Biomedical subjects

A Ahmadian

Publications and source records attributed to A Ahmadian.

12 recordsLinked to original sources

PubMed2000

Mutation detection by pyrosequencing: sequencing of exons 5-8 of the p53 tumor suppressor gene.

The ability to sequence a large number of DNA samples rapidly and accurately for detection of all possible mutations is a critical goal for the future application of DNA sequencing in routine medical diagnostics. Pyrosequencing() is a non-electrophoretic real-time DNA sequencing method that uses the luciferase-luciferin light release as the detection signal for nucleotide incorporation into target DNA. For pyrosequencing of the human p53 gene, a nested multiplex PCR method for amplification of exons 5-8 was prepared. In order to investigate the use of pyrosequencing in mutation detection, DNA samples from skin-cancer patients were used. Two forms of nucleotide dispensation strategy were used, cyclic and programmed. Bi-directional pyrosequencing was performed and the overlapping sequence data produced were assembled to determine the sequence of the gene. Reliable sequencing data were obtained with both dispensation strategies, but some advantages were obtained using the programmed nucleotide dispensation approach, such as longer and faster reads, and fewer out-of-phase problems. The accuracy of pyrosequencing for detection of p53 mutations and allele distribution was demonstrated.

Base Sequence↗

PubMed2000

Single-nucleotide polymorphism analysis by pyrosequencing.

There is a growing demand for high-throughput methods for analysis of single-nucleotide polymorphic (SNP) positions. Here, we have evaluated a novel sequencing approach, pyrosequencing, for such purposes. Pyrosequencing is a sequencing-by-synthesis method in which a cascade of enzymatic reactions yields detectable light, which is proportional to incorporated nucleotides. One feature of typing SNPs with pyrosequencing is that each allelic variant will give a unique sequence compared to the two other variants. These variants can easily be distinguished by a pattern recognition software. The software displays the allelic alternatives and allows for direct comparison with the pyrosequencing raw data. For optimal determination of SNPs, various protocols of nucleotide dispensing order were investigated. Here, we demonstrate that typing of SNPs can efficiently be performed by pyrosequencing using an automated system for parallel analysis of 96 samples in approximately 5 min, suitable for large-scale screening and typing of SNPs.

Base Sequence↗

PubMed2000

Analysis of the p53 tumor suppressor gene by pyrosequencing.

Tumor suppressor genes are implicated in cell cycle progression. Inactivation of these genes predominantly occurs through mutations and/or allelic loss that involves both alleles. With inactivation by multiple mutations in a single gene, cloning of the amplified gene is necessary to determine whether the mutations reside on one or both alleles. Using pyrosequencing, a recently developed approach based on sequencing-by-synthesis, we studied genetic variability in the p53 tumor suppressor gene and could quantify the ratio between the mutated and wild-type amplified fragments. Furthermore, this sequencing technique also allows allelic determination of adjacent mutations with no cloning of amplified fragments.

Alleles↗

PubMed1999

Context-dependent Taq-polymerase-mediated nucleotide alterations, as revealed by direct sequencing of the ZNF189 gene: implications for mutation detection.

We have recently reported on the genetic organisation of a novel Krüppel-like zinc finger, ZNF189, located to 9q22-q31. In that study we found no mutations in the coding sequence when using ZNF189 as a candidate gene for sporadic basal cell cancer and squamous cell cancer. Here, by direct sequencing of the proximal promotor of ZNF189, mutations were found to appear in a small hot-spot region in over 50% of analysed tumour samples, the majority being G to A substitutions. The hot-spot region spans a 24bp G-rich region. Repeated analyses of the original sample lysates fail to confirm each of these mutations; and frequently new mutations appear at neighbouring positions. Subsequent analysis with serial dilutions of genomic DNA and a cosmid harbouring the wild-type ZNF189 gene demonstrate that these sequence-specific alterations arise in the outer PCR-amplification when 50 copies or less of template are used. Although the mechanism of how these context-specific alterations arise is not proven, the results demonstrate a previously unreported type of PCR-mediated sequence-specific alteration that easily could have been interpreted as being of clinical relevance. The phenomena observed show that mutations detected by direct sequencing can be caused by PCR-introduced alterations. Consequently, this should be of general caution in mutation analysis of disease gene candidates when using small amounts of template, such as microdissected biopsies.

Artifacts↗

PubMed1998

Genetic instability in the 9q22.3 region is a late event in the development of squamous cell carcinoma.

Squamous cell carcinoma (SCC) of the skin represents a group of neoplasms which is associated with exposure to UV light. Recently, we obtained data suggesting that invasive skin cancer and its precursors derive from one original neoplastic clone. Here, the analysis were extended by loss of heterozygosity (LOH) analysis in the chromosome 9q22.3 region. A total of 85 samples, taken from twenty-two sections of sun-exposed sites, corresponding to normal epidermis, morphological normal cells with positive immuno-staining for the p53 protein (p53 patches), dysplasias, cancer in situ (CIS) and squamous cell carcinomas (SCC) of the skin were analysed. Overall, about 70% of p53 patches had mutations in the p53 gene but not LOH in the p53 gene or 9q22.3 region. Approximately 70% of the dysplasias showed p53 mutations of which about 40% had LOH in the p53 region but not in the 9q22.3 region. In contrast, about 65% of SCC and CIS displayed LOH in the 9q22.3 region, as well as frequent (80%) mutations and/or LOH in the p53 gene. These findings strongly suggest that alterations in the p53 gene is an early event in the progression towards SCC, whereas malignant development involves LOH and alterations in at least one (or several) tumor suppressor genes located in chromosome 9q22.3.

Carcinoma in Situ↗

PubMed1998

Cloning and characterization of ZNF189, a novel human Krüppel-like zinc finger gene localized to chromosome 9q22-q31.

A 3-kb-long cDNA encoding a Krüppel-like human zinc finger protein was isolated and mapped to chromosome 9q22-q31. The ZNF189 gene encodes a protein with 16 zinc fingers at its C-terminus and belongs to the Krüppel-associated box (KRAB)-containing group of zinc finger proteins. Four differently spliced cDNA transcripts, differing at the 5' coding region where a KRAB A repressor domain is encoded, were isolated. In addition, Northern blot analysis indicates the presence of two additional unidentified splice variants. Comparison of cDNA and genomic sequences shows that the ZNF189 gene spans approximately 11 kb and is organized into at least four exons, the large 3'-end exon coding for the complete zinc finger domain and the 3' untranslated region. ZNF189 is expressed in all tissues and cell types currently investigated, at varying levels, but with a tissue- or cell-type-restricted expression pattern for the different splice variants. ZNF189 is conserved in the genome of several mammalian species. Direct sequencing of the ZNF189 gene in microdissected tumor biopsies of sporadic basal cell carcinoma and squamous cell carcinoma reveals no mutations in the coding sequence or at exon/intron boundaries.

Amino Acid Sequence↗

PubMed1998

Clones of normal keratinocytes and a variety of simultaneously present epidermal neoplastic lesions contain a multitude of p53 gene mutations in a xeroderma pigmentosum patient.

A patient with xeroderma pigmentosum group C was extensively examined for mutations in the p53 gene in normal skin exposed to varying degrees of sunlight and in excisional biopsies of basal cell cancer, squamous cell cancer, and squamous cell dysplasia. Seventy-three samples were analyzed by microdissection of small cell clusters, followed by PCR and direct DNA sequencing. In skin taken from areas that most likely had never been exposed to the sun, no mutations were found. However, in skin exposed to the sun, we observed a multitude of mutations in the p53 gene. UV light-induced mutations were found in all types of lesions, as well as in clusters of morphologically normal epidermal cells. Twenty-nine distinct mutations were found in exons 5-8, all missense or nonsense, of which 27 (93%) were UV-specific C --> T or CC --> TT transitions at dipyrimidine sites of the nontranscribed strand. Two types of normal skin areas containing p53 mutations were observed: areas that stain strongly with p53 antibody (p53 patches) and those that do not stain. Because no silent or intron mutations were found in these cell clusters, the alterations in the p53 gene of morphologically normal cells are likely to have resulted in a selective growth advantage. The poor correlation between mutations and morphological phenotypes demonstrates that p53 mutations alone do not determine the phenotypes observed.

Adolescent↗

PubMed1998

Assessment of sequence-based p53 gene analysis in human breast cancer: messenger RNA in comparison with genomic DNA targets.

The high prevalence of p53 mutations in human cancers and the suggestion from several groups that the presence or absence of p53 mutations might have both prognostic and therapeutic consequences point to the importance of optimal methods for p53 determination. Several strategies exploring this have been described, based either on mRNA or genomic DNA as a template. However, no comparative study on the reliability of the two templates has been performed. The principal aim of this study was to study the concordance of RNA- and DNA-based direct sequencing methods in detecting p53 mutations in breast tumors. In 100 tumors, 22 mutations were detected by both methods. Furthermore, one stop mutation, two splice-site mutations, and one intron alteration were found only by genomic sequencing. In addition, the comparative study suggests that cells with missense mutations have increased steady-state concentrations of p53-specific mRNA, in contrast to cells with a gene encoding a truncated protein.

Aneuploidy↗

PubMed1997

Molecular pathology in basal cell cancer with p53 as a genetic marker.

Human basal cell cancer (BCC) has unique growth characteristics with virtual inability to metastasize. We investigated clonality and genetic progression using p53 mutations as marker. Sampling was done through microdissection of frozen immunohistochemically stained 16 microm slices of tumors. From 11 BCC tumors 78 samples were analysed. Direct DNA sequencing of exons 5-8 was performed, haplotypes were determined after cloning of p53 exons and loss of heterozygosity (LOH) ascertained by microsatellite analysis. All tumors had p53 mutations and in a majority both p53 alleles were affected, commonly through missense mutations. Microdissection of small parts (50-100 cells) of individual tumors showed BCC to be composed of a dominant cell clone and prone to genetic progression with appearance of subclones with a second and even third p53 mutation. Samples from normal immunohistochemically negative epidermis always showed wild type sequence, except for a case of previously unknown germline p53 mutation. Our analysis also included p53 immunoreactive patches i.e. morphologically normal epidermis with a compact pattern of p53 immunoreactivity. Mutations within those were never the same as in the adjacent BCC. This detailed study of only one gene thus uncovered a remarkable heterogeneity within a tumor category famous for its benign clinical behavior.

Aged↗

PubMed1997

Genomic analysis of single cells from human basal cell cancer using laser-assisted capture microscopy.

In this study, we show that direct mutational analysis of genomic DNA can be performed on single somatic cells extracted from a frozen, immunohistochemically stained tissue section using laser-assisted capture microscopy. Eighty-nine single tumor cells were separately dissected from one case of human basal cell cancer (BCC) and p53 mutations were analyzed by direct semi-automated sequencing of PCR fragments. Amplification was obtained for at least one of the two analyzed exons from approximately 50% of the single tumor cells. Identical p53 mutations were found in widely spread areas of the tumor, suggesting a clonal proliferation originating from one cell. Interestingly, comparison between results of immunohistochemistry and genetic analysis of the single cells revealed the same p53 mutations irrespective of the p53 immunoreactivity. We propose that this approach has a great potential to allow investigation of genotypic differences in single cells and more specifically to resolve important and fundamental questions determining cancer heterogeneity.

Aged↗

PubMed1997

Benign clonal keratinocyte patches with p53 mutations show no genetic link to synchronous squamous cell precancer or cancer in human skin.

Ultraviolet light, which is the major etiology of human skin cancer, will cause mutations in the p53 gene. We and others have found that such mutations occur in more than one-half of non-melanoma squamous cell cancer and precancer. Immunostaining for p53 has disclosed a characteristic compact pattern not only in cancer/precancer but also in areas of microscopically normal epidermis termed p53 patches. By microdissection, sequence analysis of the p53 gene, and analysis of loss of heterozygosity (LOH) at the site of this gene, we have now extended previous data to ascertain whether these p53 patches are precursors of simultaneously present squamous cell cancer or its morphologically recognized precancerous stages (dysplasia, carcinoma in situ). In none of 11 instances with co-existence of a p53 patch with dysplasia or in situ or invasive cancer were the mutations identical. We conclude that p53 patches, estimated to be approximately 100,000 times as common as dysplasia, have a very small or even no precancerous potential. Their common presence demonstrates that human epidermis contains a large number of p53 mutations apparently without detrimental effect. The only result of the mutation may be a clandestine benign clonal keratinocyte proliferation. The importance of p53 mutations for such benign cell multiplication on one band and malignant transformation on the other is unclear. Although the spectrum, type, and multiplicity of mutations were similar in both types of proliferative responses, there was a clear difference with respect to LOH. No LOH was found in 17 p53 patches. By contrast 11 of 30 precancers/cancers had LOH.

Carcinoma in Situ↗

PubMed1996

Human epidermal cancer and accompanying precursors have identical p53 mutations different from p53 mutations in adjacent areas of clonally expanded non-neoplastic keratinocytes.

Microdissection of biopsies with sequencing of exons 4-8 of the p53 gene permitted precise morphological identification of correlation between mutations and/or loss of heterozygosity, immunoreactivty of p53 and type of squamous neoplasia. Seventy-two specimens from ten lesions of sun-exposed sites including normal epidermis were analysed. Irrespective of p53 immunoreactivity and morphological grade dysplasia, in situ or invasive cancer, in each case, carried the identical mutation indicating that invasive skin cancer and its precursors derive from the same original neoplastic clone. Additionally, morphologically normal epidermis showed some sharply demarcated immunoreactive areas. These never had the same p53 mutation as that of the adjacent tumor, indicating that their mutations were separate events and ruling them out as common precursors of cancer. Non-immunoreactive normal epidermis did not show p53 mutations. Our findings indicate that a large fraction of keratinocytes in sun-exposed human skin carry mutations of p53 and suggest that at least two options exist for such cells (i) innocuous clonal expansion with preserved morphology and normal differentiation or (ii) malignant transformation with the p53 mutation as an early event. Suggestive evidence existed that the p53 mutations were qualitatively different in the two respective groups of lesions.

Aged↗