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At least 847 records · Page 47Linked to original sources
PubMed2004

Pyrophosphorolysis by Type II DNA polymerases: implications for pyrophosphorolysis-activated polymerization.

We find that Type II DNA polymerases can catalyze pyrophosphorolysis, the reverse reaction of DNA polymerization. This property is applied utilizing pyrophosphorolysis-activated polymerization (PAP), a method of nucleic acid amplification using serial coupling of pyrophosphorolysis and polymerization. PAP can be used for ultrarare allele detection (detection of minimal residual disease and cancer risk assessment through measurement of mutation load) and for microarray-based scanning for unknown mutations. Herein, we show that Type II DNA polymerases efficiently catalyze template-dependent pyrophosphorolysis to activate oligonucleotides blocked at their 3' termini with acyclonucleotides in which a 2-hydroxyethoxymethyl group substitutes for the 2'-deoxyribofuranosyl sugar. Type II archeon DNA polymerases Vent (exo-) and Pfu (exo-) can be utilized for PAP or a bidirectional form of PAP with acyclonucleotide-blocked oligonucleotides, but not with dideoxynucleotide-blocked oligonucleotides. In contrast, a Type I DNA polymerase, TaqFS, can utilize either acyclonucleotide-blocked or dideoxynucleotide-blocked oligonucleotides. These findings expand the potential of nascent PAP technology.

Alleles↗
PubMed2000

A sensitive scanning technology for low frequency nuclear point mutations in human genomic DNA.

Knowledge of the kinds and numbers of nuclear point mutations in human tissues is essential to the understanding of the mutation mechanisms underlying genetic diseases. However, nuclear point mutant fractions in normal humans are so low that few methods exist to measure them. We have now developed a means to scan for point mutations in 100 bp nuclear single copy sequences at mutant fractions as low as 10(-6). Beginning with about 10(8) human cells we first enrich for the desired nuclear sequence 10,000-fold from the genomic DNA by sequence-specific hybridization coupled with a biotin-streptavidin capture system. We next enrich for rare mutant sequences 100-fold against the wild-type sequence by wide bore constant denaturant capillary electrophoresis (CDCE). The mutant-enriched sample is subsequently amplified by high fidelity PCR using fluorescein-labeled primers. Amplified mutant sequences are further enriched via two rounds of CDCE coupled with high fidelity PCR. Individual mutants, seen as distinct peaks on CDCE, are then isolated and sequenced. We have tested this approach by measuring N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced point mutations in a 121 bp sequence of the adenomatous polyposis coli gene (APC) in human lymphoblastoid MT1 cells. Twelve different MNNG-induced GC-->AT transitions were reproducibly observed in MNNG-treated cells at mutant fractions between 2 x 10(-6) and 9 x 10(-6). The sensitivity of this approach was limited by the fidelity of Pfu DNA polymerase, which created 14 different GC-->TA transversions at a mutant fraction equivalent to approximately 10(-6) in the original samples. The approach described herein should be general for all DNA sequences suitable for CDCE analysis. Its sensitivity and capacity would permit detection of stem cell mutations in tissue sectors consisting of approximately 10(8) cells.

Adenomatous Polyposis Coli↗
PubMedDate not supplied

Scanning of beta-globin gene for identification of beta-thalassemia mutation in Romanian population.

Beta-thalassemia is uncommon (0.5%) in the Romanian population, but it must be considered in the differential diagnosis of hypochromic anemia. The molecular characterization of beta-thalassemia is absolutely necessary for molecular diagnosis, as well as any genetic epidemiological study in this region. Molecular analyses consist of mutation detection by molecular scanning of beta-globin gene. This gene has 3 exons and 2 introns, involved in beta-thalassemic pathogenesis. Clinical application of DNA analysis on beta-thalassemic chromosomes allowed characterization of 29 persons with different beta-thalassemia mutations among 58 patients with anemia. The experimental strategy was based on sequential PCR amplification of most of the beta-globin gene and running on denaturing gradient gel electrophoresis of amplification products. Definitive characterization of mutations in samples identified with shifted DGGE patterns was performed ARMS-PCR and/or PCR-restriction enzyme analysis methods. Eight different beta-thalassemia alleles were identified, the most common being IVS I-110 (G-A) and cd 39 (C-T). Comparison of overall frequency of mutations in the neighboring countries, shows that these results are in the frame of overall distribution of these mutations in Mediterranean area, especially in Greece and in Bulgaria. Molecular diagnosis is useful for differentiating mild from severe alleles, for genetic counseling, as well as for mutation definition in carriers, identified by hematological analysis necessary for prenatal testing and genetic counseling.

Adolescent↗
PubMed1998

Rapid detection of genetic mutations using the chemiluminescent hybridization protection assay (HPA): overview and comparison with other methods.

The detection of genetic mutations is of paramount importance for the study, diagnosis, and treatment of human genetic disease. Methods of detection generally fall into one of two categories: those to scan for unknown mutations and those to detect known mutations. This review focuses on methods for the detection of known mutations. The hybridization protection assay (HPA) is described in detail. The HPA method utilizes short oligonucleotide probes covalently labeled with a highly chemiluminescent acridinium ester (AE). The assay format is completely homogeneous, requiring no physical separation steps, and can rapidly and sensitively detect all single-base mismatches as well as multiple mismatches, insertions, deletions, and genetic translocations. When very low copy number targets are assayed, HPA is coupled with transcription-mediated amplification (TMA), an isothermal method that amplifies DNA or RNA targets. Other methods that are described for the detection of known mutations include hybridization with sequence-specific oligonucleotides, hybridization to oligonucleotide arrays, allele-specific amplification, ligase-mediated detection, primer extension, and restriction fragment analysis. The advantages and limitations of each of these methods are discussed. Methods to scan for unknown mutations are briefly described.

Base Pair Mismatch↗
PubMed2006

Roles of human parainfluenza virus type 3 bases 13 to 78 in replication and transcription: identification of an additional replication promoter element and evidence for internal transcription initiation.

The genomic promoter of human parainfluenza virus type 3 (HPIV3) contains multiple cis-elements controlling transcription and replication. Previous work showed that regions 1 to 12 and 79 to 96 were critical in promoting replication of an HPIV3 minireplicon, while the intergenic sequence and N gene start signal (IS/Ngs, bases 49 to 61) were important for transcription. Because these data were collected primarily using point mutations, not every base from position 1 to 96 was analyzed, and some important control elements may have been missed. To clarify the role of bases 13 to 78 in transcription and replication, a series of mutations were made which collectively scanned this entire region. Mutation of bases 13 to 28 resulted in markedly decreased HPIV3 minireplicon replication, indicating these bases constitute an additional cis-element involved in the synthesis of the HPIV3 antigenomic RNA. The position dependence of the IS/Ngs was also examined. Analysis of mutants in which the IS/Ngs was shifted 5' or 3' showed that this segment could be moved without significantly disrupting transcription initiation. Additional mutants which contained two successive IS/Ngs segments were created to test whether the polymerase accessed the gene start signal by proceeding along the template 3' to 5' or by binding internally at the gene start signal. Based on analysis of the double gene start mutants, we propose a model of internal transcription initiation in which the polymerase enters the template at approximately the location of the natural N gene start but then scans the template bidirectionally to find a gene start signal and initiate transcription.

Genome, Viral↗
PubMed2000

Complete molecular scanning of the human Fas gene: mutational analysis and linkage studies in families with type I diabetes mellitus. The Danish Study Group of Diabetes in Childhood and The Danish IDDM Epidemiology and Genetics Group.

AIMS/HYPOTHESIS: The human Fas gene (FAS) on chromosome 10q24.1 encoding a cell surface receptor involved in apoptosis was evaluated as a candidate susceptibility gene for Type I (insulin-dependent) diabetes mellitus. Apoptosis mediated by Fas is important in maintaining peripheral self-tolerance and in down-regulating the immune response and could have a role in immune-mediated beta-cell destruction. METHODS: We did a molecular scan of the entire human FAS (promoter, exons 1-9 including exon-intron boundaries and the 3'UTR) using single strand conformational polymorphism-heteroduplex analysis. RESULTS: We identified 15 mutations, of which 11 are new. Of these a g-1194A-->T and a g-295Ains give rise to alterations of transcription-factor-binding consensus sequences for c-Myb, SP-1 and NF-kappa B, respectively. A total of 1068 people from a Danish family collection comprising 138 Type I diabetic sib-pair families (289 affected and 121 unaffected offspring) and 103 Type I diabetic parent-offspring multiplex families (103 affected and 112 unaffected offspring) were typed for the three most frequent polymorphisms with high heterozygosity indices and for a FAS microsatellite. Haplotypes were established and data analysed using the extended transmission disequilibrium test, ETDT. CONCLUSION/INTERPRETATION: We found no overall evidence for linkage of the FAS polymorphisms to Type I diabetes. We conclude that it is unlikely that the Fas gene does contribute to genetic susceptibility for Type I diabetes.

Adult↗
PubMed1999

Superiority of denaturing high performance liquid chromatography over single-stranded conformation and conformation-sensitive gel electrophoresis for mutation detection in TSC2.

We evaluated denaturing high pressure liquid chromatography (DHPLC) as a scanning method for mutation detection in TSC2, and compared it to conformation-sensitive gel electrophoresis (CSGE) and single-stranded conformation polymorphism analysis (SSCP). The first 20 exons of TSC2 were amplified from 84 TSC patients and screened initially by CSGE and then by DHPLC. Optimization of DHPLC analysis of each exon was carried out by design of primers with minimum variation in the melting temperature of the amplicon, and titration of both elution gradient and temperature. CSGE analysis identified 40 shifts (21 unique) in the 84 patients and 20 exons. All of these variants were detected by DHPLC, and an additional 27 changes (14 unique) were identified. Overall 15 of 28 (54%) unique single base substitutions were detected by CSGE; all were detected by DHPLC. 25 definite or probable mutations were found in these 84 patients (30%) in exons 1-20 of TSC2. In a subsequent blinded analysis of 15 samples with 18 distinct TSC2 sequence variants originally detected by SSCP in another centre, all variants were detected by DHPLC except one where the variation occurred within the primer. Ten other (7 unique) sequence variants were detected in these samples which had not been detected by SSCP. Overall, 11 of 16 (69%) unique single base substitutions were detected by SSCP; all were detected by DHPLC. We conclude that DHPLC is superior to both CSGE and SSCP for detection of DNA sequence variation in TSC2, particularly for single base substitution mutations.

Chromatography, High Pressure Liquid↗
PubMed2001

Skin biopsy immunostaining with a Notch3 monoclonal antibody for CADASIL diagnosis.

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy) is a small-artery disease of the brain caused by NOTCH3 mutations that lead to an abnormal accumulation of NOTCH3 within the vasculature. We aimed to establish whether immunostaining skin biopsy samples with a monoclonal antibody specific for NOTCH3 could form the basis of a reliable and easy diagnostic test. We compared the sensitivity and specificity of this method in two groups of patients suspected of having CADASIL with complete scanning of mutation-causing exons of NOTCH3 (in a retrospective series of 39 patients) and with limited scanning of four exons that are mutation hotspots (prospective series of 42 patients). In the retrospective series skin biopsy was positive in 21 (96%) of the 22 CADASIL patients examined and negative in all others; in the prospective series, seven of the 42 patients had a positive skin biopsy whereas only four had a mutation detected by limited NOTCH3 scanning. Our immunostaining technique is highly sensitive (96%) and specific (100%) for diagnosis of CADASIL.

Adult↗
PubMed1996

Scanning mutagenesis reveals a similar pattern of mutation sensitivity in transmembrane sequences M4, M5, and M6, but not in M8, of the Ca2+-ATPase of sarcoplasmic reticulum (SERCA1a).

Scanning mutagenesis was performed on all amino acids in transmembrane sequences M5, M6, and M8, which, together with M4, make up the Ca2+ binding domain of the Ca2+-ATPase of sarcoplasmic reticulum (SERCA1a). When these transmembrane sequences were displayed on a helical net, examination of the effects of 101 novel point mutations and 95 prior mutations carried out on 92 transmembrane amino acids revealed "patches" of sensitivity to mutation in M4, M5, and M6 but not in M8. The patches of mutation-sensitive residues spanned 6 of the 7 tiers of the helical net and covered about 240 degrees at their widest point in tiers 3 or 4 and 140 degrees in tiers 2 and 5. A contiguous column of mutation-insensitive hydrophobic amino acids was found in M4 and M6 and in tiers 4 to 7 of M5. A six-residue motif, (E/D)GLPA(T/V) in tiers 3 and 4 of M4 and M6 with Ca2+-binding residues Glu309 and Asp800 as the first residue, was highlighted by mutation sensitivity. Elements of the motif could also be discerned in M5, but reading in the C-terminal to N-terminal direction. Mutation sensitivity in tier 5 of M4 mirrored mutation sensitivity of tier 5 in M6, although the amino acid sequences were not similar. The motif or its counterpart was found in a region in M4, M5, and M6 that is made up of tiny or small amino acids but is bounded by tiers with a larger percentage of bulky amino acids. Tiers 3, 4, and 5 of M4, M5, and M6 contain Ca2+ binding and affinity mutations, E1P to E2P block mutations and E2P dephosphorylation mutations, indicating an important role for these central tiers in Ca2+ binding and in the conformational changes that accompany Ca2+ translocation. Analysis of M8 revealed only a single mutation-sensitive residue, the Ca2+-binding amino acid, Glu908. This residue and a mutation-insensitive residue, Ala912, were the only vestiges of the motif that was found in M4 and M6. Additional mutations to Glu908 provided further evidence for its role in Ca2+ binding. Since mutation of M8 failed to identify residues involved in blocking conformational changes or altering Ca2+ affinity, it is apparent that M8 plays a peripheral role in Ca2+ binding and translocation in comparison with M4, M5, and M6.

Amino Acid Sequence↗
PubMed1990

Detection of point mutations associated with genetic diseases by an exon scanning technique.

A major challenge in genetics is identifying the basis of human heritable disease. We describe an "exon scanning" technique which surveys exons in genomic DNA for sequence alterations. By hybridizing genomic DNA to RNA probes derived from cDNAs, we can use RNase A to survey entire coding regions, comprising exons spread across extensive regions of genomic DNA, for mutations associated with genetic disease. Exon scanning of the beta-globin locus in the DNA of patients with 12 different hemoglobinopathies detected all of the culpable single base substitutions and deletions, but not single base insertions. Our analysis also revealed unsuspected polymorphisms and corrected a diagnosis originally based on hemoglobin electrophoresis. Exon scanning of the ornithine aminotransferase gene in a gyrate atrophy patient detected and localized a mutation in the sixth exon. Subsequent PCR amplification and sequencing characterized this as a missense mutation (proline----glutamine). Exon scanning of genomic DNA for sequence alterations, in combination with PCR amplification and sequencing, should be a generally useful strategy for evaluating suspect genes in disorders of unknown etiology, as well as for clinical diagnosis.

Base Sequence↗
PubMed2004

Comprehensive molecular diagnosis of mitochondrial disorders: qualitative and quantitative approach.

Mitochondrial disorders can be caused by mutations in nuclear or mitochondrial encoded genes. Point mutations and large deletions in mitochondrial DNA (mtDNA) are responsible for a small portion of the molecular defects in the mitochondrial oxidative phosphorylation system. A significant number of molecular defects of respiratory chain disorders are probably due to mutations in nuclear genes. Molecular diagnosis of mitochondrial disorders has been difficult because of broad genetic and clinical heterogeneity. Mutational analysis of common point mutations of mtDNA such as A3243G, A8344G, and T8993G/C is routinely performed. However, many patients who clearly have clinical manifestations and muscle pathology consistent with oxidative phosphorylation deficiency do not have detectable common mtDNA point mutations. A more comprehensive mutation screening method, temporal temperature gradient gel electrophoresis, was used to scan for unknown mutations in the entire mitochondrial genome. Novel mutations have been discovered but only account for a small portion of patients with suspected mitochondrial RC disorders. Real-time quantitative PCR analysis was used to measure cellular mtDNA content. Abnormal levels of mtDNA were found in many patients with respiratory chain disorders. Molecular analysis revealed that mutations in the thymidine phosphorylase gene are not seen in young patients with severe mtDNA depletion who do not demonstrate clinical features of mitochondrial neurogastrointestinal encephalomyopathy. It was also noted that an increase in the size or number of mitochondria was not necessarily associated with an increase in mtDNA content. On the contrary, in some cases the mtDNA was depleted. Respiratory activity in patients with a defective mitochondrial genome due to either point mutations or deletions may be compensated by amplification of mtDNA. Therefore, a comprehensive molecular analysis of mitochondrial respiratory chain disorders should include qualitative identification of the mutation and quantitative measurement of both the degree of mutant heteroplasmy and the total amount of mtDNA.

DNA Mutational Analysis↗
PubMed2002

Mutation searching in colorectal cancer studies: experience with a denaturing high-pressure liquid chromatography system for exon-by-exon scanning of tumour suppressor genes.

AIMS: In hereditary colorectal cancer (CRC) disorders such as familial adenomatous polyposis and hereditary non-polyposis colon cancer, the identification of germline mutations greatly assists in the clinical management of families. In addition, study of somatic mutations in the cancers themselves (both hereditary and sporadic) has been fundamental in the elucidation of the initiation and progression of CRC. Many of the genes underlying CRC development are large; hence mutation screening is a time-consuming and labour-intensive process requiring a rapid and accurate alternative to gel-based systems such as single-strand confirmational polymorphism (SSCP) or denaturing gradient gel electrophoresis (DGGE). Here we report our progress using denaturing gradient high-pressure liquid chromatography (DHPLC) in the screening of the mismatch repair genes MLH1 and MSH2 and in screening the APC and HPP1 tumour suppressor genes for mutations. METHODS: Genomic DNA was amplified using intronic primer sets spanning individual exons in the gene(s) under study. PCR products were subjected to DHPLC and the resultant chromatographs were compared with those of normal controls and aberrant peaks identified. Amplified products with aberrant peaks in the study samples underwent manual sequencing to confirm the presence of sequence variants. RESULTS: The proportion of amplified fragments showing aberrant peaks (hits) ranged from 18 to 30% and in the case of every gene, more than 80% of these could be confirmed as a sequence variant by manual sequencing. The highest rate was found in HPP1, where all hits were found to be sequence variants, and the lowest rate was found in MSH2, where manual sequencing failed to find a sequence variant in 17% of the hits attained. Mutations varied in their nature from directly truncating through splice variants to missense and deletion mutations. Traces for each mutation displayed unique shapes and both deletions and single base changes were equally dramatic. During the mutation scanning many polymorphisms presented as aberrant peaks, as would be expected. Importantly, the same polymorphism gave an identical chromatographic tracing between individuals, opening the possibility to identify common polymorphisms on pattern recognition alone. There remains, though, the possibility that rare pathogenic variants may assume an identical shape. CONCLUSIONS: The results indicate that DHPLC is a sensitive and efficient technique for screening of DNA for sequence variants. Given that polymorphisms comprised the largest proportion of variants found in each gene (66-100%), excluding these by pattern recognition would markedly reduce the amount of sequencing required.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Secondary structures in the capsid protein coding sequence and 3' nontranslated region involved in amplification of the tobacco etch virus genome.

The 3'-terminal 350 nucleotides of the tobacco etch potyvirus (TEV) genome span the end of the capsid protein (CP)-coding sequence and the 3' nontranslated region (NTR). The CP-coding sequence within this region contains a 105-nucleotide cis-active element required for genome replication (S. Mahajan, V. V. Dolja, and J. C. Carrington, J. Virol. 70:4370-4379, 1996). To investigate the sequence and secondary structure requirements within the CP cis-active region and the 3' NTR, a systematic linker-scanning mutagenesis analysis was done. Forty-six mutations, each with two to six nucleotide substitutions, were introduced at consecutive hexanucleotide positions in the genome of a recombinant TEV strain expressing a reporter protein (beta-glucuronidase). Genome amplification activity of each mutant in the protoplast cell culture system was measured. Mutations that severely debilitated genome amplification were identified throughout the CP-coding cis-active sequence and at several distinct locations within the 3' NTR. However, based on a computer model of RNA folding, mutations that had the most severe effects mapped to regions that were predicted to form base-paired secondary structures. Linker-scanning mutations predicted to affect either strand of a base-paired structure within the CP-coding cis-active sequence, a base-paired structure between two segments of the CP-coding cis-active sequence and a contiguous 14-nucleotide segment of the 3' NTR, and a base-paired structure near the 3' terminus of the 3' NTR inactivated genome amplification. Compensatory mutations that restored base pair interactions in each of these regions restored amplification activity, although to differing levels depending on the structure restored. These data reveal that the 3' terminus of the TEV genome consists of a series of functionally discrete sequences and secondary structures and that the CP-coding sequence and 3' NTR are coadapted for genome amplification function through a requirement for base pair interactions.

Base Sequence↗
PubMed1991

Regulatory elements mediating transcription of the human Ha-ras gene.

In order to identify transcriptional regulatory elements controlling the expression of the human Ha-ras gene and to quantitatively assess the role of each element, we made mutations of the transcriptional regulatory region, including 5' and internal deletions, linker scanning and replacement mutations, and combinations of these mutations all fused to the bacterial chloramphenicol acetyltransferase gene. The promoter activity of each of these mutants was determined by measuring the transient expression of chloramphenicol acetyltransferase activity after transfection into human epithelial HeLa cells. We found that the most important regulatory region consists of two closely linked but functionally independent elements, the non-consensus GC-II element, CGGGCGGGC, centered at position -153 from the major transcription start site cluster and a new element, CCGGAA, centered at position -161 directly upstream from GC-II. In addition, there are two functional regulatory elements which make minor contributions to the full promoter activity; a double CCAAT NF-I binding site at position -88 and an unidentified upstream element between positions -199 and -252. Aside from GC-II, the GC boxes, of which there are a total of six between positions -185 and +85, make little or no contribution to Ha-ras promoter activity when individual mutations are tested in growing HeLa cells. The three potential AP2 sites and a weak single NF-I binding site make no contribution. The basal promoter region extending to position -75 from the major start site cluster has no independent activity in this TATA-less gene.

Base Sequence↗
PubMed2004

Molecular scanning of the betacellulin gene for mutations in type 2 diabetic patients.

Betacellulin (BTC), a member of the epidermal growth factor (EGF) family, is an important factor in the growth and/or differentiation of pancreatic beta cells. In this point of view, we determined the transcriptional start site of the human BTC gene and screened the protein-coding region for mutations. The transcriptional start site was located 347 bp upstream from the translational initiation codon. After screening the protein coding exons (exons 1-5), we identified two novel missense mutations, Cys (TGC) to Gly (GGC) at codon 7 (C7G) and Leu (TTG) to Met (ATG) at codon 124 (L124M), and a single nucleotide substitution (-31c/t) in the intron 2. The C7G was located in the signal peptide and the L124M in the transmembrane domain and this Leu at codon 124 was conserved among human, bovine, rat, and mouse. The frequencies of these variants, however, were similar between type 2 diabetic patients (n = 228) and non-diabetic control subjects (n = 170). These data suggest that genetic variations in the protein-coding region of the human BTC gene are unlikely to be a major contributor to development of type 2 diabetes.

Aged↗
PubMed2003

Genomewide scan for hand osteoarthritis: a novel mutation in matrilin-3.

Osteoarthritis (OA) is the most common human joint disease, characterized by loss and/or remodeling of joint synovium, cartilage, and bone. Here, we describe a genomewide linkage analysis of patients with idiopathic hand OA who were carefully phenotyped for involvement of either or both the distal interphalangeal (DIP) joints and the first carpometacarpal (CMC1) joints. The best linkage peaks were on chromosomes 4q and 3p and on the short arm of chromosome 2. Genomewide significance was reached for a locus on chromosome 2 for patients with affected CMC1 joints (LOD = 4.97); this locus was also significant for patients with OA in both CMC1 and DIP joints (LOD = 4.44). The peak LOD score at this locus coincides with a gene, MATN3, encoding the noncollagenous cartilage extracellular matrix protein, matrilin-3. Subsequent screening of the genomic sequence revealed a missense mutation, of a conserved amino acid codon, changing threonine to methionine in the epidermal growth factor-like domain in matrilin-3. The missense mutation cosegregates with hand OA in several families. The mutation frequency is slightly more than 2% in patients with hand OA in the Icelandic population and has a relative risk of 2.1.

Amino Acid Sequence↗
PubMed2000

Adrenocorticotropin deficiency in combined pituitary hormone deficiency patients homozygous for a novel PROP1 deletion.

Incomplete differentiation of the anterior pituitary (AP) hormone-secreting cells can result in combined pituitary hormone deficiency (CPHD), in which patients display deficiencies in GH and at least one other AP hormone. The majority of familial CPHD cases are due to mutations in the pituitary transcription factor PROP1 (Prophet of Pit1). We have scanned for PROP1 mutations in a large consanguineous Indian CPHD pedigree and identified a novel 13-bp deletion in exon 2 that is predicted to generate a null allele. Assessment of GH, TSH, gonadotropin, and PRL levels in homozygous affected individuals indicated impaired production of these hormones by the AP. Interestingly, two of the affected subjects also displayed cortisol deficiency, which was progressive in one of these patients. This phenotypic feature is not normally associated with CPHD resulting from PROP1 mutation. These data show that PROP1 mutations can result in panhypopituitarism, the most severe form of AP deficiency, in which the production of all hormones is compromised and support a role for PROP1 in the maintenance and/or differentiation of all five hormone-secreting cell types. From a clinical perspective, these data indicate that the presence of an impaired pituitary-adrenal axis in CPHD patients does not exclude the possibility of an underlying PROP1 gene defect.

Adolescent↗
PubMed2001

Missense polymorphism in the human carboxypeptidase E gene alters enzymatic activity.

Carboxypeptidase E (CPE) is involved in the biosynthesis of peptide hormones and neurotransmitters, including insulin. One of the features of type 2 diabetes mellitus (T2DM) is an elevation in the proinsulin level and/or proinsulin/insulin molar ratio, suggesting that mutations in proinsulin processing enzymes may contribute to the development of T2DM. We scanned CPE for mutations in a collection of Ashkenazi T2DM families and identified five novel single nucleotide polymorphisms (SNPs). An SNP in the 283(rd) codon, c.847C>T, changes arginine to tryptophan (R283W). The residue Arg283 is conserved among CPE orthologs as well as most enzymatically active metallocarboxypeptidases. Of the 272 Ashkenazi T2DM pedigrees screened, we found four families segregating R283W. Within these four families, patients who inherited one copy of this variant had much earlier age of onset for T2DM. The R283W CPE protein cleaves peptide substrates with substantially lower efficiencies and is less stable at elevated temperature. In addition, the R283W CPE variant has a narrower pH optimum and is much less active at pH 6.0-6.5, indicating that the R283W CPE variant would be substantially less active than wild type CPE in the trans-Golgi network and immature secretory vesicles where the enzyme functions in vivo. To summarize, we uncovered a rare non-conservative missense mutation in CPE and demonstrated that the mutant protein has altered enzymatic properties. We predict that this mutant could cause hyperproinsulinism and diabetes in the homozygous state.

5' Untranslated Regions↗