Clinical trio genome sequencing facilitates the interpretation of variants in cancer predisposition genes in paediatric tumour patients.

The prevalence of pathogenic and likely pathogenic (P/LP) variants in genes associated with cancer predisposition syndromes (CPS) is estimated to be 8-18% for paediatric cancer patients. In more than half of the carriers, the family history is unsuspicious for CPS. Therefore, broad genetic testing could identify germline predisposition in additional children with cancer resulting in important implications for themselves and their families. We thus evaluated clinical trio genome sequencing (TGS) in a cohort of 72 paediatric patients with solid cancers other than retinoblastoma or CNS-tumours. The most prevalent cancer types were sarcoma (n = 26), neuroblastoma (n = 15), and nephroblastoma (n = 10). Overall, P/LP variants in CPS genes were identified in 18.1% of patients (13/72) and P/LP variants in autosomal-dominant CPS genes in 9.7% (7/72). Genetic evaluation would have been recommended for the majority of patients with P/LP variants according to the Jongmans criteria. Four patients (5.6%, 4/72) carried P/LP variants in autosomal-dominant genes known to be associated with their tumour type. With the immediate information on variant inheritance, TGS facilitated the identification of a de novo P/LP in NF1, a gonadosomatic mosaic in WT1 and two pathogenic variants in one patient (DICER1 and PALB2). TGS allows a more detailed characterization of structural variants with base-pair resolution of breakpoints which can be relevant for the interpretation of copy number variants. Altogether, TGS allows comprehensive identification of children with a CPS and supports the individualised clinical management of index patients and high-risk relatives.

Reduced cell size, chromosomal aberration and altered proliferation rates are characteristics and confounding factors in the STHdh cell model of Huntington disease.

Huntington disease is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the gene encoding the huntingtin protein. Expression of the mutant protein disrupts various intracellular pathways and impairs overall cell function. In particular striatal neurons seem to be most vulnerable to mutant huntingtin-related changes. A well-known and commonly used model to study molecular aspects of Huntington disease are the striatum-derived STHdh cell lines generated from wild type and huntingtin knock-in mouse embryos. However, obvious morphological differences between wild type and mutant cell lines exist, which have rarely been described and might not have always been considered when designing experiments or interpreting results. Here, we demonstrate that STHdh cell lines display differences in cell size, proliferation rate and chromosomal content. While the chromosomal divergence is considered to be a result of the cells' tumour characteristics, differences in size and proliferation, however, were confirmed in a second non-immortalized Huntington disease cell model. Importantly, our results further suggest that the reported phenotypes can confound other study outcomes and lead to false conclusions. Thus, careful experimental design and data analysis are advised when using these cell models.

Generation of induced pluripotent stem cells derived from a 77-year-old healthy woman as control for age related diseases.

Induced pluripotent stem cells (iPSCs) hold great promise to model diseases, where the disease affected cell type is difficult to access. A major obstacle for the development of disease models is the lack of well characterized control iPSCs from old people not affected by such a disease. Furthermore, gene-editing approaches often require iPSCs from healthy donors, where pathogenic mutations can be inserted if patient material is not available. Here, we report the generation of an iPSC line (16423 #6) from a 77-year-old woman, who did not display any disease symptoms at the time, when the skin biopsy was taken.

A mouse bone marrow stromal cell line with skeletal stem cell characteristics to study osteogenesis in vitro and in vivo.

Bone marrow stromal cells (BMSCs) are composed of progenitor and multipotent skeletal stem cells, which are able to differentiate in vitro into osteocytes, adipocytes, and chondrocytes. Mouse BMSCs (mBMSCs) are a versatile model system to investigate factors involved in BMSC differentiation in vitro and in vivo as a variety of transgenic mouse models are available. In this study, mBMSCs were isolated and osteogenic differentiation was investigated in tissue culture and in vivo. Three out of seven independent cell isolates showed the ability to differentiate into osteocytes, adipocytes, and chondrocytes in vitro. In vitro multipotency of an established mBMSC line was maintained over 45 passages. The osteogenic differentiation of this cell line was confirmed by quantitative polymerase chain reaction (qPCR) analysis of specific markers such as osteocalcin and shown to be Runx2 dependent. Notably, the cell line, when transplanted subcutaneously into mice, possesses full skeletal stem cell characteristics in vivo in early and late passages, evident from bone tissue formation, induction of vascularization, and hematopoiesis. This cell line provides, thus, a versatile tool to unravel the molecular mechanisms governing osteogenesis in vivo thereby aiding to improve current strategies in bone regenerative therapy.

Amniotic bands as a cause of congenital anterior staphyloma.

BACKGROUND: Congenital anterior staphyloma is a rare, complex malformation syndrome of the anterior segment. Only a few reports on associated systemic malformations have been published. We herein present a rare manifestation of congenital anterior staphyloma (CAS) combined with amniotic band disruption syndrome (ABS). PATIENT AND METHODS: Shortly after birth, a massive enlargement of the left eye was observed in a female child. Furthermore, an extensive bilateral congenital cleft lip and cleft alveolar ridge with oblique facial cleft extending into the left medial canthal region, coloboma(s) of the left eyelids, extensive adhesions between lids and eye bulb, as well as circumferential grooves, clubfeet, and terminal transverse defects in both hands and feet were present. Due to severe progression of eye bulb protrusion with thinning of the sclera, enucleation of the left eye was performed at the age of 3 years in order to prevent complications including perforation of the globe and with the aim of improving cosmetic aspects. RESULTS: Histopathological examination of the enucleated eye disclosed findings typical of congenital anterior staphyloma, including massive corneal staphylomatic deformation with superficial vascularization and elapsed corneoscleral margin, destruction of Bowman's layer, absence of Descemet's layer, corneal endothelium, and angle structures. The lens was only partially formed, and had mainly dissolved. The neural retina appeared normal. The optic nerve disc revealed a pronounced excavation. Facial clefts, lid colobomas, congenital constriction bands, and amputation of distal limbs match ABS. This malformation complex develops in early pregnancy, probably prior to 35 days post conception. CONCLUSION: This is the first report on an association of these two rare complex congenital malformations, congenital anterior staphyloma and amniotic band syndrome. The anterior staphyloma was unilateral, and related to facial clefts and lid coloboma in the area adjacent to the anterior staphyloma. Furthermore, the systemic deformities are clearly due to the amniotic bands, and the timing of the development of both complex malformations seems to be similar. All findings suggest that the presence of amniotic bands is a causative factor for all observed abnormalities including anterior staphyloma.

Characterisation of the cell line HC-AFW1 derived from a pediatric hepatocellular carcinoma.

Current treatment of paediatric hepatocellular carcinoma (HCC) is often inefficient due to advanced disease at diagnosis and resistance to common drugs. The aim of this study was to generate a cell line derived from a paediatric HCC in order to expand research in this field. We established the HC-AFW1 cell line from a liver neoplasm of a 4-year-old boy through culturing of primary tumor specimens. The cell line has been stable for over one year of culturing and has a doubling time of 40 h. The tumour cells have an epithelial histology and express HCC-associated proteins such as Alpha-fetoprotein (AFP), Glypican 3, E-cadherin, CD10, CD326, HepPar1 and Vimentin. Forty-nine amino acids in exon 3 of ß-Catenin that involve the phosphorylation sites of GSK3 were absent and ß-Catenin is detectable in the cell nuclei. Cytogenetic analysis revealed large anomalies in the chromosomal map. Several alterations of gene copy numbers were detected by genome-wide SNP array. Among the different drugs tested, cisplatin and irinotecan showed effective inhibition of tumour cell growth in a proliferation assay at concentrations below 5 µg/ml. Subcutaneous xenotransplantation of HC-AFW1 cells into NOD/SCID mice resulted in fast growing dedifferentiated tumours with high levels of serum AFP. Histological analyses of the primary tumour and xenografts included national and international expert pathological review. Consensus reading characterised the primary tumour and the HC-AFW1-derived tumours as HCC. HC-AFW1 is the first cell line derived from a paediatric HCC without a background of viral hepatitis or cirrhosis and represents a valuable tool for investigating the biology of and therapeutic strategies for childhood HCC.

Visualization of xenotransplanted human rhabdomyosarcoma after transfection with red fluorescent protein.

BACKGROUND/AIMS: Discosoma sp red fluorescent protein (DsRed2) is a newly developed marker for in vivo labeling studies in different biologic systems. After vector transfection, DsRed2 is expressed in mammalian cells and can be detected by fluorescence microscopy. The aims of this study were to establish a DsRed2-transfected human rhabdomyosarcoma (RMS) cell line and to perform a xenotransplantation on nude mice to use imaging as a tool for further basic research studies on this neoplasm. PROCEDURE: The human alveolar RMS cell line Rh30 was transfected with the pDsRed2-N1 vector by lipofection. The DsRed2-positive cells were sorted out by fluorescence-activated cell sorting analysis 96 hours after transfection and selected in culture with G418. Expression of DsRed2 messenger RNA was assessed using single-cell reverse transcriptase polymerase chain reaction after laser microdissection. Transfected and parental cells were characterized cytologically, cytogenetically, immunohistochemically, and in vivo after subcutaneous injection in NMRI (nu/nu) nude mice. RESULTS: After vector transfection, a pure and stable DsRed2-positive cell line was established by monoclonal growth of the cells. Reverse transcriptase polymerase chain reaction revealed constant expression of DsRed2 messenger RNA in fluorescencing cells. There was no difference between transfected and parental cells by means of cell morphology and desmin expression. Clonal cells (1 x 10(6)) were used for xenotransplantation. Tumors were visualized noninvasively through the skin of the mice using specific emission and excitation filters. Tumor vascularization and vessel growth could be discriminated from tumor tissue using this imaging system. CONCLUSION: This is the first report on successful transfection of an RMS cell line with red fluorescent protein followed by xenotransplantation into nude mice. This model can serve as an imaging tool for in vivo studies investigating tumor biology and metastases of human RMS.