Comprehensive TP53-denaturing gradient gel electrophoresis mutation detection assay also applicable to archival paraffin-embedded tissue.

A comprehensive mutation detection assay is described for the entire coding region and all splice site junctions of TP53. The assay is based on denaturing gradient gel electrophoresis, which follows either multiplex polymerase chain reaction (PCR) applied to DNA extracted from fresh or frozen tissue samples or nested PCR applied to DNA extracted from paraffin-embedded tissue samples. In both instances, the analysis can be performed under a single set of conditions. When testing the assay on DNA from cultured lung cancer cell lines and from paraffin-embedded Dukes C colorectal carcinomas, significant TP53 mutations were observed at high frequencies in 15 of 16 lung cancer cell lines (94%) and in 21 of 30 paraffin-embedded tissue samples of Dukes C colorectal carcinomas (70%). A substantial proportion of these significant mutations occurred outside the evolutionary conserved region of TP53 in 4 of 16 lung cancer cell lines (25%) and in 11 of 30 paraffin-embedded colorectal carcinomas (37%). This underscores the importance of a comprehensive TP53 mutation analysis in those instances that TP53 mutation is taken into account for diagnostic and prognostic purposes.

High prevalence of the Cys282Tyr HFE mutation facilitates an improved diagnostic service for hereditary haemochromatosis in South Africa.

OBJECTIVE: The aim of the study was to investigate the molecular basis of hereditary haemochromatosis (HH) in South Africa in order to establish a reliable, cost-effective molecular diagnostic service for this potentially lethal disorder. DESIGN: DNA samples of patient and control groups were screened for two common haemochromatosis (HFE) gene mutations. The local frequencies of mutations C282Y and H63D were determined and the DNA results correlated with biochemical parameters. SETTING: Patients were referred from private practitioners, health workers and pathologists for a molecular diagnosis of HH at the University of Stellenbosch Medical School. Twenty-two of the 244 referrals were clinically diagnosed with HH, while the remaining patients were family members of the probands or unrelated subjects referred solely on the basis of an abnormal iron profile. RESULTS: Seventeen of the 22 patient referrals (77%) diagnosed with HH were homozygous for the C282Y mutation, 3 (14%) were compound heterozygotes for mutations C282Y and H63D, and 2 patients (9%) did not exhibit either mutation. Screening of 458 control individuals from the general South African population demonstrated a carrier frequency of approximately 17% for the C282Y mutation among whites, implying that up to 1 out of every 115 South Africans of European descent may be homozygous for this founder-type mutation. Among 64 healthy blood donors of mixed ancestry, we detected 2 individuals heterozygous and 1 homozygous for the C282Y mutation. CONCLUSIONS: The detection of mutations C282Y and H63D at a high frequency in the majority of affected South African patients facilitates accurate pre-clinical and confirmatory diagnosis of HH in South Africa. Early detection by DNA screening and subsequent treatment by repeated phlebotomy can prevent disease onset in affected individuals. DNA diagnosis is particularly applicable to a common genetic disease such as HH, which is underdiagnosed and potentially lethal, but treatable.

Mapping of novel regions of DNA gain and loss by comparative genomic hybridization in esophageal carcinoma in the Black and Colored populations of South Africa.

Esophageal cancer (EC) is the leading cause of cancer death in the Black male population in South Africa. Although several oncogenes and tumor suppressor genes have previously been found altered in this cancer, many novel genes remain to be identified. To identify the chromosomal location of these unknown genes, we have analyzed DNA of 29 South African EC patients by comparative genomic hybridization. Frequent loss occurred at chromosome 1p (52%), 4p (52%), 18q (48%), 19p (52%), 19q (55%), and 22q (41%). The most common gains were detected at 1q (41%), 2q (52%), 3q (72%), 5p (31%), 7p (48%), 7q (45%), 8q (55%), and Xq (69%). High level amplification was detected at 2q24-33, 6p21.1-q14, 7p12-q21, 7q11.2-31, 8q22-24, 8q13-qter, 13q21-34, and at 13q32-34. The present comparative genomic hybridization study opens the way for additional targeted studies on these particular chromosomal regions to identify the specific genes involved in the higher susceptibility to specific subtypes of esophageal carcinoma in different geographical regions. The loss of 8p (28%) and Xp (17%) in tumors of male individuals may provide clues to the basis of the sex-biased frequency of occurrence of EC favoring men.

A nonsense mutation (Arg-196-Term) in exon 6 of the human TP53 gene identified in small cell lung carcinoma.

In a search for mutations of the TP53 tumour suppressor gene in lung cancer samples from gold miners in the Witwatersrand, South Africa, using heteroduplex and single strand conformation polymorphism (SSCP) analysis, a nonsense mutation was found in exon 6, consisting of a C to T transition and resulting in chain termination of the TP53 gene. The mutation occurred in a small cell lung cancer sample and is the first reported codon 196 TP53 mutation in both radon-associated and small cell lung cancer (SCLC) material.

Report on a molecular diagnostic service for familial hypercholesterolemia in Afrikaners.

The development of DNA-based methods for the direct detection of specific low density lipoprotein receptor (LDLR) gene mutations enabled us to establish a molecular diagnostic service for familial hypercholesterolemia (FH). This specialised service is of particular relevance and can be applied in the Afrikaner population of South Africa, where a founder gene effect increased the prevalence of FH to about 5-10 times greater than that found in most other population groups. Three point mutations in the LDLR gene were shown to account for approximately 90% of all Afrikaner FH cases. We report on the results obtained in 354 Afrikaner hyperlipidemics, from 274 unrelated families, referred for mutation screening-during the four-year period following the elucidation of the molecular basis of FH in this South African population group. By screening for the three founder-related LDLR gene mutations, approximately 50% of referrals were diagnosed as having FH. Presymptomatic diagnosis of FH by DNA analysis overcame the difficulties involved in the clinical diagnosis of some heterozygous cases. We could offer appropriate genetic counselling to FH patients, in addition to optimal clinical management by clinicians who referred the patients. Genetic testing has made early diagnosis of FH, including prenatal diagnosis, a reality.

Combined (team) approach to hemangioma management: arteriography with superselective embolization plus YAG laser/sapphire-tip resection.

Thirteen patients have been treated over a 2-year period utilizing a team approach between the radiology and surgical services. Initial diagnostic evaluation is directed at determining the size and anatomic extent of the hemangioma; clinical assessment of coagulation and hemodynamic status is also performed. Arteriography with superselective embolization precedes the surgical procedure, usually by 24 hours. The YAG laser with contact sapphire-tip scalpels is then utilized for resection and reconstruction of the hemangioma. Total removal of hemangioma was achieved in 9 of 13 patients, and partial or subtotal removal was achieved in the remainder. Blood loss and complications were minimal.

Molecular characterisation of a low-frequency mutation in exon 8 of the human low-density lipoprotein receptor gene.

The prevalence of familial hypercholesterolaemia (FH), an autosomal dominant disease characterised by raised low-density lipoprotein (LDL) cholesterol levels, is at least five times higher in the white Afrikaner population than in most other population groups in the world. A founder gene effect has been suggested to explain this abnormally high frequency. Detection of a polymorphic Stu I site in the 5' region of the LDL receptor gene and association of both restriction fragment length polymorphism alleles with FH in Afrikaners, indicated the existence of at least two founder members for the disease in this population. DNA from a hetero-allelic FH homozygote from this South African group has been analysed through genomic cloning and sequencing. The DNA polymorphic site is caused by a single guanine to adenine transition within exon 8 of the LDL receptor gene and can be used in the determination of haplotype-associated defects.

The identification of two low-density lipoprotein receptor gene mutations in South African familial hypercholesterolaemia.

Two point mutations were discovered in the low-density lipoprotein genes of patients with familial hypercholesterolaemia (FH). Defective genes were cloned and/or amplified by the polymerase chain reaction (PCR) method and the DNA sequences determined. A guanine to adenine base transition in exon 4 was found to be the molecular defect in 20% of cases of FH in the Afrikaner population. A second mutation, a guanine to adenine base substitution in exon 9, was identified in two homozygous FH individuals. Restriction enzyme analysis of PCR-amplified DNA from blood and tissue samples now permits accurate diagnosis of these mutations.

Fanconi's anemia: a cytogenetic study on lymphocyte and bone marrow cultures utilizing 1,2:3,4-diepoxybutane.

Fanconi's anemia (FA) is an autosomal recessive genetic trait characterized by congenital abnormalities, pancytopenia with a late onset, and increased chromosome instability. A great deal of heterogeneity exists in the disease, making an early correct diagnosis very difficult. Previously chromosome instability was used as a diagnostic tool but was found to be unreliable. Auerbach et al. have described the use of a difunctional alkylating agent, 1,2:3,4-diepoxybutane (DEB), in lymphocyte, fibroblast, and amniotic fluid cultures for the accurate diagnosis of homozygotes and heterozygotes for the FA gene. We report here the findings on lymphocyte and bone marrow cultures from 18 FA homozygotes and 17 family members. Statistical analysis of the results with DEB at different concentrations showed a significant increase in induced chromosome breakage rates for homozygotes and heterozygotes when compared to those for a control group. The bone marrow cultures gave similar results.