Congenital Adrenal Hyperplasia with Non-functional Mutations in Both Alleles in a Clinically Unaffected Infant.

BACKGROUND: Results in neonatal screening programs aiming at detection of congenital adrenal hyperplasia (CAH) can only report elevated levels of 17-hydroxy-progesterone (17-OHP), without being able to differentiate presence or absence of salt loss. AIM: To predict presence or absence of salt loss in newborn infants with CAH. METHODS: The first specimen of suspected CAH in samples sent from People's Democratic Republic of Laos (Lao PDR) was investigated for known mutations in CAH associated with salt loss. RESULTS: Molecular genetic diagnosis revealed mutations associated with loss of function in both alleles; however, the infant was clinically unaffected even without any corticosteroid substitution therapy. CONCLUSIONS: Although molecular genetic methods can theoretically predict loss of function in CAH, our infant was clinically unaffected even without therapy at 6 years of age. We speculate that in CAH, remaining enzyme activity can be sufficiently high, despite the presence of loss of function mutations, which do not affect infants clinically.

New model for gastroenteropancreatic large-cell neuroendocrine carcinoma: establishment of two clinically relevant cell lines.

Recently, a novel WHO-classification has been introduced that divided gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) according to their proliferation index into G1- or G2-neuroendocrine tumors (NET) and poorly differentiated small-cell or large-cell G3-neuroendocrine carcinomas (NEC). Our knowledge on primary NECs of the GEP-system is limited due to the rarity of these tumors and chemotherapeutic concepts of highly aggressive NEC do not provide convincing results. The aim of this study was to establish a reliable cell line model for NEC that could be helpful in identifying novel druggable molecular targets. Cell lines were established from liver (NEC-DUE1) or lymph node metastases (NEC-DUE2) from large cell NECs of the gastroesophageal junction and the large intestine, respectively. Morphological characteristics and expression of neuroendocrine markers were extensively analyzed. Chromosomal aberrations were mapped by array comparative genomic hybridization and DNA profiling was analyzed by DNA fingerprinting. In vitro and in vivo tumorigenicity was evaluated and the sensitivity against chemotherapeutic agents assessed. Both cell lines exhibited typical morphological and molecular features of large cell NEC. In vitro and in vivo experiments demonstrated that both cell lines retained their malignant properties. Whereas NEC-DUE1 and -DUE2 were resistant to chemotherapeutic drugs such as cisplatin, etoposide and oxaliplatin, a high sensitivity to 5-fluorouracil was observed for the NEC-DUE1 cell line. Taken together, we established and characterized the first GEP large-cell NEC cell lines that might serve as a helpful tool not only to understand the biology of these tumors, but also to establish novel targeted therapies in a preclinical setup.

Impact of the 3D microenvironment on phenotype, gene expression, and EGFR inhibition of colorectal cancer cell lines.

Three-dimensional (3D) tumor cell cultures grown in laminin-rich-extracellular matrix (lrECM) are considered to reflect human tumors more realistic as compared to cells grown as monolayer on plastic. Here, we systematically investigated the impact of ECM on phenotype, gene expression, EGFR signaling pathway, and on EGFR inhibition in commonly used colorectal cancer (CRC) cell lines. LrECM on-top (3D) culture assays were performed with the CRC cell lines SW-480, HT-29, DLD-1, LOVO, CACO-2, COLO-205 and COLO-206F. Morphology of lrECM cultivated CRC cell lines was determined by phase contrast and confocal laser scanning fluorescence microscopy. Proliferation of cells was examined by MTT assay, invasive capacity of the cell lines was assayed using Matrigel-coated Boyden chambers, and migratory activity was determined employing the Fence assay. Differential gene expression was analyzed at the transcriptional level by the Agilent array platform. EGFR was inhibited by using the specific small molecule inhibitor AG1478. A specific spheroid growth pattern was observed for all investigated CRC cell lines. DLD-1, HT-29 and SW-480 and CACO-2 exhibited a clear solid tumor cell formation, while LOVO, COLO-205 and COLO-206F were characterized by forming grape-like structures. Although the occurrence of a spheroid morphology did not correlate with an altered migratory, invasive, or proliferative capacity of CRC cell lines, gene expression was clearly altered in cells grown on lrECM as compared to 2D cultures. Interestingly, in KRAS wild-type cell lines, inhibition of EGFR was less effective in lrECM (3D) cultures as compared to 2D cell cultures. Thus, comparing both 2D and 3D cell culture models, our data support the influence of the ECM on cancer growth. Compared to conventional 2D cell culture, the lrECM (3D) cell culture model offers the opportunity to investigate permanent CRC cell lines under more physiological conditions, i.e. in the context of molecular therapeutic targets and their pharmacological inhibition.

KRAS p.G13D mutations are associated with sensitivity to anti-EGFR antibody treatment in colorectal cancer cell lines.

PURPOSE: Targeted therapies using the anti-EGFR antibodies panitumumab (Pmab) or cetuximab (Cmab) are currently restricted to patients with metastatic colorectal adenocarcinoma whose tumours do not show a mutation in KRAS. However, recent retrospective studies indicated that patients with tumours mutated in codon 13 of KRAS may benefit from treatment with Cmab in contrast to patients with tumours mutated in KRAS codon 12. METHODS: To study the functional impact of the subtype of KRAS mutations on the efficiency of EGFR-targeted therapies, we correlated the KRAS mutation status of 15 colorectal carcinoma cell lines with the in vitro sensitivity of these cells to Cmab/Pmab. Mutations in the potential predictive biomarkers BRAF and PIK3CA as well as protein expression of EGFR and PTEN were also determined. RESULTS: Four out of seven KRAS-mutated cell lines were characterised by the p.G13D mutation. Treatment of these cells using Cmab/Pmab induced a significant growth inhibition in contrast to cell lines showing a KRAS mutation at codon 12 or 61. Out of the eight KRAS wild-type cell lines, five were insensitive to Cmab/Pmab. These cell lines were characterised either by BRAF mutation or by absence of EGFR or PTEN protein expression. CONCLUSIONS: Since KRAS p.G13D-mutated tumour cells may respond to EGFR-targeted therapy, we suggest including subtype analysis of KRAS mutations in prospective clinical trials. In KRAS wild-type tumour cells, BRAF mutations and loss of EGFR or PTEN expression may lead to resistance to EGFR-targeted therapy and should be considered as additional negative predictive biomarkers.

Severe generalised rhabdomyolysis with fatal outcome associated with isotretinoin.

Isotretinoin is considered to be a safe and effective therapy in otherwise therapy-resistant acne. Elevated serum creatine phosphokinase values with or without muscle-related symptoms in isotretinoin-treated patients have been reported and interpreted as benign phenomena, lethal cases have not been described yet. We present the case of a 20-year-old male who died from severe generalised rhabdomyolysis associated with isotretinoin treatment.

Discovery of TP53 splice variants in two novel papillary urothelial cancer cell lines.

BACKGROUND: Using a novel cell culture technique, we established two new cell lines, BC44 and BC61, from papillary urothelial carcinoma and analyzed them for genetic changes typical of this tumor type. METHODS AND RESULTS: Karyotyping revealed aneuploid karyotypes with loss of chromosome 9 and rearranged chromosome 5p. Molecular analysis showed CDKN2A deletions but wild-type PIK3CA. BC61 contained a G372C FGFR3 mutation. TP53 was not mutated in either cell line and BC61 expressed normal full-length protein. In contrast, BC44 exclusively expressed cytoplasmic and nuclear p53Δ40 and 133 isoforms from the alternative promoter P2 as revealed by Western blotting, immunocytochemistry and PCR. The only discernible difference in TP53 in BC44 was homozygosity for the deletion allele of the rs17878362 polymorphism in the P2 promoter. Expression of p53 isoforms was also detected in a few other urothelial carcinoma cell lines and tumor cultures and in 4 out of 28 carcinoma tissues. CONCLUSION: In urothelial cancers, TP53 is typically inactivated by mutations in one allele and loss of the wildtype allele and more frequently in invasive compared to papillary carcinomas. We show that some urothelial carcinomas may predominantly or exclusively express isoforms which are not detected by commonly used antibodies to epitopes located in the p53 TA amino-terminal region. Expression of these isoforms may constitute a further mode of p53 inactivation in urothelial carcinoma. Our findings raise the question to which extent this mechanism may compromise wildtype p53 function in papillary tumors in particular, where point mutations in the gene are rare.

MtDNA typing of single-sperm cells isolated by micromanipulation.

Some sexual assault crimes constitute a problem for the legal institutions confronted with the DNA analysis of such cases. Often, sperm cells are found in the victim's vaginal tract during medical examination but their successful genotyping is compromised by the huge excess of the victim's epithelial cells as well as by the degradation of genomic DNA present in sperm cells as a consequence of female immune response. Mitochondrial DNA present in the mid-piece of sperm cells might be useful in some specific cases in order to differentiate the donors of a semen sample. The high number of copies per cell and its circular nature that may confer some protection from the action of exonucleases make it more suitable for cases where few cells are available and/or the DNA is degraded. We have developed a novel strategy for typing mtDNA from single-sperm cells. Specific amplification of male mitochondrial DNA is ensured by use of sequence specific primers designed on the basis of mitochondrial single nucleotide polymorphisms existent throughout the control region. The strategy was applied to single-sperm cells isolated by micromanipulation from slides smeared with vaginal swabs taken immediately after sexual intercourse of voluntary couples. After sequencing the PCR products, it was possible to obtain a match between the DNA sequence from the buccal swab and the DNA sequence of the single sperm-cell, for each voluntary man. With this new strategy, the problem of contamination with DNA from the victim observed when using universal primers was completely overtaken. This method will probably allow the resolution of multiple-rapist crimes, where the collected sperm cells can be separately typed.

[Autoerotic fatalities in Greater Dusseldorf]. / Autoerotische Todesfälle im Grossraum Düsseldorf.

Autoerotic fatalities in the Greater Dusseldorf area correspond to the relevant medicolegal literature. Our results included exclusively young to middle-aged, usually single men who were found dead in their city apartments. Clothing and devices used showed a great variety. Women's or fetish clothing and complex shackling or hanging devices were disproportionately frequent. In most cases, death occurred due to hanging or ligature strangulation. There was no increased incidence of underlying psychiatric disorders. In most of the deceased no or at least no remarkable alcohol intoxication was found. Occasionally, it may be difficult to reliably differentiate autoerotic accidents, accidents occurring in connection with practices of bondage & discipline, dominance & submission (BDSM) from natural death, suicide or homicide.

[Identification of an unknown body by means of osteosynthesis material]. / Identifizierung einer Unbekannten Leiche Anhand von Osteosynthesematerial.

The present case describes the identification of an unknown corpse recovered from water by means of two osteosynthesis plates with serial numbers. This method of identification is compared with the pros and cons of other possibilities of identification using medical findings. Although in Western Europe osteosynthesis material is normally removed some months after the operation, autoptic exposure of a suspicious bone may occasionally allow valid, fast and convenient identification of the body, especially if osteosynthesis material with different serial numbers was used.

Molecular characterization of commonly used cell lines for bone tumor research: a trans-European EuroBoNet effort.

Usage of cancer cell lines has repeatedly generated conflicting results provoked by differences among subclones or contamination with mycoplasm or other immortal mammalian cells. To overcome these limitations, we decided within the EuroBoNeT consortium to characterize a common set of cell lines including osteosarcomas (OS), Ewing sarcomas (ES), and chondrosarcomas (CS). DNA fingerprinting was used to guarantee the identity of all of the cell lines and to distinguish subclones of osteosarcoma cell line HOS. Screening for homozygous loss of 38 tumor suppressor genes by MLPA revealed deletion of CDKN2A as the most common event (15/36), strictly associated with absence of the CDKN2A (p16) protein. Ten cell lines showed missense mutations of the TP53 gene while another set of nine cell lines showed mutations resulting in truncation of the TP53 protein. Cells harboring missense mutations expressed high levels of nuclear TP53, while cell lines with nonsense mutations showed weak/absent staining for TP53. TP53(wt) cell lines usually expressed the protein in 2-10% of the cells. However, seven TP53(wt) osteosarcomas were negative for both mRNA and protein expression. Our analyses shed light on the correlation between immunohistochemical and genetic data for CDKN2A and TP53, and confirm the importance of these signaling pathways. The characterization of a substantial number of cell lines represents an important step to supply research groups with proven models for further advanced studies on tumor biology and may help to make results from different laboratories more comparable.

Allele frequencies for twelve STR loci in two Bulgarian populations: Bulgarians and Karakachani.

The allele frequencies of 12 STRs (D2S1338, D3S1358, D5S818, D8S1179, D16S539, D18S51, D19S433, D21S11, FGA, TH01, TPOX, VWA) from two Bulgarian population samples are presented: ethnic Bulgarians and Karakachani. The descent of Karakachani is not known. They are regarded as descendants of an older Balkan population, or that they may have nearer genetic relationships to the Aromuns. Using the allele frequencies of the 12 STR polymorphisms, the genetic relationships of Bulgarians and Karakachani to other Balkan populations have been studied. The results are compared with other population data, including two Aromun populations. While the Bulgarians show nearer relationships to other South Slavonian populations, there are no hints for nearer relationships between the Karakachani and other Balkan populations, especially the Aromuns. So, based on the 12 STRs, there is no indication for a closer relationship between Karakachani and Aromuns.

Hodgkin disease-like posttransplantation lymphoproliferative disorder of donor origin in a renal allograft recipient.

Posttransplantation lymphoproliferative disorder (PTLD) develops in 1.6% of renal allograft recipients. More than 90% are of recipient origin. There are only a few reports of Hodgkin disease-like PTLD in allograft patients. We report the case of a Hodgkin disease-like PTLD of donor origin in a 16-year-old renal allograft recipient. Fourteen months after transplantation, an increasing inhomogeneous structure in the hilar region of the transplanted kidney became apparent and was excised. Histological examination showed Hodgkin- and Sternberg-Reed-like cells. Immunostaining showed CD20-positive and CD15-negative cells and Epstein-Barr virus (EBV) involvement (EBV-encoded small nonpolyadenylated RNA and EBV-determined nuclear antigen 2). DNA fingerprinting analysis proved the lymphoma to be of donor origin. Treatment consisted of nephrectomy, discontinuation of immunosuppression therapy, and local radiation. Three years after lymphoma removal, the patient was still without relapse and underwent retransplantation with stable function of the second allograft for more than 2 years now.

Genetic studies in south Balkan populations.

Within a study of the genetics of Balkan populations, four DNA-STR systems and 19 classical markers were examined in seven samples: Romanians (two groups), Albanians, Greeks and Aromuns (three groups). The results for the DNA-STR systems have been compared with data from the literature. The results show four clear separated groups: sub-Saharan black populations, North-African, Japanese and European populations. The large Balkan populations, except the Greek sample, are genetically more homogenous than the Aromun populations. A second Neighbor-joining tree based on all 23 analyzed systems, show a particular trend of the Aromun groups, which indicates a particular genetic structure.