Germline mutations of the BRCA1 gene in breast and ovarian cancer families provide evidence for a genotype-phenotype correlation.

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.

Entropic exponents of grafted lattice stars.

The surface entropic exponents of half-space lattice stars grafted at their central nodes in a hard wall are estimated numerically using the PERM algorithm. In the square half-lattice the exact values of the exponents are verified, including Barber's scaling relation and a generalization for 2-stars with one and two surface loops respectively. This is the relation γ_{211}=2γ_{21}-γ_{20}, where γ_{21} and γ_{211} are the surface entropic exponents of a grafted 2-star with one and two surface loops, respectively, and γ_{20} is the surface entropic exponent with no surface loops. This relation is also tested in the cubic half-lattice where surface entropic exponents are estimated up to 5-stars, including many with one or more surface loops. Barber's scaling relation and the relation γ_{3111}=γ_{30}-3γ_{31}+3γ_{311} are also tested, where the exponents {γ_{31},γ_{311},γ_{3111}} are of grafted 3-stars with one, two, or three surface loops, respectively, and γ_{30} is the surface exponent of grafted 3-stars.

Phase diagrams of confined square lattice linked polygons.

The phase diagrams of two models of two confined and dense two-dimensional ring polymers are examined numerically. The ring polymers are modeled by square lattice polygons in a square cavity and are placed to be either unlinked or linked in the plane. The phase diagrams of the two models are found to be a function of the placement of the ring polymers and include multicritical points where first-order and continuous phase boundaries meet. We estimate numerically the critical exponents associated with the phase boundaries and the multicritical points.

Numerical estimates of square lattice star vertex exponents.

We implement parallel versions of the generalized atmospheric Rosenbluth methods and Wang-Landau algorithms for stars and for acyclic uniform branched networks in the square lattice. These are models of monodispersed branched polymers, and we estimate the star vertex exponents σ_{f} for f stars, and the entropic exponent γ_{G} for networks with comb and brush connectivity in two dimensions. Our results verify the predicted (but not rigorously proven) exact values of the vertex exponents and we test the scaling relation [B. Duplantier, J. Stat. Phys. 54, 581 (1989)JSTPBS0022-471510.1007/BF01019770]γ_{G}-1=[under ∑]f≥1m_{f}σ_{f}for several acyclic branched networks in two dimensions.

Reply to "Comment on 'Osmotic pressure of compressed lattice knots' ".

The free energy of a model of uniformly weighted lattice knots of length n and knot type K confined to a lattice cube of side length L-1 is given by F_{L}(ϕ)=-1/Vlogp_{n,L}(K), where V=L^{3} and where ϕ=n/V is the concentration of monomers of the lattice knot in the confining cube. The limiting free energy of the model is F_{∞}(ϕ)=lim_{L→∞}F_{L}(ϕ) and the limiting osmotic pressure of monomers leaving the lattice knot to become solvent molecules is defined by Π_{∞}(ϕ)=ϕ^{2}d/dϕ[F_{∞}(ϕ)/ϕ]. I show that, under very mild assumptions, the functions P_{L}(ϕ)=ϕ^{2}d/dϕ[F_{L}(ϕ)/ϕ]|_{n} and Π_{L}(ϕ)=ϕ^{2}d/dϕ[F_{L}(ϕ)/ϕ]|_{L} are finite-size approximations of Π_{∞}(ϕ).

Osmotic pressure of compressed lattice knots.

A numerical simulation shows that the osmotic pressure of compressed lattice knots is a function of knot type, and so of entanglements. The osmotic pressure for the unknot goes through a negative minimum at low concentrations, but in the case of nontrivial knot types 3_{1} and 4_{1} it is negative for low concentrations. At high concentrations the osmotic pressure is divergent, as predicted by Flory-Huggins theory. The numerical results show that each knot type has an equilibrium length where the osmotic pressure for monomers to migrate into and out of the lattice knot is zero. Moreover, the lattice unknot is found to have two equilibria, one unstable, and one stable, whereas the lattice knots of type 3_{1} and 4_{1} have one stable equilibrium each.

Mean field analysis of algorithms for scale-free networks in molecular biology.

The sampling of scale-free networks in Molecular Biology is usually achieved by growing networks from a seed using recursive algorithms with elementary moves which include the addition and deletion of nodes and bonds. These algorithms include the Barabási-Albert algorithm. Later algorithms, such as the Duplication-Divergence algorithm, the Solé algorithm and the iSite algorithm, were inspired by biological processes underlying the evolution of protein networks, and the networks they produce differ essentially from networks grown by the Barabási-Albert algorithm. In this paper the mean field analysis of these algorithms is reconsidered, and extended to variant and modified implementations of the algorithms. The degree sequences of scale-free networks decay according to a powerlaw distribution, namely P(k) ∼ k-γ, where γ is a scaling exponent. We derive mean field expressions for γ, and test these by numerical simulations. Generally, good agreement is obtained. We also found that some algorithms do not produce scale-free networks (for example some variant Barabási-Albert and Solé networks).

Novel mutations identified using a comprehensive CCR5-denaturing gradient gel electrophoresis assay.

BACKGROUND: Most mutations detected for the gene for CC chemokine receptor 5 (CCR5) are either relatively specific to different population groups or rarely observed in Africans. OBJECTIVES: To develop a comprehensive mutation detection assay for the entire coding region of CCR5 and to identify novel mutations that may play a role in genetic susceptibility to HIV-1 infection, within the diverse South African population. DESIGN: The study cohort consisted of 103 HIV-seropositive patients and 146 HIV-seronegative controls of predominantly African descent. METHODS: A mutation detection assay for the entire coding region of CCR5 was designed; this included amplification of part of the coding region of CCR2. The assay was based on denaturing gradient gel electrophoresis (DGGE) and allowed the complete analysis of samples from 10 individuals per denaturing gel. RESULTS: The use of the CCR5-DGGE assay led to the identification of seven novel and six previously reported mutations. All novel mutations, including a common polymorphism at codon 35, occurred exclusively in non-Caucasians, indicating possible African origin. CONCLUSION: A comprehensive DGGE mutation detection assay has been developed for the entire coding region of CCR5. Application of this assay resulted in the identification of novel CCR5 mutations, which may have a significant effect on the normal functioning of CCR5 and thus contribute to host variability and susceptibility to HIV-1 infection and/or progression to AIDS within this population.

Detection and subtyping of human herpesvirus-8 in renal transplant patients before and after remission of Kaposi's sarcoma.

BACKGROUND: Kaposi's sarcoma (KS) is a complication of renal transplantation. If the human herpesvirus-8 (HHV-8) causes KS, the virus should be present in all KS lesions and be drastically reduced or cleared from involved tissue on remission of the KS. METHODS: Fourteen renal transplant patients with cutaneous KS, including autopsy material from two cases, were investigated for the presence of HHV-8. A second skin biopsy was taken from 11 survivors, after remission of KS, from normal skin in the same anatomical region as the first biopsy. Remission was induced by reduction or cessation of immunosuppression. A peripheral blood sample was collected simultaneously with the repeat biopsy. A nested polymerase chain reaction assay was used to detect HHV-8 DNA in the biopsy tissue and peripheral blood mononuclear cells followed by direct sequencing of polymerase chain reaction product to detect any nucleotide changes. RESULTS: HHV-8 DNA was detected in all the cutaneous KS and all the visceral KS samples, as well as a number of KS-free organs including the thyroid, salivary gland, and myocardium that have not been described before. Mutations in the viral DNA could be demonstrated in all patients. The mutations found were related more to that seen in AIDS-KS cases than that found in African endemic KS cases. HHV-8 sequences could be detected in follow-up frozen skin biopsies of five patients but were negative in the equivalent formalin-fixed specimens. Viral DNA was also detected in 2 of 11 peripheral blood mononuclear cell samples collected at the time of the follow-up skin biopsies. CONCLUSION: Reduction or withdrawal of immunosuppression allows the immune system to recover sufficiently to reduce viral replication with subsequent viral persistence and low grade viral replication that coincides with clinical remission of the KS lesions. This provides further evidence for the important etiological role played by HHV-8 in the pathogenesis of posttransplant KS.

Characterization and phylogenetic analysis of South African HIV-1 subtype C accessory genes.

To acquire new knowledge about the genetic diversity and potential impact on vaccine strategies of HIV-1 subtype C in South Africa, we have characterized the vif, vpr, and vpu genes of 15 isolates. Phylogenetic analysis of the genomic fragment encompassing these genes revealed subtype C subclusters, suggesting close relatedness with subtype C strains from other geographic locations and excluded isolation of South African strains. The putative T155 phosphorylation site in the C terminal of Vif was absent in all subtype C sequences. Variation in the predicted amino acid sequences of the three genes further showed strong correlation with other subtype C sequences.

Identification of env subtypes in fourteen HIV type 1 isolates from south Africa.

PIP: All subtypes of HIV-1 have been identified in Africa. The envelope glycoprotein of the HIV-1 is the most variable region of the virus, with the third variable region, including the V3 loop, a major target of vaccine research. This paper reports the identification of the HIV-1 subtypes present in 14 viral strains, isolated between 1984 and 1992 in South Africa. HIV-1 strains were isolated routinely at Tygerberg Hospital in the Western Cape region, with genomic DNA isolated from virus-infected cultures. After presenting the distance calculations and describing the tree constructions and bootstrap analysis, the authors emphasize the need for ongoing molecular epidemiological analysis of HIV-1 subtypes to track the current epidemic in South Africa. More rapid methods will facilitate subtyping to monitor the circulation and spread of HIV-1 subpopulations in the country.^ieng

Genetic analysis of the complete gag and env genes of HIV type 1 subtype C primary isolates from South Africa.

South Africa has one of the fastest growing HIV-1 epidemics, with an estimated 4.7 million people infected. To better understand the genetic diversity of this epidemic and its potential impact on vaccine development, we have cloned and sequenced the complete gag and env genes of 13 primary virus isolates. Phylogenetic analysis of our sequences and 69 complete env genes from the Los Alamos and GenBank databases revealed multiple subclusters within subtype C. The V3 loop region was relatively conserved in all our strains when compared with other subtypes, but the region immediately downstream was highly variable. No intersubtype recombinant forms were observed when comparing the gag and env sequences. Characterization of the complete gag and env genes enabled us to select specific strains for further vaccine development.

HIV type 1 V3 domain serotyping and genotyping in Gauteng, Mpumalanga, KwaZulu-Natal, and Western Cape Provinces of South Africa.

More than 20.8 million people are living with HIV/AIDS in sub-Saharan Africa, with southern Africa the worst affected area and accounting for one of the fastest growing AIDS epidemics worldwide. Samples from 81 patients, including 25 from KwaZulu-Natal, 26 from Gauteng, 5 from Mpumalanga, and 25 from Western Cape Province, were serotyped using a competitive V3 peptide enzyme immunoassay (cPEIA). Viral RNA was also isolated from serum and the V3 region amplified by reverse transcriptase polymerase chain reaction (RT-PCR) to obtain a 240-bp product for direct sequencing of 29 samples. CLUSTAL W was used to make multiple sequence alignments. Distance calculation, tree construction methods, and bootstrap analysis were done using TREECON. Subtype C-like V3 loop sequences predominate in all provinces tested in South Africa. Discordant sero- and genotype results were observed in one patient only. The correlation between sero- and genotyping was 96% (24 of 25) in KwaZulu-Natal and 100% in Gauteng and Mpumalanga. In Western Cape Province 18% of patients were identified as sero/genotype B and 82% as sero/genotype C. Our data show that results of the second-generation V3 cPEIA correlated well with V3 sequencing and would be a rapid and affordable screening test to monitor the explosive southern African HIV-1 epidemic.

Detection of southern African human immunodeficiency virus type 1 subtypes by polymerase chain reaction: evaluation of different primer pairs and conditions.

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.

PIP: During 1984-92, in South Africa, virologists isolated HIV-1 from HIV/AIDS patients at hospitals in the Western Cape. Two virologists from the University of Stellenbosch Hospital in Tygerberg selected 22 virus strains, belonging to HIV-1 subtypes B, C, and D, to study in order to develop a specific and sensitive polymerase chain reaction (PCR) protocol using env, gag, and LTR primers. They used five different primer pairs to prepare cell lysates from the HIV-infected cultured cells: gag SK38/SK39, gag 22/SK39, gag a/b, gag c/d (nested), env SK68/SK69, and LTR SK29/SK30. The virologists evaluated eight different PCR profiles: one profile each for the three gag primer pairs, three profiles for the env, and two profiles for the LTR primer pairs. They changed the number of PCR cycles, time per cycle, and/or annealing temperature in each profile. The PCR profile for a specific primer pair that detected one copy of proviral plasmid DNA after dot-blot hybridization was considered the optimum PCR profile. Gag primer pairs detected HIV-1 DNA in all 22 samples. The env primer pair did not detect most HIV-1 subtype C samples. When the researchers increased the number of cycles and time per cycle, the env primer pair achieved 100% sensitivity. When they decreased the annealing temperature and increased the individual cycle times, the LTR primer pairs detected all samples. These findings support that optimization of a PCR assay is necessary to achieve high assay sensitivity, specificity, and reproducibility and that PCR sensitivity should be considered seriously when interpreting PCR results for HIV diagnosis.

Human papillomavirus DNA in oral squamous cell carcinomas from an African population sample.

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.

Prevalence of EBV in oral squamous cell carcinomas in young patients.

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.

Correlation between p53 gene mutation, p53 protein labeling and PCNA expression in oral squamous cell carcinomas.

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.

FHIT RNA and protein expression in oral squamous cell carcinomas.

BACKGROUND: To investigate the possible role of FHIT, a possible tumour suppressor gene, in oral carcinogenesis, we examined 17 oral squamous cell carcinomas (OSCCs) for genetic alterations. MATERIALS AND METHODS: Fresh tissue was obtained during surgery, snap-frozen in liquid nitrogen and stored at -70 degrees C. Nested PCR amplification to examine the integrity of FHIT mRNA was performed on the reverse transcribed complementary DNA obtained from the frozen normal and tumour tissue. Immunohistochemistry was done on formal in-fixed paraffin-embedded tissue protein from the same cases using a polyclonal antiserum against the full length Fhit. RESULTS: Twelve out 17 (71%) OSCCs showed reduced or absent Fhit protein and half of the cases with reduced Fhit protein exhibited aberrant RT-PCR products. CONCLUSION: Immunohistochemical detection of Fhit protein expression in OSCCs is the more sensitive method to determine the status of Fhit in these tumours, in agreement with previous studies of other tumour types.

Warthin's tumour is not an Epstein-Barr virus related disease.

BACKGROUND: The histogenesis of Warthin's tumour (WT) is controversial. A possible role for Epstein-Barr virus (EBV) has been suggested. MATERIALS AND METHODS: Twenty formaldehyde-fixed, paraffin-embedded blocks of WT from the parotid gland were examined for the presence of EBV. In situ hybridisation was performed using EBV encoded small nuclear RNAs (EBER1/2) probes labelled with fluorescein isothiocyanate. An EBV-positive P3HR-1 cell line processed to paraffin wax was used as a positive control and a brain section as negative control. RESULTS: EBER1/2 could not be found in the neoplastic epithelial cells in any of the tumours nor in the adjacent normal parotid tissues. Individual positive lymphocytes were present in 7 tumours. CONCLUSIONS: These results indicated that EBV is not involved in the pathogenesis of WT.