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Study Objective: To assess clinically South African patients with the putative diagnosis of Prader-Willi syndrome (PWS) and confirm this diagnosis by DNA/molecular analysis., Design: Prospective, nationally based, combined clinical and laboratory study., Main Results: Thirty-seven patients with a putative diagnosis of PWS were examined by clinical geneticists. Only 13 (35.1%) of these patients had the diagnosis of PWS confirmed by molecular analysis, and all 13 PWS patients had positive scores using the PWS consensus diagnostic criteria of Holm et al. The clinical features of the remaining 24 (64.9%) non-PWS patients were analysed and 23 did not have the neonatal, infantile and childhood features necessary to warrant consideration of a diagnosis of PWS; neither did they obtain a positive score according to Holm et al.'s criteria., Conclusion: PWS was confirmed in only 35% of South African patients with a putative PWS diagnosis, confirming that this condition is overdiagnosed and that the clinical diagnosis is difficult. Clinically, the diagnostic criteria of Holm et al. are of great assistance in making the diagnosis, but it remains essential to confirm the diagnosis by molecular analysis.

Mutations in the p53 gene have been reported in head and neck carcinomas. We determined the p53 mutation profile in 55 oral squamous cell carcinomas (OSCCs) from a black African population sample. DNA from all the patients were investigated using PCR amplification of the p53 gene (exons 5-9), followed by heteroduplex single-stranded conformational polymorphism (HEX-SSCP) analysis on the PCR products. Direct sequencing was performed on cases where mutations were identified. The results showed mutations in 13 of 55 (23.6%) tumours. Eleven of 13 (85%) were single base pair substitutions (9 transitions and 2 transversions), and 2 were deletions. Two novel mutations were identified: a large 63-base pair deletion, and a single base pair substitution. The mutations in our study occurred outside the head and neck tumour hot spot region (codons 238-248).

Background: The prevalence of oral squamous cell carcinoma (OSCC) among the Black community in South Africa is unacceptably high. The association between p53 protein, and PCNA overexpression and the presence of p53 gene mutations was evaluated., Materials and Methods: One hundred and ten formalin-fixed, paraffin-embedded blocks of OSCC were selected for immunohistochemical studies for p53 protein and PCNA expression using the DO-7 and PC10 monoclonal antibodies, respectively. DNA was extracted from fifty-five blocks and exons 5 to 9 of the p53 gene were amplified with nested primers, thereafter sequencing was performed to confirm the presence of mutations detected by single stranded conformational polymorphism., Results: Fifty-six cases (51%) showed p53 expression, while fourteen mutations (25%) were detected. A significant difference was found between the PCNA index in p53 positive and p53 negative tumors while the mean PCNA index for the tumors with p53 mutations was not significantly different from the tumors without mutations., Conclusions: No association between p53 protein overexpression and p53 gene mutations could be demonstrated.

The aetiology and detection of human herpes virus type 8 (HHV-8) DNA sequences in Kaposi's sarcoma (KS) is a matter of intense investigation. We report on the detection of HHV-8 DNA and sequence polymorphism in different clinicopathological subtypes of cutaneous KS samples from South Africa. The diagnosis was confirmed by histological examination in all cases. Six patients had classic KS (CKS), 3 epidemic KS (EKS), and 3 iatrogenic KS (IKS). A nested polymerase chain reaction (PCR) assay was used to detect HHV-8 DNA in cell lysates, prepared from formalin fixed, paraffin embedded sections. We investigated polymorphism in the HHV-8 DNA using single-stranded conformational polymorphism (SSCP) analysis on the PCR products, followed by direct sequencing. HHV-8 DNA was detected in all the patients with KS, irrespective of the clinicopathological subtype. Direct sequencing was performed on 5 selected cases and showed single base pair substitutions in all. The spectrum of mutations was similar to those described previously. No correlation was found between the different types of KS and sequence variation. The results support the hypothesis that HHV-8 is strongly associated with different clinicopathological subtypes of KS and confirm the occurrence of HHV-8 in patients with CKS, EKS, and IKS in South Africa.

The effects of the riminophenazine antimicrobial agents clofazimine and B669 as well as those of lysophosphatidylcholine (LPC), on microbial K(+)-transporting systems were investigated in a range of Gram-positive and Gram-negative bacteria using 42K and 86Rubidium (86Rb) as tracers. Exposing the Gram-positive bacteria to 0.1-10 mg/L of the drugs resulted in a dose-related inhibition of uptake of both radiolabelled cations due primarily to the inhibition of their influx which was prevented by pretreating the microorganisms with 25 mg/L alpha-tocopherol (vitamin E) which forms a complex with lysophospholipids. In contrast, Gram-negative bacteria were resistant to riminophenazine-mediated inhibition of K(+)-transport, with only one of four well-characterised K(+)-transport system mutants of Escherichia coli, namely Kup, being affected by the antimicrobial agents. The selective antimicrobial activity of riminophenazines against Gram-positive bacteria is probably achieved by lysophospholipid-mediated inactivation of K(+)-transport, while Gram-negative microorganisms possess several K(+)-transport systems which are either inaccessible and/or insensitive to lysophospholipids. Thus, K(+)-transport systems may represent novel targets for antimicrobial agents.

We report on the subtyping of South African primate T-cell lymphotropic virus type I (PTLV-I) strains by investigating the LTR region using sequence analysis and restriction fragment length polymorphism (RFLP) techniques. DNA from either uncultured peripheral blood mononuclear cells (PBMCs); cultured PBMC or cell lines of eight human T-cell lymphotropic virus type I (HTLV-I); and two simian T-cell lymphotropic virus type I (STLV-I) strains (Cercopithecus aethiops pygerythrus) were amplified by polymerase chain reaction (PCR), cloned, and sequenced. The samples originated from different geographical regions in South Africa. Phylogenetic relationships were estimated using the neighbor-joining method. The South African HTLV-I strains were of Cosmopolitan origin and similar to each other. RFLP analysis confirmed this subtyping. A divergence of 0.3 to 1.6% between the Cosmopolitan strains was observed, while the divergence between the HTLV-I and STLV-I strains ranged from 6.3 to 7%. The STLV-I strains were closely related to that of a chimpanzee, providing evidence of interspecies transmission.

Background: The incidence of oral squamous cell carcinoma (OSCC) is on the increase in developing countries., Materials and Methods: Formalin fixed paraffin embedded blocks of OSCCs from a Black South African population sample of peri-urban and rural origin were selected as follows: Group 1 - 57 OSCCs with a mean age of 59 years; Group 2 - 43 OSCCs all cases younger than 40 years; Group 3 - 46 OSCCs with blocks containing only tumour tissue without any normal epithelium and Group 4, a control group of 38 non-neoplastic epithelial lesions. Type specific primers were used in a standard PCR to amplify a segment of the E6 region of HPV 6, 11, 16 and 18., Results: HPV 11 and 16 DNA were found in one sample each from groups 1 and 2 respectively., Conclusion: HPV is not an etiologic factor in the development of OSCC in the population studied.

Mutations in the BRCA1 gene, discovered in 1994, are associated with an 80-90% lifetime risk of breast cancer. We have analysed 60 families with a history of breast and/or ovarian cancer for germline mutations in BRCA1. Twenty-two different mutations were detected in 32 families (53%), of which 14 are previously unreported. We observed a significant correlation between the location of the mutation in the gene and the ratio of breast to ovarian cancer incidence within each family. Our data suggest a transition in risk such that mutations in the 3' third of the gene are associated with a lower proportion of ovarian cancer. Haplotype analysis supports previous data which suggest some BRCA1 mutation carriers have common ancestors; however, we have found at least two examples where recurrent mutations appear to have arisen independently.

The purpose of the study was to develop a specific and sensitive PCR protocol using env, gag and LTR primer pairs to detect HIV-1 subtypes present in the Western Cape, South Africa. Twenty-two virus strains, belonging to HIV-1 subtypes B, C and D, were randomly selected for PCR evaluation. Cell lysates prepared from these virus-infected cultured cells were tested using 5 different primer pairs: gag SK38/SK39; gag 22/SK39; gag a/b, gag c/d (nested); env SK68/SK69 and LTR SK29/SK30. Eight different PCR profiles were evaluated: one profile each for the 3 gag primer pairs, 3 profiles for the env and 2 profiles for the LTR primer pairs. The number of PCR cycles, time per cycle and/or annealing temperature were changed in each profile. The optimum PCR profile for a specific primer pair was defined as that which detected one copy of proviral plasmid DNA after dot-blot hybridisation. Gag primer pairs detected HIV-1 DNA in all 22 samples. With the env primer pair, suboptimal conditions failed to detect most of the HIV-1 subtype C samples. By increasing the number of cycles and time per cycle, a 100% sensitivity was achieved. With the LTR primer pair all samples were detected by decreasing the annealing temperature and increasing the individual cycle times. This confirms that once PCR conditions are optimised, all HIV-1 subtypes in our study could be detected using different PCR primer pairs.

Certain health risks have been associated with recreational exposure to faecally polluted water. Canoeing in certain South African waters is considered to be a high risk activity with regard to schistosomiasis, gastroenteritis and possibly hepatitis. In a cross-sectional study, a serosurvey was conducted amongst canoeists to ascertain whether or not they had a higher seroprevalence to hepatitis A virus, Norwalk virus and Schistosoma spp. than non-canoeists. In comparisons between the two groups, a significant association could not be demonstrated between canoeing and antibody response to hepatitis A and Norwalk viruses (P-values for age-adjusted chi 2 were 0.083 and 0.219 respectively), but a significant association could be demonstrated between canoeing and the antibody response to Schistosoma spp. (P < 0.001; age-adjusted).

The seroprevalence for antibodies to HIV-1, HTLV-1, hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis E virus (HEV) were determined in a large group of Mozambican refugees living in Swaziland. Serum samples were collected from a total of 398 refugees located in the two camps (Ndzevane and Malindza). The prevalence for antibodies in the two camps were as follows: Ndzevane: 1.2% (HIV-1); 2.8% (HTLV-1); 0.3% (HCV); 4% (HEV) and 66% for any HBV marker. Malindza: 10.8% (HIV-1); 5.4% (HTLV-1); nil (HCV); 2% (HEV) and 65.7% for any HBV marker. The difference in the HIV-1 seroprevalence between the two camps was statistically highly significant. The phenomenon is possibly related to the location of the Malindza camp in the northern most populous area of Swaziland, resulting in more frequent contact between refugees and the local Swazi population.

Background: Recent studies reported a difference in the age distribution of oral squamous cell carcinoma (OSCC) between Black and White South Africans with OSCC more prevalent in Black patients under the age of 50 compared to Whites., Materials and Methods: Paraffin embedded blocks of OSCC were divided into two groups: one with a mean age of 56.2 years and the second group all younger than 40 years of age. A control group of 30 non-neoplastic intraoral lesions were selected. A standard PCR reaction was used to amplify the BAM H1 W-fragment of the EBV., Results: EBV DNA was demonstrated in 11/45 (24%) cases from the first group and in 11/45 (24%) cases from the second group. EBV DNA was present in 11/30 (37%) cases from the control group., Conclusions: This study showed that the prevalence of EBV in OSCC was not influenced by the age of the patient.

Intra-oral carcinoma is the third most common malignancy among men in developing countries, and carries a high mortality rate, particularly in Africa, where patients often present initially with lesions at an advanced stage. The present study was undertaken to determine the prevalence of human papillomavirus (HPV) DNA in oral squamous carcinoma in the west of the Northern Transvaal, an area where a large number of new cases has been diagnosed over the past few years. Paraffin blocks from 66 cases (51 men, 15 women; mean age 58.7 years) of oral squamous carcinoma were randomly selected. Blocks contained samples of both tumour and adjacent normal epithelium. The presence of HPV antigen was established by means of immunocytochemistry and HPV DNA by in situ hybridisation with radiolabelled probes for HPV-6, 11, 16 and 18. Immunocytochemistry for viral antigen was negative in all the specimens. HPV-18 was detected in normal epithelium adjacent to the tumour in one case only. It appears from our study that HPV is of limited importance in oral squamous cell carcinogenesis in the population studied.

We have isolated a novel cDNA that maps distal to BRCA1 at 17q12-q21. The total sequence predicts a protein of 576 amino acids with three conserved regions: a 90-amino-acid repeat domain, a SH3 (src homology region 3) motif, and a guanylate kinase domain. These conserved regions are shared among members of the discs-large family of proteins that include human p55, a membrane protein expressed in erythrocytes, rat PSD-95/SAP90, a synapse protein expressed in brain, Drosophila dIg-A, a septate junction protein expressed in various epithelia, and human and mouse ZO-1 and canine ZO-2, two tight junction proteins. dIg-A has been shown to act as a tumor suppressor, and the other members may all be involved in signal transduction through specialized membrane domains with highly organized cytoskeletons and thus are potential tumor suppressors. Since allelic loss has been reported in the 17q12-q21 region in breast and ovarian cancer and it appears that BRCA1 is not the target of the losses, we looked for somatic alterations in DLG2 in sporadic breast tumors. No evidence for mutation was found, making it unlikely that DLG2 is involved in sporadic breast cancer.

The purpose of this study was to determine the presence of Epstein-Barr virus (EBV) DNA in oral squamous cell carcinoma (OSCC) patients from a Black African population. Formalin fixed paraffin embedded blocks of OSCC of two randomly selected groups were investigated. Group 1 consisting of 57 blocks containing OSCC with a fragment of normal appearing adjacent/overlying epithelium. Group 2 consisted of 48 blocks containing only OSCC tissue without any normal appearing epithelium. The control group consisted of 38 non-malignant, non-viral associated lesions. A standard polymerase chain reaction (PCR) was used to amplify the Bam HI W-fragment using a nested primer set. EBV DNA was demonstrated in 14/57 (25%) blocks from Group 1, in 13/48 (27%) blocks from Group 2 and in 16/38 (42%) blocks from the control group. No evidence for a direct role of EBV in the process of malignant transformation of intraoral epithelial cells was found in this study.

This study was undertaken to determine the inter-observer reproducibility of the invasive cell grading method on oral squamous cell carcinomas and to correlate this with the DNA ploidy status and Langerhans cell (LC) population. Fifty formalin-fixed paraffin-embedded blocks that contained both tumor and adjacent normal epithelium were randomly selected. DNA ploidy analysis was performed on a flow cytometer and the LC population was determined using an immunohistochemical technique with anti-S100 and anti-HLADR primary antibodies. The inter-observer correlation of the total malignancy score and individual morphologic features was highly significant. Thirty-two of the 50 tumors were graded as poorly differentiated and 18/50 as moderately differentiated. Thirteen of 25 poorly differentiated tumors had an aneuploid DNA content compared with 9/18 of the moderately differentiated group. No statistical differences in the LC counts between the poorly and moderately differentiated and aneuploid and diploid carcinomas were found. This study showed that the invasive cell grading method is reproducible but no correlation was found between the grading results and the ploidy status or LC count.

Between 1984 and 1990, virus was routinely isolated and serum collected from patients diagnosed at hospitals in the Western Cape as suffering from AIDS or AIDS-related conditions (ARC). From these, 17 virus strains were selected at random for sequencing and molecular characterisation of the env gene. The strains were previously characterised as belonging to HIV-1 subtypes B, C and D. The purpose of the present study was to evaluate retrospectively the serological diagnosis of HIV-1 in these 17 South African patients. Thirteen anti-HIV screening assays, including 7 rapid/simple test devices (RTDs), 4 enzyme-linked immunosorbent assays (EIAs) and 2 Western immunoblot assays were evaluated. Using commercial EIAs, 16 serum samples were HIV antibody-positive and these results were confirmed by Western immunoblot analysis. Serum from one terminal AIDS patient was found negative with all the serological tests. Some RTDs gave false negative antibody reactions on specimens from patients infected with subtype D strains. To investigate the false negative antibody reactions, the polymerase chain reaction (PCR) was used to amplify, clone and sequence proviral DNA from the immunodominant gp41 region from 7 of the HIV-1 strains. Two patients, both subtype D strains (D214 and D482) with false negative results in the RTDs, showed a significant amino acid substitution, i.e., substitution of a histidine residue for leucine at env position 607. It was concluded that although there were false negative RTD reactions on patients with HIV-1 subtype D strains, the commercial EIAs tested are sensitive and are able to detect patients infected with HIV-1 subtypes B, C and D that are present in South Africa.

A series of 10 oesophageal and 10 laryngeal squamous carcinomas was examined by means of immunocytochemistry and in situ DNA hybridisation to demonstrate human papillomavirus (HPV) infection. Changes in the epithelium adjacent to the carcinoma were found in 5 of 10 oesophageal and 7 of 10 laryngeal carcinomas. Viral antigens could not be detected with immunocytochemistry in any of the specimens. HPV 6, 11 and 16 were detected in 3 oesophageal specimens. In one of these, HPV 16 was detected in normal as well as malignant cells. HPV 7 was detected for the first time in a laryngeal carcinoma. Our results confirm previous reports of possible HPV involvement in the pathogenesis of aerodigestive carcinomas.

Human papillomavirus (HPV) infection is implicated in squamous cell carcinogenesis. Oesophageal carcinoma has a high incidence in certain geographical regions and, using different methods of detection, HPVs have been found in these tumours. HPV 6 and 11 are frequently detected in laryngeal papillomas, benign lesions which rarely become malignant. HPVs have been detected in squamous carcinoma of the larynx but more frequently, especially HPV 16, in verrucous carcinomas a distinct variant of squamous carcinoma. An increased density of Langerhans cells, important in immunosurveillance in squamous epithelium, has been associated with a more favourable patient prognosis in laryngeal and other carcinomas.

HPV type 18 DNA was identified in an intrabony ameloblastoma using radiolabelled in situ hybridization. The viral DNA was found in a verrucous lesion in a cystic area of the tumor. The absence of HPV DNA in other epithelial areas of the ameloblastoma is suggestive of a secondary infection. HPV is not considered to be an etiological factor in the pathogenesis of this ameloblastoma.

In this study the formation of DNA single-strand breaks in MNL in close proximity to activated phagocytes, or in contact with added H2O2 and/or HOCl, were evaluated. Neutrophils activated by phorbol myristate acetate (PMA), induced DNA-strand breaks in neighboring lymphocytes which increased after 1-2 h incubation in a repair medium. These DNA-strand breaks could be prevented by the addition of catalase or substitution of the neutrophils with cells from a patient with chronic granulomatous disease. Inclusion of the myeloperoxidase (MPO) inhibitor, sodium azide (NaN3), to the system was associated with less damage after 1-2 h incubation and a faster repair rate. Exposure of MNL to added reagent H2O2 (12-100 microM) was also accompanied by DNA damage. Addition of reagent HOCl (3-25 microM) did not induce any DNA-strand breaks. However, when combined with H2O2 (12.5 microM), HOCl increased H2O2-mediated DNA damage and compromised the repair process. Interactions between the phagocyte-derived reactive oxidants H2O2 and HOCl are probably involved in the etiology of inflammation-related cancer.

The use of the chacma baboon (Papio ursinus) in radiobiological investigations justifies special attention because it answers many of the criteria of parallelism to the human. The present study was undertaken to establish whether in vitro gamma-radiation effects in chacma baboon and human lymphocytes are comparable. The sensitive and rapid nucleoid sedimentation technique was employed to evaluate in vitro DNA superstructure, damage and repair in readily obtainable radiosensitive peripheral lymphocytes. Dose-response curves after 60Co gamma-irradiation were obtained, and by applying single-hit kinetics of the target theory, an estimation of molecular masses of the supercoiled domains was made. The baboon and human lymphocytes produced analogous results, while an ethidium bromide intercalation study also revealed a similarity in average DNA superhelical density. Lymphocyte DNA repair after 0.5-4.0 Gy gamma-irradiation and repair times from 0.5 to 5.0 h were evaluated. The repair data obtained from baboon and human cells after 2.0 Gy irradiation compared favourably in extent of DNA repair as well as the profiles of the kinetic curves. These findings indicate that the chacma baboon would be a useful and relevant model for further in vivo radiobiological studies on lymphocytes. The effects of sedimentation conditions and advantages of using vertical-tube rotors in the nucleoid sedimentation technique are also discussed.

Enriched human B-lymphocyte and T-lymphocyte subpopulations were isolated by means of two Percoll density gradient centrifugation steps. Strand-break repair kinetics of 60Co gamma-irradiation-damaged DNA were obtained by applying a modified nucleoid sedimentation technique. Although no variations in the strand-break repair patterns of B-cells and T-cells within an individual could be detected, variations between individuals were evident. Changes in DNA supercoiling during the repair process were studied by means of ethidium bromide intercalation. The supercoiled conformation of DNA in peripheral lymphocyte subpopulations displayed similar undulating patterns within an individual, variations occurring between individuals. The results of this study indicate that alterations to the higher-order structure (supercoiling) of DNA are integral to the repair process.

1. DNA repair was measured in 3 Gy gamma-irradiated human peripheral lymphocyte subpopulations by means of nucleoid sedimentation. 2. The influence of the antibiotic, novobiocin (an inhibitor of inter alia topoisomerase II) on the repair process was investigated. 3. Repair of 33 37% of the DNA lesions induced by gamma-irradiation in enriched B lymphocyte fractions, was retarded by novobiocin. 4. Repair in enriched T lymphocyte fractions was unaffected by novobiocin.

1. DNA repair was measured in 3 Gy gamma-irradiated human peripheral lymphocyte subpopulations by means of nucleoid sedimentation. 2. The influence of aphidicolin (an inhibitor of DNA polymerase) on the repair process was investigated. 3. Repair of 40-44% of the DNA lesions induced by gamma-irradiation was blocked by aphidicolin. 4. Enriched B- and T-lymphocyte fractions were affected by aphidicolin to the same extent.