Micaela Poetsch

Significant genetic differentiation between Poland and Germany follows present-day political borders, as revealed by Y-chromosome analysis

To test for human population substructure and to investigate human population history we have analysed Y-chromosome diversity using seven microsatellites (Y-STRs) and ten binary markers (Y-SNPs) in samples from eight regionally distributed populations from Poland (n=913) and 11 from Germany (n=1,215). Based on data from both Y-chromosome marker systems, which we found to be highly correlated (r=0.96), and using spatial analysis of the molecular variance (SAMOVA), we revealed statistically significant support for two groups of populations: (1) all Polish populations and (2) all German populations. By means of analysis of the molecular variance (AMOVA) we observed a large and statistically significant proportion of 14% (for Y-SNPs) and 15% (for Y-STRs) of the respective total genetic variation being explained between both countries. The same population differentiation was detected using Monmonier’s algorithm, with a resulting genetic border between Poland and Germany that closely resembles the course of the political border between both countries. The observed genetic differentiation was mainly, but not exclusively, due to the frequency distribution of two Y-SNP haplogroups and their associated Y-STR haplotypes: R1a1*, most frequent in Poland, and R1*(xR1a1), most frequent in Germany. We suggest here that the pronounced population differentiation between the two geographically neighbouring countries, Poland and Germany, is the consequence of very recent events in human population history, namely the forced human resettlement of many millions of Germans and Poles during and, especially, shortly after World War II. In addition, our findings have consequences for the forensic application of Y-chromosome markers, strongly supporting the implementation of population substructure into forensic Y chromosome databases, and also for genetic association studies.

PTEN/MMAC1 in malignant melanoma and its importance for tumor progression

A novel tumor suppressor gene, PTEN/MMAC1, on 10q23, displayed a number of mutations in solid tumors as gliomas and breast cancer. Aberrations of the long arm of chromosome 10 have been frequently detected in tumor progression of malignant melanoma of the skin by a variety of methods including cytogenetic analysis, fluorescence in situ hybridization and loss of heterozygosity analysis. Compared to previous studies, which propose an involvement of PTEN/MMAC1 in malignant melanoma mostly on the basis of data derived from cell lines and metastases, we analyzed a broader spectrum of exclusively patient derived tumor tissue by PCR and direct sequencing analysis of PTEN/MMAC1. Here, we present data of 25 primary melanomas (8 superficial spreading melanomas, 17 nodular melanomas) and 25 metastases of 41 patients. Neither loss of the complete gene nor a whole exon nor any nonsense mutations could be demonstrated. However, we detected several polymorphisms and some mutations in the introns, and in two metastatic tumors mutations with an amino acid change. Our results obtained from tissue samples underline that mutations of PTEN/MMAC1 are not an essential event in the onset of malignant melanoma of the skin, but could have an impact on tumor progression.

Different risk factors in basaloid and common squamous head and neck cancer.

Objectives/Hypothesis: The prevalence of human papillomavirus (HPV), herpes simplex virus (HSV), cigarette smoking and alcohol abuse was compared between two histological subgroups of head and neck cancer.

Down-regulation of the metastasis suppressor protein KAI1/CD82 correlates with occurrence of metastasis, prognosis and presence of HPV DNA in human penile squamous cell carcinoma

In penile squamous cell carcinoma (PSCC), the outcome largely depends on early detection and resection of inguinal lymph node metastases. We investigated the role of metastasis suppressor protein kang ai 1 (KAI1)/cluster of differentiation 82 (CD82), which is known to be of prognostic significance for a wide variety of cancers. Moreover, we analysed the tumours for human papillomavirus (HPV) DNA and loss of heterozygosity at the 11p11.2 locus. Tissue samples of 30 primary PSCCs were investigated immunohistochemically using an anti-KAI1/CD82 polyclonal antibody. The expression was assessed according to the degree of KAI1/CD82-positive tumour cells as positive, decreased or negative. The presence of HPV6/11, HPV16 and HPV18 DNA was analysed by polymerase chain reaction. All patients with decreased or negative expression of KAI1/CD82 in primary lesions had lymph node metastases (pu2009=u20090.0002). Patients with positive KAI1/CD82 expression showed a significant better prognosis for survival compared to the other groups (pu2009=u20090.0042). Presence of HPV DNA was associated with decreased or negative KAI1/CD82 expression. Lacking or decreased expression of metastasis suppressor gene KAI1/CD82 appears to be a prognostic parameter for the occurrence of lymph node metastases in PSCC. Our study suggests an association of decreased KAI1/CD82 expression with tumour progression, development of metastases and disease-specific death.

Frameshift mutations of RIZ , but no point mutations in RIZ1 exons in malignant melanomas with deletions in 1p36

Recently, the retinoblastoma protein interacting zinc finger gene RIZ has been proposed as a candidate for the tumor suppressor locus on 1p36, because of the common loss of RIZ1 RNA in human tumors. In addition, frameshift mutations of this gene have been demonstrated in a variety of tumors with microsatellite instability. Since alterations in this region have been described in malignant melanomas, we investigated DNA of paraffin-embedded sections from 16 typical naevi, 19 atypical naevi, 33 primary melanoma lesions and 25 metastases and DNA from four melanoma cell lines by PCR and direct sequencing analysis of RIZ. Frameshift mutations in the RIZ gene were found in 17% of melanoma samples and 8.6% of naevi, but we could not demonstrate any missense mutations in the exons of RIZ1. No LOH of the RIZ gene nor any microsatellite instability in six dinucleotide markers or in the mononucleotide repeats IGRIIR, hMSH3, and hMSH6 could be demonstrated in the samples with RIZ frameshift mutations. Although our results do not explain the high rate of deletions in 1p36 found in this tumor, they assign RIZ a potential role in the multi-step tumor forming process of malignant melanoma of the skin.

Expression of AP-2α, c-kit, and cleaved caspase-6 and -3 in naevi and malignant melanomas of the skin. A possible role for caspases in melanoma progression?

Progression of melanoma is associated with loss of the transcription factor AP‐2α and tyrosine‐kinase receptor c‐kit. However, the mechanisms by which these two proteins are down‐regulated have not been fully elucidated. Fifty non‐selected melanomas comprising ten superficial spreading melanomas (five exhibiting a radial growth phase and five a vertical growth phase), ten primary nodular melanomas, 30 melanoma metastases, and 16 naevi were investigated by direct sequencing analysis of the AP‐2α and c‐kit genes and by immunohistochemistry for the respective proteins. Because it has recently been demonstrated that AP‐2α is preferentially cleaved by caspase‐6 and to a lesser extent by caspase‐3, immunohistochemistry for the cleaved (activated) forms of caspase‐6 (c‐casp‐6) and caspase‐3 (c‐casp‐3) was carried out. No mutations were identified in the c‐kit gene, but three different point mutations were demonstrated in the activation motif of AP‐2α in four tumours: one vertical growth phase superficial spreading melanoma, one nodular melanoma, and two metastases. Immunohistochemistry revealed progressive loss of the AP‐2α and c‐kit proteins in primary melanomas and metastases when compared with naevi. The decrease of both markers was more accentuated in the dermal component of all primary tumours, with c‐kit more affected than AP‐2α. All invasive melanomas and metastases expressed c‐casp‐6. c‐casp‐3 was expressed by 83% of the metastases and in the dermal component of one nodular melanoma. These findings suggest that the loss of AP‐2α protein expression during the progression of melanoma could be related to mutation of the gene in only a small number of tumours, whereas the expression and activation of caspases, most prominently caspase‐6, may be an important factor for the down‐regulation of AP‐2α protein. Furthermore, this study supports recent data that the activation of caspases does not inevitably result in apoptosis, but may also contribute to tumour progression in melanomas. Copyright

An Increased Frequency of Numerical Chromosomal Abnormalities and 1p36 Deletions in Isolated Cells from Paraffin Sections of Malignant Melanomas by Means of Interphase Cytogenetics

At present, little information is available on tumor and stage-specific chromosomal aberrations in malignant melanoma. Therefore, we applied fluorescence in situ hybridization on isolated interphase cells from paraffin sections of 25 cases of malignant melanomas, comprising 17 primary tumors (PTs) and 8 metastases (MTs) in various anatomical sites. We used centromeric probes for chromosomes 1, 7, 9, 10, 11, 12, 15, 17, 18, X, and Y and a midisatellite probe localized in 1p36. Four of the PTs and 5 of the MTs showed polyploidy for all applied probes. The most frequent type of numerical aberration was an overrepresentation of chromosomes 1 (3 PTs, 5 MTs) and 7 (3 PTs, 1 MT), and an underrepresentation of chromosomes 9 (3 PTs) and 10 (6 PTs, 5 MTs). The Y chromosome was lost in all male tumors. In addition, we observed monosomy 11, 12, 15, 17 or 18, and trisomy 12 or 17. Only 1 PT showed no aberrations for any applied DNA probe. A deletion in the near-telomeric region of 1p36 was found surprisingly often (9 PTs, 7 MTs). Our results suggest that the loss of gene(s) in this region is an important event in the pathogenesis of malignant melanoma of the skin.

Mitochondrial DNA instability in malignant melanoma of the skin is mostly restricted to nodular and metastatic stages.

Ultraviolet (UV) radiation is thought to be a major contributor to the development of sporadic malignant melanoma of the skin. It may induce alterations in genomic or mitochondrial DNA (mtDNA), especially C to T or CC to TT changes. Mutations or other alterations in mtDNA have been reported in a variety of human cancers and may be due to different mechanisms. In this study, we have attempted to elucidate whether aberrations in the mtDNA of melanoma are due to UV radiation or other factors by investigating two parts of the mitochondrial D-loop and two mitochondrial genes, as well as looking for the Δ4977 mtDNA deletion and mtDNA duplications, in 61 primary malignant melanomas and neighbouring normal skin tissue (in 70% of primary tumours; otherwise, corresponding blood samples). Point mutations were a rare feature, occurring in only seven tumour samples and never as a C to T change, whereas mtDNA instability in the D-loop (mtMSI) was found in 13% of primary nodular tumours and 20% of metastases. A de novo Δ4977 mtDNA deletion was demonstrated in 10% of melanomas; in 20% of patients, mtDNA duplications and/or the Δ4977 mtDNA deletion was detectable. Our data indicate that mtDNA alterations in malignant melanoma are not induced by UV radiation. In addition, point mutations and mtMSI were mostly a feature of nodular and metastatic melanoma samples.

Mitochondrial DNA in the central european population: Human identification with the help of the forensic mt-DNA D-Loop-Base Database

Sequencing of mtDNA is an advanced method for the individualisation of traces. Disadvantages of this method are expensive and time-consuming analysis and evaluation procedures as well as the necessary stock of population-genetic data which is still insufficient. Central European institutes of forensic medicine from Germany, Austria, and Switzerland have been working together since the beginning of 1998 to establish a mtDNA database. The aim is to build up a large stock of forensically established data and provide population-genetic data for frequency investigations, which will serve as a basis for expert opinions and scientific research. Good data quality is ensured by using original sequences only. Ring tests, which have been conducted to enhance analytical reliability, revealed a high correspondence rate of the analytical results obtained by the individual member institutes. Today 1410 sequences are available for comparison, of which 1285 sequences in the HV1 and HV2 regions cover the full ranges from 16051 to 16365 and from 73 to 340 (according to Anderson). The major part is formed by Central European sequences comprising 1256 data sets from Germany, Austria, and Switzerland. Today the database contains sequences from a total of 12 European, six African and three Asian countries including 100 sequences from Japan. This paper is aimed at discussing the individualisation potentials of mtDNA as well as the possibilities and limits of ethnic differentiation by means of pairwise sequence differences on the basis of the data stock available.

Significance of the small subtelomeric area of chromosome 1 (1p36.3) in the progression of malignant melanoma: FISH deletion screening with YAC DNA probes

Abstractu2002The short arm of chromosome 1 (1p), especially the subtelomeric region of 1p36, is a common site for abnormalities in malignant melanoma of the skin. In a recent study nodular melanomas displayed deletions of 1p36 in an augmented percentage of cases. To evaluate the dimension of these deletions and to study their significance for the progression of malignant melanoma we analyzed seven melanoma cell lines, 32 primary tumors, and 32 metastatic tumors by fluorescence in situ hybridization with the DNA probe D1Z2 in 1p36.3 and eight YAC DNA probes hybridizing to 1p36, 1p32, 1p31, and 1p21. All cell lines, 91% of the metastatic tumors and 63% of nodular melanomas showed a deletion of 1p36.3. In the YAC hybridization experiments, the most frequent deletions were found in 1p36 in all cell lines, in 13% of nodular melanoma, and in 44% of metastatic tumors. Deletions in 1p36 were mostly confined to a rather small area near the locus D1Z2. The frequent occurrence of this deletion in melanomas with a high metastatic potential and the abundant accumulation of this deletion in metastasis point to genes located on 1p36, which might be of significance for the metastatic capability of malignant melanoma.