TUNEL assay and SCSA determine different aspects of sperm DNA damage.

For the determination of sperm DNA damage, different assays are used. However, no further distinction is made and the literature generally speaks about DNA damage. Thus, this study aimed at comparing the sperm chromatin structure assay (SCSA) and the TUNEL assay. In 79 patients, sperm DNA damage was determined flow cytometrically using the SCSA and the TUNEL assay. Moreover, normal sperm morphology was evaluated according to strict criteria. A statistical comparison of the two methods was performed using standard correlations, Bland and Altman plots, Passing-Bablok regressions and concordance correlation. Results show a significant difference between P- and G-pattern morphology only for the mean channel fluorescence of the SCSA. Spearman's rank correlations between the different parameters of both assays, SCSA and TUNEL, revealed significant associations between the parameters of the assays. However, when applying Bland and Altman plots, Passing-Bablok regression and concordance correlation results showed that these methods are not comparable. These different techniques determine different aspects of sperm DNA damage, i.e. 'real' DNA damage for the TUNEL assay and 'potential' DNA damage in terms of susceptibility to DNA denaturation for the SCSA. Thus, one should clearly distinguish between the different assays, not only practically and methodologically but also linguistically.

Ultrastructural investigation of Zimmermann-Laband syndrome.

Zimmermann-Laband syndrome (ZLS) is a very rare autosomal dominant inherited condition characterized by 3 major clinical findings of which gingival hyperplasia are always present. The great heterogenicity of the syndrome is illustrated by the numerous variable clinical findings described in the literature. The purpose of the study was to examine a patient diagnosed with ZLS and to describe possible new characteristics of this rare syndrome, including the ultrastructural morphology using a transmission electron microscope (TEM) of the gingival and dermal fibroblasts. The ultrastrucutral morphology as has not previously been described in the literature. Tissue was collected from the alveolar ridge and skin of the forearm for TEM. TEM studies indicated the presence of prominent fibroblasts situated among numerous regular dense connective tissue bundles. Genetic analysis showed a new chromosomal insertion, ins(12;8)(p11.2;q11.2q24.3), suggesting that the gene responsible for the syndrome lies on chromosome 8.

Cavernous sinus anatomy as a basis for interpretation of the clinical picture and radiological investigations in a case of Entomophthorales infection.

Several cranial nerves traverse the cavernous sinus producing the typical symptom complex seen during cavernous venous sinus thrombosis in Mucorales infection. Fungi of the order Entomophthorales display different pathological and histological characteristics although belonging to the same class of fungi. A case is presented, wherein the anatomy of the cavernous sinus forms the basis in explaining the presenting symptoms of a patient with Entomophthorales infection. The anatomical explanation for the presenting neurological symptoms is confirmed by radiological investigations and further supports the diagnosis of Entomophthorales infection.

Detection of villous conidia of Conidiobolus coronatus in a blood sample by scanning electron microscopy investigation.

Conidiobolus coronatus is a major insect pathogen belonging to the fungal order Entomophthorales, causing a rare subcutaneous infection of the nasofacial region, resulting in swelling of predominantly the nose, mouth, and perinasal tissue. Later in the course of the infection firm, painless, subcutaneous nodules develop that are attached to the underlying tissues but not to the skin. No morphological studies are available in the literature on the morphology of C. coronatus in vivo and all morphological studies have been conducted on in vitro cultures. Here the authors report on the ultrastructural pathology as seen with a scanning electron microscope (SEM) of villous conidia of C. coronatus, detected in a 37-year-old woman who presented to the casualty department at Pretoria Academic Hospital, South Africa with left-sided facial pain and headache. The diagnosis of C. coronatus was confirmed by LightCycler real-time flourescence PCR technique. Research shows that typically diagnosis of the pathogen is established only on histological examination, and in over 85% of cases cultures for the causative organism is negative. This pathogen has not previously been found in a blood sample and the authors present for the first time the morphology of C. coronatus in blood using the SEM.

Rhino-orbitocerebral entomophthoramycosis.

Conidiobolus coronatus is recognized as a human pathogen causing subcutaneous fungal infection of the face in immunocompetent patients. The disease process is usually benign. We report, what we believe to be the first case of intracranial extension of C. coronatus producing rhino-orbitocerebral syndrome, and subsequent dissemination of C. coronatus in an immunocompetent patient.

Allelic variation in the promoter region of the LDL receptor gene: analysis of an African-specific variant in the FP2 cis-acting regulatory element.

DNA samples of 2303 individuals from nine different population groups were screened for variant -175g-->t in the promoter region of the low-density lipoprotein receptor (LDLR) gene. The -175g-->t variant detected at carrier frequencies of 3-10% in different African population groups was absent in the Caucasian and Asian (Chinese) individuals studied. In contrast to previous findings in Black South Africans where this polymorphism predominated in patients with familial hypercholesterolaemia (FH), it occurred at a significantly lower frequency in hypercholesterolaemics from the recently admixed Coloured population of South Africa compared with population-matched controls (P<0.0001). Haplotype and mutation analysis excluded the likelihood that this finding is due to association with a specific disease-related mutation in FH patients, although reversal of the positive association with FH observed in the Black population may, at least in part, be due to admixture linkage disequilibrium. Transient transfection studies in HepG2 cells demonstrated that the -175t allele is associated with a non-significant decrease ( approximately 7%) of LDLR transcription in the absence of sterols. The data presented in this study raise the possibility that the -175g-->t polymorphism may have subtle effects that become clinically important within certain genetic and/or environmental contexts.

Predominance of a 6 bp deletion in exon 2 of the LDL receptor gene in Africans with familial hypercholesterolaemia.

In South Africa, the high prevalence of familial hypercholesterolaemia (FH) among Afrikaners, Jews, and Indians as a result of founder genes is in striking contrast to its reported virtual absence in the black population in general. In this study, the molecular basis of primary hypercholesterolaemia was studied in 16 Africans diagnosed with FH. DNA analysis using three screening methods resulted in the identification of seven different mutations in the coding region of the low density lipoprotein (LDLR) gene in 10 of the patients analysed. These included a 6 bp deletion (GCGATG) accounting for 28% of defective alleles, and six point mutations (D151H, R232W, R385Q, E387K, P678L, and R793Q) detected in single families. The Sotho patient with missense mutation R232W was also heterozygous for a de novo splicing defect 313+1G-->A. Several silent mutations/polymorphisms were detected in the LDLR and apolipoprotein B genes, including a base change (g-->t) at nucleotide position -175 in the FP2 LDLR regulatory element. This promoter variant was detected at a significantly higher (p<0.05) frequency in FH patients compared to controls and occurred in cis with mutation E387K in one family. Analysis of four intragenic LDLR gene polymorphisms showed that the same chromosomal background was identified at this locus in the four FH patients with the 6 bp deletion. Detection of the 6 bp deletion in Xhosa, Pedi, and Tswana FH patients suggests that it is an ancient mutation predating tribal separation approximately 3000 years ago.

Mutation -59c-->t in repeat 2 of the LDL receptor promoter: reduction in transcriptional activity and possible allelic interaction in a South African family with familial hypercholesterolaemia.

The low-density lipoprotein receptor (LDLR) plays a major role in cholesterol homeostasis. Mutations in the regulatory region of the LDLR gene, although rare, have been shown to alter transcriptional activity of the gene and can cause familial hypercholesterolaemia (FH). In this study, a transition (c-->t) was identified at nucleotide position -59 within repeat 2 of the LDLR promoter in a South African FH patient of mixed ancestry. By screening 17 family members of the index case for this promoter mutation, two additional single base changes (-124c-->t and-175g-->t) were identified, located at recently described cis- acting regulatory sequences of the LDLR promoter. Both the-59c-->t and the-124c-->t transitions were identified in the normocholesterolaemic son of the index patient. Reporter plasmids containing the normal and mutant promoter fragments were constructed by directional cloning. Transcription studies using a luciferase reporter system demonstrated that the-59c-->t mutation significantly reduces promoter activity in both the presence and absence of sterols ( approximately 40% of normal activity), while the-124c-->t variant increases transcription ( approximately 160%) of the LDLR gene. The intra-familial phenotypic variability observed amongst individuals with the-59c-->t mutation can probably be ascribed to allelic interaction, suggesting that variation in the LDLR promoter region may contribute significantly to the phenotypic expression of FH-related mutations in populations where these mutations prevail.

Ethnic variation and in vivo effects of the -93t-->g promoter variant in the lipoprotein lipase gene.

Recently, a (t-->g) transition at nucleotide -93 in the lipoprotein lipase (LPL) gene promoter has been observed in Caucasians. Here, we have compared the frequency of the -93g carriers in three distinct populations (Caucasians, South African Blacks, and Chinese). The carrier frequency in the Caucasian population was 1.7% (4/232), which was in contrast to the South African Black population, which had a frequency for this allele of 76.4% (123/161) of the individuals tested. This transition was not observed in the Chinese population under study. Near complete linkage disequilibrium between the -93g and the previously described D9N mutation was observed in the Caucasian population but not in South African Blacks. To further assess the ancestral origins of these DNA changes, DNA haplotyping using a CA repeat 5' to these substitutions was performed. The -93t allele was associated with only a few specific dinucleotide repeat sizes. In contrast, the -93g allele occurred on chromosomes with many different repeat lengths. The broad distribution of repeats on -93g carrying chromosomes, their high frequency in the South African Black population, and the conservation of the -93g allele among different species may suggest that the -93g allele is the ancestral allele on which a transition to t and the D9N mutations arose. The very high frequency of the -93g allele distinct from the N9 allele in a cohort of Black South Africans allowed us to specifically assess the phenotypic effects of the -93g allele on lipids. Individuals homozygous for the g allele at -93 showed mildly decreased triglycerides compared with individuals homozygous for the t allele (1.14 +/- 0.66 mmol/L versus 0.82 +/- 0.3; P = .04). Thus, the -93g allele in this cohort is associated with low plasma triglyceride levels.