MtDNA diversity of Ghana: a forensic and phylogeographic view.

West Africa is characterized by a migration history spanning more than 150,000 years. Climate changes but also political circumstances were responsible for several early but also recent population movements that shaped the West African mitochondrial landscape. The aim of the study was to establish a Ghanaian mtDNA dataset for forensic purposes and to investigate the diversity of the Ghanaian population sample with respect to surrounding populations. We sequenced full mitochondrial control regions of 193 Akan people from Ghana and excluded two apparently close maternally related individuals due to preceding kinship testing. The remaining dataset comprising 191 sequences was applied as etalon for quasi-median network analysis and was subsequently combined with 99 additional control region sequences from surrounding West African countries. All sequences were incorporated into the EMPOP database enriching the severely underrepresented African mtDNA pool. For phylogeographic considerations, the Ghanaian haplotypes were compared to those of 19 neighboring populations comprising a total number of 6198 HVS1 haplotypes. We found extensive genetic admixture between the Ghanaian lineages and those from adjacent populations diminishing with geographical distance. The extent of genetic admixture reflects the long but also recent history of migration waves within West Africa mainly caused by changing environmental conditions. Also, evidence for potential socio-economical influences such as trade routes is provided by the occurrence of U6b and U6d sequences found in Dubai but also in Tunisia leading to the African West Coast via Mauritania and Senegal but also via Niger, Nigeria to Cameroon.

Allele frequencies of 11 X-chromosomal loci in a population sample from Ghana.

Eleven X-chromosomal short tandem repeats (STRs) from two multiplex PCR approaches (DXS6807, DXS8378, DXS7132, DXS6800, DXS9898, DXS7424, DXS101, DXS7133, HPRTB, DXS8377, and DXS7423), located in four different X-chromosomal linkage groups, were typed in a population sample from Ghana, Africa. After genotyping unrelated men (129) and women (114) from the Ashanti population, forensic efficiency parameters such as polymorphism information content and mean exclusion chance were calculated. A deviation from the Hardy-Weinberg equilibrium could not be found. The investigation of 11 father-daughter and seven mother-son meioses revealed no mutations in any STR analyzed. Our data were compared with European, African-American, and Asian populations from the literature.

Amelogenin sex determination by pyrosequencing of short PCR products.

We developed an assay, which allows the sex determination of human DNA samples by pyrosequencing of short PCR products. A 48/45-bp stretch including primers of the amelogenin gene with a 3-bp insertion on the Y chromosome was chosen for analysis. In an initial study, we correctly typed 50 male and 50 female DNA samples from unrelated donors. First experiments with forensic samples, which failed in conventional analyses, indicate that this approach might be an advantage when dealing with degraded DNA.

NHE3 in the human brainstem: implication for the pathogenesis of the sudden infant death syndrome (SIDS)?

Previous studies have demonstrated an inverse correlation between the degree of respiratory drive and NHE3 mRNA expression in the brainstem of awake rabbits. Here we show that the levels of NHE3 mRNA extractable from kryo-conserved tissue are highly variable also in the human brainstem. As an insufficient drive to breath may be a final event causing sudden infant death, we compared the expression of NHE3 mRNA in a collective of children who died from non-natural causes to an equal number of SIDS victims. Evaluation of signals from NHE3 RT-PCR showed higher values for the SIDS collective than for the control group. We suggest that the level of NHE3 expression in brainstem tissue may contribute to the vulnerability of infants for SIDS.

Genetic investigation of the TSPYL1 gene in sudden infant death syndrome.

BACKGROUND: Sudden infant death syndrome (SIDS) constitutes the most frequent cause of death in the postperinatal period in Germany. Recently, a lethal phenotype characterized by sudden infant death with dysgenesis of the testes syndrome (SIDDT) was identified to be caused by loss of function mutations in the TSPYL1 gene. PURPOSE: The study's purpose was to reveal a possible role of TSPYL1 in SIDS. METHODS: DNA samples of 126 SIDS cases and 261 controls were investigated. RESULTS: We found five sequence variations, each of them causing an amino acid substitution. No Hardy Weinberg disequilibrium and no significant difference in allele frequencies between patients and controls were observed for any variation. In one female patient a p.F366L amino acid polymorphism was found heterozygous, which could not be displayed in controls. A pathogenic implication of this substitution, which is conserved in primates and rodents, cannot be ruled out completely. Because SIDDT is the result of homozygous TSPYL1 mutations, this heterozygous exchange cannot solely explain the sudden death in this child. The reported mutation associated with SIDDT (457_458insG) was not detectable in our cohort. CONCLUSION: No association of sequence variations in the TSPYL1 gene and SIDS has been found in a German cohort. Genetic analysis of TSPYL1 seems to be of limited significance in the differential diagnosis of SIDS without dysgenesis of the testes.

Protocols for ancient DNA typing.

Molecular analysis of fossil and archaeological remains has been established as a powerful tool in providing new insight in phylogenetic investigations. The overlapping set of molecular modifications and degradation that forensic samples share with archaeological specimen suggests the application of similar technical approaches to the respective biological material. Polymerase chain reaction is the molecular technique of choice for the retrieval of specimen deoxyribonucleic acid (DNA) molecules. Because of intrinsic sensitivity, potential contaminations from exogenous DNA sources must be monitored through the entire process by the introduction of multiple blank controls. Cloning and sequencing of polymerase chain reaction products often is the only way to discriminate between contaminations and endogenous sequences as well as to identify variable positions from nucleotide modifications/DNA polymerase errors. Phylogenetic analysis and investigations of the pattern of substitutions are an additional and necessary step to validate the retrieved sequence. Comparison with available related samples (modern or extinct) is critical to correctly validate the results and to avoid artifactual data.

Tumor classification based on gene expression profiling shows that uveal melanomas with and without monosomy 3 represent two distinct entities.

Uveal melanoma is the most common intraocular malignancy. About 50% of patients die of metastases, which almost exclusively originate from primary tumors that have lost one chromosome 3 (monosomy 3). To gain insight into the biological mechanisms that underlie the various metastasizing potential of uveal melanoma, we have determined gene expression levels in 20 primary tumors using oligonucleotide microarrays containing 12500 probe sets. The expression measurements of those 7902 genes that were expressed in more than 10% of tumors were analyzed using two different statistical approaches. We used a modified Wilcoxon rank-sum test to identify genes differentially expressed between tumors with and without monosomy 3. Seven genes showed complete loss of expression in tumors with monosomy 3 but were expressed in tumors with disomy 3. Two of them, CHL1 and fls485, are located within or close to the uveal melanoma susceptibility locus UVM2 at 3p25. However, mutation analysis of both genes in eight tumors with monosomy 3 did not reveal structural or epigenetic alteration. To identify tumor classes, we performed unsupervised hierarchical cluster analysis; this approach separated uveal melanomas into two groups. We found that this classification is strikingly robust because, when tested by "resampling," the same grouping is obtained from 47 of 50 subsamples of genes. In clusterings of the three remaining subsamples, the grouping of only one tumor does not conform with the original classification. Excluding this tumor, cluster analyses of subsamples containing as few as 300 randomly chosen genes consistently result in the same classification, thus indicating that the difference between the two tumor classes is pervasive. Interestingly, all of the tumors in one of the groups have disomy 3, whereas all of the others have monosomy 3. Our findings suggest that there are two distinct entities of uveal melanoma that were previously unrecognized because they are not obviously distinguishable by clinicopathological features.

Methylation analysis of several tumour suppressor genes shows a low frequency of methylation of CDKN2A and RARB in uveal melanomas.

We have investigated the frequency of methylation of several tumour suppressor genes in uveal melanoma. As the loss of one copy of chromosome 3 (monosomy 3), which is found in about half of these tumours, is tightly associated with metastatic disease, a special emphasis was laid on genes located on this chromosome, including the fragile histidine triad (FHIT), von Hippel-Lindau (VHL), beta-catenin (CTNNB1), activated leukocyte cell adhesion molecule (ALCAM) and retinoic acid receptor-beta2 (RARB) genes. In addition, the methylation patterns of the CpG-rich regions 5' of the E-cadherin (CDH1), p16/cyclin-dependent kinase inhibitor 2 A (CDKN2A) and retinoblastoma (RB1) genes were analysed by bisulphite genomic sequencing or methylation-specific PCR (MSP). Furthermore, the SNRPN and D15S63 loci, which are located in the imprinted region of chromosome 15, were included in the study. Aberrant methylation was detected in nine of 40 tumours analysed: The imprinted SNRPN and D15S63 loci were hypermethylated in three tumours, all of which retained both copies of chromosome 3. Methylated RARB alleles were detected in three tumours, whereas in three other tumours CDKN2A was found to be methylated. As we did not find RARB and CDKN2A preferentially methylated in tumours with monosomy 3, which is a significant predictor of metastatic disease, we suggest that these genes may play a causative role in the formation of uveal melanoma but not in the development of metastases.