Genome wide noninvasive prenatal testing detects microduplication of the distal end of chromosome 15 in a fetus: a case report.

BACKGROUND: Noninvasive prenatal testing (NIPT) is the most recent modality widely used in prenatal diagnostics. Commercially available NIPT has high sensitivity and specificity for the common fetal chromosomal aneuploidies. As future advancements in NIPT sequencing technology are becoming promising and more reliable, the ability to detect beyond aneuploidies and to expand detection of submicroscopic genomic alterations, as well as single-gene disorders might become possible. CASE PRESENTATION: Here we present a case of a 34-year-old pregnant woman, G2P1, who had NIPT screening which detected a terminal microduplication of 10.34 Mb on the long arm of chromosome 15 (15q26.1q26.3). Subsequent prenatal diagnostic testing including karyotype, microarray and fluorescence in situ hybridization (FISH) analyses were performed. Microarray testing confirmed and particularized a copy number gain of 10.66 Mb of the distal end of the long arm of chromosome 15. The G-banding cytogenetic studies yielded results consistent with unbalanced translocation between chromosome 15 and 18. To further characterize the abnormality involving the long arm of chromosome 18 and to map the genomic location of the duplicated 15q more precisely, FISH analysis using specific sub-telomeric probes was performed. FISH analysis confirmed that the extra duplicated segment of chromosome 15 is translocated onto the distal end of the long arm of chromosome 18 at band 18q23. Parental karyotype and FISH studies were performed to see if this unbalanced rearrangement was inherited from a healthy balanced translocation carrier versus being a de novo finding. Parental chromosomal analysis provided no evidence of a rearrangement between chromosome 15 and chromosome 18. The final fetal karyotype was reported as 46,XX,der(18)t(15;18)(q26.2;q23)dn. CONCLUSIONS: In this case study, the microduplication of fetal chromosome 15q26.1q26.3 was accurately detected using NIPT. Our results suggest that further refinements in NIPT have the potential to evolve to a powerful and efficient screening method, which might be used to detect a broad range of chromosomal imbalances. Since microduplications and microdeletions are a potential reportable result with NIPT, this must be included in pre-test counseling. Prenatal diagnostic testing of such findings is strongly recommended.

Gene-rich chromosomal regions are preferentially localized in the lamin B deficient nuclear blebs of atypical progeria cells.

More than 20 mutations in the gene encoding A-type lamins (LMNA) cause progeria, a rare premature aging disorder. The major pathognomonic hallmarks of progeria cells are seen as nuclear deformations or blebs that are related to the redistribution of A- and B-type lamins within the nuclear lamina. However, the functional significance of these progeria-associated blebs remains unknown. We have carried out an analysis of the structural and functional consequences of progeria-associated nuclear blebs in dermal fibroblasts from a progeria patient carrying a rare point mutation p.S143F (C428T) in lamin A/C. These blebs form microdomains that are devoid of major structural components of the nuclear envelope (NE)/lamina including B-type lamins and nuclear pore complexes (NPCs) and are enriched in A-type lamins. Using laser capture microdissection and comparative genomic hybridization (CGH) analyses, we show that, while these domains are devoid of centromeric heterochromatin and gene-poor regions of chromosomes, they are enriched in gene-rich chromosomal regions. The active form of RNA polymerase II is also greatly enriched in blebs as well as nascent RNA but the nuclear co-activator SKIP is significantly reduced in blebs compared to other transcription factors. Our results suggest that the p.S143F progeria mutation has a severe impact not only on the structure of the lamina but also on the organization of interphase chromatin domains and transcription. These structural defects are likely to contribute to gene expression changes reported in progeria and other types of laminopathies.

Hematogenous dissemination of glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most frequent and aggressive brain tumor in adults. The dogma that GBM spread is restricted to the brain was challenged by reports on extracranial metastases after organ transplantation from GBM donors. We identified circulating tumor cells (CTCs) in peripheral blood (PB) from 29 of 141 (20.6%) GBM patients by immunostaining of enriched mononuclear cells with antibodies directed against glial fibrillary acidic protein (GFAP). Tumor cell spread was not significantly enhanced by surgical intervention. The tumor nature of GFAP-positive cells was supported by the absence of those cells in healthy volunteers and the presence of tumor-specific aberrations such as EGFR gene amplification and gains and losses in genomic regions of chromosomes 7 and 10. Release of CTCs was associated with EGFR gene amplification, suggesting a growth potential of these cells. We demonstrate that hematogenous GBM spread is an intrinsic feature of GBM biology.

Diagnostic accuracy of random massively parallel sequencing for non-invasive prenatal detection of common autosomal aneuploidies: a collaborative study in Europe.

OBJECTIVE: The objective of this study is to validate the diagnostic accuracy of a non-invasive prenatal test for detecting trisomies 13, 18, and 21 for a population in Germany and Switzerland. METHODS: Random massively parallel sequencing was applied using Illumina sequencing platform HiSeq2000. Fetal aneuploidies were identified using a median absolute deviation based z-score equation. A bioinformatics algorithm based on guanine-cytosine normalization was applied after the data were unblinded. Results of massively parallel sequencing and invasive procedures were compared. RESULTS: Overall, 40/42 samples were correctly classified as trisomy 21-positive, including a translocation trisomy 21 [46,XY,der(13;21),+21] and a structural aberration of chromosome 21 [46,XX,rec(21)dup(21q)inv(21)(p12q21.1)] but not including a low percentage mosaic trisomy 21 [47,XY,+21/46,XY], [sensitivity: 95.2%; one-sided lower confidence limit: 85.8%]; 430/430 samples were correctly classified as trisomy 21-negative (specificity: 100%; one-sided lower CL: 99.3%). Using a new bioinformatics algorithm with guanine-cytosine normalization, detection of trisomy 21 was facilitated, and five of five trisomy 13 cases and eight of eight trisomy 18 cases were correctly identified. CONCLUSION: Our newly established non-invasive prenatal test allows detection of fetal trisomies 13, 18, and 21 with high accuracy in a population in Germany and Switzerland.

Heterogeneity of epidermal growth factor receptor status and mutations of KRAS/PIK3CA in circulating tumor cells of patients with colorectal cancer.

BACKGROUND: Molecular characterization of circulating tumor cells (CTCs) is pivotal to increasing the diagnostic specificity of CTC assays and investigating therapeutic targets and their downstream pathways on CTCs. We focused on epidermal growth factor receptor (EGFR) and genes relevant for its inhibition in patients with colorectal cancer (CRC). METHODS: We used the CellSearch® system for CTC detection in peripheral blood samples from 49 patients with metastatic CRC (mCRC) and 32 patients with nonmetastatic CRC (nmCRC). We assessed EGFR expression in 741 CTCs from 27 patients with mCRC and 6 patients with nmCRC using a fluorescein-conjugated antibody with the CellSearch Epithelial Cell Kit. DNA of a single CTC isolated by micromanipulation was propagated by whole-genome amplification and analyzed by quantitative PCR for EGFR gene amplification and sequencing for KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog), BRAF (v-raf murine sarcoma viral oncogene homolog B1), and PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α) mutations. RESULTS: At least 2 CTCs were detected in 24 of 49 patients with mCRC and 7 of 32 patients with nmCRC. In 7 of 33 patients, CTCs with increased EGFR expression were identified. Heterogeneity in EGFR expression was observed between CTCs from the same patient. EGFR gene amplification was found in 7 of 26 CTCs from 3 patients. The investigated BRAF gene locus was not mutated in 44 analyzed CTCs, whereas KRAS mutations were detected in 5 of 15 CTCs from 1 patient and PIK3CA mutations in 14 of 36 CTCs from 4 patients. CONCLUSIONS: Molecular characterization of single CTCs demonstrated considerable intra- and interpatient heterogeneity of EGFR expression and genetic alterations in EGFR, KRAS, and PIK3CA, possibly explaining the variable response rates to EGFR inhibition in patients with CRC.