centers, causing a typical yfh1 delta phenotype. These results demonstrate that fxn functions like Yfh1p, providing experimental support to the hypothesis that FRDA is a disorder of mitochondrial iron homeostasis.

Cell Line↗
PubMed2000

Glucocorticosteroids stimulate the activity of the promoters of peripheral myelin protein-22 and protein zero genes in Schwann cells.

To better understand the mechanism by which glucocorticosteroids (GLUC) could enhance myelination in the PNS, cultured rat Schwann cells were transiently transfected with reporter constructs in which luciferase expression was controlled by the promoter region of either the peripheral myelin protein-22 (PMP22) or the protein zero (P(0)) genes. GLUC stimulated the activity of the P(0) promoter and the PMP22 promoters 1 and 2. The effect of GLUC was specific as estradiol and testosterone did not activate the promoters. The antagonist RU486 did not abolish the effect of GLUC, but instead stimulated promoter activities by itself. In the mammary carcinoma cell line 34i, which expresses GLUC receptors, GLUC did not stimulate the P(0) and PMP22 promoters while the promoter of the mouse mammary tumor virus was strongly activated. Thus, the activation by GLUC of the promoter activities of two peripheral myelin protein genes is Schwann cell-specific.

Age Factors↗
PubMed2000

Very late-onset Friedreich ataxia despite large GAA triplet repeat expansions.

BACKGROUND: Most patients with Friedreich ataxia (FRDA) have abnormal GAA triplet repeat expansions in both X25 genes. The size of the GAA expansion in the shorter of the 2 expanded alleles correlates significantly with parameters of clinical severity and is inversely related to the age at onset. OBJECTIVES: To describe the clinical and molecular genetic findings in a patient with very late-onset FRDA and to review the literature. PATIENT AND METHODS: A 58-year-old white woman with mild progressive gait disturbance of 15 years' duration whose examination revealed mild incoordination was analyzed for mutations in the X25 gene. A combination of long-range polymerase chain reaction and genomic Southern blot analyses were used to identify GAA expansions in intron 1 of the X25 gene. To uncover evidence of somatic variability in triplet repeat length, DNA isolated from several tissue samples was similarly analyzed. Single-strand conformational polymorphism analysis was used to screen for mutations spanning the entire coding sequence of frataxin and all intron-exon junctions of the X25 gene. RESULTS: DNA isolated from blood leukocytes revealed GAA triplet repeat expansions in both X25 genes, which were estimated to contain 835 and 1200 repeats. Similar expansions were detected in DNA isolated from lymphoblasts, fibroblasts, buccal cells, and sural nerve, with estimated mean (+/- SD) lengths of the shorter and longer expansions being 854 (+/-69) and 1283 (+/-72) triplets, respectively. A review of reported cases of late-onset Friedreich ataxia (25-39 years) and very late-onset Friedreich ataxia (> or =40 years) demonstrated that this is the first instance of a patient presenting with very late-onset FRDA despite carrying more than 800 GAA repeats in both expanded X25 alleles. CONCLUSIONS: This unique case of very late-onset FRDA highlights a limitation in our ability to accurately predict the phenotype in FRDA based solely on the size of the GAA expansion. Other genetic or environmental factors may significantly modify disease severity in FRDA.

Age of Onset↗
PubMed2000

Clinical, radiographic, and genetic evaluation of a novel form of autosomal-dominant oligodontia.

A frameshift mutation recently identified within the paired domain of the transcription factor, PAX9, has been linked to a unique form of oligodontia in a single, multigenerational family (Stockton et al., 2000). We now describe the phenotypic and segregation analyses of this remarkable kindred, the initial approach taken to identify a candidate gene involved in this form of oligodontia, and the power of this single-family pedigree to generate significant linkage in a genome search. Of the 43 family members enrolled in this study, 21 individuals were affected with several congenitally missing permanent teeth. The pattern of inheritance of the oligodontia trait suggested the involvement of a single gene bearing a dominant mutation. To various degrees, affected members lacked permanent first, second, and third molars in all four quadrants. Several individuals with missing molars also lacked second premolars- most commonly, maxillary second premolars and mandibular central incisors. To the best of our knowledge, this pattern of non-syndromic, familial tooth agenesis has not been previously described in the literature. Since a missense mutation in the homeobox gene, MSX1, was previously linked to tooth agenesis in a single family lacking second premolars and third molars, we performed a mutational analysis of MSX1 by PCR. The absence of a mutation in exons 1 and 2 of MSX1 suggested that allelic mutations in the coding region of MSX1 are not associated with this phenotypically distinct form of oligodontia. Computer simulation of linkage analysis further proved that this pedigree alone was sufficient to generate a significant result for a total genome scan.

Adolescent↗
PubMed1999

Hemizygosity for the COP9 signalosome subunit gene, SGN3, in the Smith-Magenis syndrome.

Smith-Magenis syndrome (SMS) is a multiple congenital anomaly/mental retardation syndrome associated with an interstitial deletion of chromosome band 17p11.2. The critical region is extremely gene-rich and spans approximately 1.5-2.0 Mb of DNA. Here we report the localization and partial characterization of the gene for subunit 3 of the COP9 signalosome, SGN3. SGN3 maps to the distal portion of the SMS critical interval, between SREBF1 and cCI17-638. We assessed the potential effect of haploinsufficiency of SGN3 in SMS patient lymphoblastoid cell lines through transfection studies and western analysis. Our results indicate that the COP9 signalosome assembles properly in these cells and appears to have normal expression and a kinase function intact. However, because the role of the COP9 signalosome in embryogenesis or differentiation is still uncertain, we cannot rule out the involvement of this gene in the Smith-Magenis syndrome.

Abnormalities, Multiple↗
PubMed1999

Somatic sequence variation at the Friedreich ataxia locus includes complete contraction of the expanded GAA triplet repeat, significant length variation in serially passaged lymphoblasts and enhanced mutagenesis in the flanking sequence.

The vast majority of Friedreich ataxia patients are homozygous for large GAA triplet repeat expansions in intron 1 of the X25 gene. Instability of the expanded GAA repeat was examined in 23 chromosomes bearing 97-1250 triplets in lymphoblastoid cell lines passaged 20-39 times. Southern analyses revealed 18 events of significant changes in length ranging from 69 to 633 triplets, wherein the de novo allele gradually replaced the original over 1-6 passages. Contractions and expansions occurred with equal frequency and magnitude. This behavior is unique in comparison with other large, non-coding triplet repeat expansions [(CGG)(n)and (CTG)(n)] which remain relatively stable under similar conditions. We also report a rare patient who, having inherited two expanded alleles, showed evidence of contracted GAA repeats ranging from nine to 29 triplets in DNA from two independent peripheral blood samples. The GAA triplet repeat is known to adopt a triplex structure, and triplexes in transcribed templates cause enhanced mutagenesis. The poly(A) tract and a 135 bp sequence, both situated immediately upstream of the GAA triplet repeat, were therefore examined for somatic mutations. The poly(A) tract showed enhanced instability when in cis with the GAA expansion. The 135 bp upstream sequence was found to harbor a 3-fold excess of point mutations in DNA derived from individuals homozygous for the GAA triplet repeat expansion compared with normal controls. These data are likely to have important mechanistic and clinical implications.

Base Sequence↗
PubMed1998

Human 12(R)-lipoxygenase and the mouse ortholog. Molecular cloning, expression, and gene chromosomal assignment.

Expressed sequence tag information was used to clone the full-length sequence for a new human lipoxygenase from the B cell line CCL-156. A related mouse sequence with 83% nucleotide identity to the human sequence was also cloned. The human lipoxygenase, when expressed via the baculovirus/insect cell system produced an approximately 80-kDa protein capable of metabolizing arachidonic acid to a product identified as 12-hydroxyeicosatetraenoic acid by mass spectrometry. Using chiral phase-high performance liquid chromatography, the product was identified as >98% 12(R)-hydroxyeicosatetraenoic acid as opposed to the S-stereoisomer formed by all other known mammalian lipoxygenases. The single copy human 12(R)-lipoxygenase gene was localized to the chromosome 17p13 region, the locus where most other lipoxygenase genes are known to reside. By reverse transcription-polymerase chain reaction, but not by Northern blot, analysis the 12(R)-lipoxygenase mRNA was detected in B cells and adult skin. However, the related mouse lipoxygenase mRNA was highly expressed in epidermis of newborn mice and to a lesser extent in adult brain cortex. By in situ hybridization the mouse lipoxygenase gene was demonstrated to be temporally and spatially regulated during embryogenesis. Expression was induced at embryonic day 15.5 in epidermis, nasal epithelium, and surface of the tongue. These results broaden the mammalian lipoxygenase family to include a 12(R)-lipoxygenase whose biological function remains to be determined.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1998

Smith-Magenis syndrome resulting from a de novo direct insertion of proximal 17q into 17p11.2.

We report on a de novo intrachromosomal rearrangement of chromosome 17 in a patient with Smith-Magenis syndrome (SMS). This 11-year-old boy had short stature, midfacial hypoplasia, and behavioral problems characteristic of this syndrome. Cytogenetic analysis showed that the proximal long arm of a chromosome 17 (q11.2-q21.3) was inserted into its short arm at p11.2, resulting in an apparent deletion of the SMS critical region [ins(17)(p11.2q11.2q21.3)]. Fluorescence in situ hybridization studies (FISH) demonstrated that the inserted segment included both the ERBB2 and RARA loci, and dual color hybridizations defined the insertion as direct, with ERBB2 located more proximally on the short arm of the der(17). The resulting deletion of the short arm included loci c130G3, D17S258, FLI, and D17S29, while the more proximal loci, D17S446 and D17S58, remained apparently unaffected and in their native locations. The CMT1A locus also remained in its native location on the short arm of the metacentric der(17) chromosome. A de novo intrachromosomal insertional rearrangement of chromosome 17 in a case of SMS has not been reported previously and further illustrates the instability of this chromosomal region.

Abnormalities, Multiple↗
PubMed1998

Gene for topoisomerase III maps within the Smith-Magenis syndrome critical region: analysis of cell-cycle distribution and radiation sensitivity.

Smith-Magenis syndrome (SMS) is caused by an interstitial deletion of chromosome band 17p11.2 averaging 4-5 Mb. This deletion is likely to contain a large number of genes, each of which could potentially contribute toward the clinical phenotype. We report that the gene for topoisomerase III (hTOP3) is commonly deleted in SMS patients and maps between D17S447 and D17S258 on the short arm of chromosome 17. Cellular studies of SMS patient lymphoblasts and their respective parental cell lines were undertaken to determine the consequences of haploinsufficiency of hTOP3. Our studies indicate that hemizygosity for hTOP3 does not appreciably affect cell-cycle kinetics or activation of ionizing radiation-sensitive cell-cycle checkpoints. Furthermore, the induction of apoptosis in response to ionizing radiation in SMS and parental cells was similar. Our studies suggest that haploinsufficiency of hTOP3 does not have a major impact on the behavior of cells from SMS patients and may not play a significant role in the SMS phenotype.

Abnormalities, Multiple↗
PubMed1998

A mild case of Friedreich ataxia: lymphocyte and sural nerve analysis for GAA repeat length reveals somatic mosaicism.

Friedreich ataxia (FRDA) is an autosomal recessive, neurodegenerative disease, characterized by progressive gait and limb ataxia, dysarthria, lower-limb areflexia, Babinski sign, loss of position and vibration senses, cardiomyopathy, and carbohydrate intolerance. It is the most common inherited ataxia, and is associated with a GAA triplet repeat expansion in the first intron of the X25 gene on the long arm of chromosome 9. We present a case whose clinical diagnosis was initially confounded by the mildness of the ataxic phenotype and a family history of multiple sclerosis. Evaluation of the X25 gene revealed that the patient was homozygous for the GAA triplet repeat expansion, pathognomonic of FRDA. Investigation of her sural nerve biopsy revealed a significantly smaller expansion size, constituting the first direct demonstration of somatic mosaicism involving the nervous system in FRDA. We speculate that a similar contraction in pathologically affected tissues could be the molecular basis for the mildness of the ataxia.

Adult↗