Novel Algorithms for Improved Sensitivity in Non-Invasive Prenatal Testing.

Non-invasive prenatal testing (NIPT) of cell-free DNA in maternal plasma, which is a mixture of maternal DNA and a low percentage of fetal DNA, can detect fetal aneuploidies using massively parallel sequencing. Because of the low percentage of fetal DNA, methods with high sensitivity and precision are required. However, sequencing variation lowers sensitivity and hampers detection of trisomy samples. Therefore, we have developed three algorithms to improve sensitivity and specificity: the chi-squared-based variation reduction (χ2VR), the regression-based Z-score (RBZ) and the Match QC score. The χ2VR reduces variability in sequence read counts per chromosome between samples, the RBZ allows for more precise trisomy prediction, and the Match QC score shows if the control group used is representative for a specific sample. We compared the performance of χ2VR to that of existing variation reduction algorithms (peak and GC correction) and that of RBZ to trisomy prediction algorithms (standard Z-score, normalized chromosome value and median-absolute-deviation-based Z-score). χ2VR and the RBZ both reduce variability more than existing methods, and thereby increase the sensitivity of the NIPT analysis. We found the optimal combination of algorithms was to use both GC correction and χ2VR for pre-processing and to use RBZ as the trisomy prediction method.

Preimplantation genetic diagnosis for X;autosome translocations: lessons from a case of misdiagnosis.

UNLABELLED: Preimplantation genetic diagnosis (PGD) is offered to couples carrying a reciprocal translocation in an attempt to increase their chance of phenotypically normal offspring. For the selection of embryos that are balanced for the translocation chromosomes, it is critical to use a combination of DNA probes that can take account of all the segregation patterns of the particular translocation. The frequency of the different segregation types differs depending on the chromosomes involved, the location of the breakpoints and the number of chiasmata and the sex of the carrier. We report on a case of misdiagnosis after PGD-fluorescence in situ hybridization in a female translocation 46,X,t(X;5)(q13;p14) carrier. Transfer of two embryos diagnosed as balanced for the translocation chromosomes resulted in a singleton pregnancy that miscarried at 8 weeks' gestational age. The unbalanced karyotype of the fetus was consistent with 3:1 segregation resulting in tertiary trisomy for the derivative chromosome 5: 47,XX,+der(5)t(X;5)(q13;p14)mat. Based on additional molecular cytogenetic studies of fetal tissue and the initially investigated blastomeres, we concluded that the misdiagnosis was most probably due to a technical error, i.e. a partial hybridization failure or co-localization of the Xq/Yq subtelomere probe signals. No evidence for a normal cell line (mosaicism) was found in the fetus, which could have explained the discrepancy. This case demonstrates the importance of using two diagnostic probes or testing 2 cells to detect translocation products with potentially viable imbalance. X;autosome translocations are a special case due to the added complication of X chromosome inactivation and particular caution is advised when designing a PGD strategy. TRIAL REGISTRATION NUMBER: not applicable.

Chromosomes 1 and 12 abnormalities in pediatric germ cell tumors by interphase fluorescence in situ hybridization.

Chromosome studies of pediatric germ cell tumors (GCTs) show differences in abnormalities dependent on age, sex, tumor location, and histology. Previous studies suggest that loss of 1p is associated with a malignant phenotype, while amplification of 12p, a common finding in adult testicular GCTs, is uncommon in pediatric GCTs. Fifty-three pediatric GCTs were analyzed for 1p36 loss and 12p amplification by G-banding and dual-color interphase FISH with probes for the centromere and short arm of chromosomes 1 or 12. Twelve tumors with loss of 1p36 were identified. No deletion was detected in tumors with nonmalignant histology, such that there was a significant association of 1p loss with malignancy in these tumors (P = 0.00115). Five of 18 tumors from male patients had amplification of 12p, consistent with G-band results. Combined analysis of our data with those in the literature revealed a significant correlation of 12p amplification with patient age (P = 0.000196). Amplification of 12p was only seen in one of 35 tumors from female patients. Five female GCTs had numerical abnormalities of chromosome 12, and two tumors showed complete lack of 12p. This spectrum of abnormalities differs from what is seen in the male tumors, providing further evidence for different etiologies of GCTs between the sexes.

Genetic reflection of glioblastoma biopsy material in xenografts: characterization of 11 glioblastoma xenograft lines by comparative genomic hybridization.

OBJECT: Human tumors implanted as subcutaneous xenografts in nude mice are widely used for the study of tumor biology and therapy. Validation of these models requires knowledge of the genetic makeup of the xenografts. The aim of this study was to establish whether chromosomal imbalances in 11 xenograft lines derived from human glioblastomas multiforme (x-GBMs) are similar to those found in GBM biopsy samples. The authors also studied genetic stability during serial passaging of three xenograft lines. METHODS: Chromosomal imbalances in x-GBMs were detected using comparative genomic hybridization (CGH). The authors compared the CGH results in x-GBMs with those in the original GBMs (o-GBMs) that were used to establish three of the xenograft lines and with the GBM biopsy results reported in the literature (1-GBMs). In three xenograft lines two different passages were analyzed. CONCLUSIONS: The results show that the chromosomal imbalances in x-GBMs are similar to those in o-GBMs and 1-GBMs, indicating that the GBM xenograft lines used were valid models from a genetic point of view. The CGH analysis of two different passages of three xenograft lines indicates that x-GBMs (like 1-GBMs) show intratumoral genetic heterogeneity and do not acquire chromosomal imbalances as a result of serial passaging.

Ring chromosomes in a malignant mesenchymoma.

We report, for the first time, the cytogenetic and molecular genetic constitution of a human mesenchymoma. As in several other soft tissue sarcomas, supernumerary ring and rod-shaped marker chromosomes were observed next to an otherwise normal diploid karyotype. Comparative genomic in situ hybridization and whole chromosome painting experiments revealed that chromosome 1q21-q25 and 12q14-q15 sequences were amplified, and that these sequences resided on the supernumerary marker chromosomes. We assume that, in this malignant mesenchymoma, the observed chromosomal anomalies may be associated with its well differentiated liposarcomatous component.

Involvement of 3q21 in nodular fasciitis.

Cytogenetic data on nodular fasciitis are sparse. We present a case of this lesion and discuss our results in view of previously reported findings in nodular fasciitis and other benign mesenchymal lesions.

Fluorescence in situ hybridization analysis of chromosome 12 anomalies in semen cells from patients with carcinoma in situ of the testis.

Carcinoma in situ (CIS) of the testis is the precursor of seminomas and non-seminomatous germ cell tumours of the adult testis. A marked cytogenetic anomaly, the isochromosome of the short arm of chromosome 12 [i(12p)], has been demonstrated in over 80 per cent of all histological varieties of testicular germ cell tumours (TGCTs). In the remaining group of i(12p)-negative TGCTs, an overrepresentation of chromosome 12p sequences has been found. The i(12p) chromosome and overrepresentation of 12p sequences in CIS cells have also been reported. In order to establish whether numerical and/or structural aberrations of chromosome 12 can be found in CIS cells exfoliated into seminal fluid, semen specimens from ten patients with CIS lesions were investigated using bicolour double fluorescence in situ hybridization (FISH). The two DNA probes used, p alpha 12H8 and YAC 5, specifically detect the centromeric region of chromosome 12 and a subregion, p11.2-p12.1, on the short arm of chromosome 12, respectively. Ejaculates of ten azoospermic or oligozoospermic infertile males, presumably CIS-free, were used as negative controls. Nuclei exhibiting three or more chromosome 12 signals were found to be present in a significantly larger number in the patient samples than in the control samples. Nuclei with five or more chromosome 12 signals were observed in eight out of the ten patients. Morphologically similar arrangements to i(12p) were observed in some of the ejaculates. These results demonstrate the potential of FISH in the early detection of CIS and TGCTs in males at high risk.

Isolation of osteosarcoma-associated amplified DNA sequences using representational difference analysis.

Comparative genomic hybridization analysis of a primary osteosarcoma and its metastasis revealed two regions of DNA amplification, one at 17p11.2-12 and one at 19q12-13. Subsequent representational difference analysis of the primary tumor resulted in the isolation of two distinct tumor-amplified DNA fragments originating from chromosome 19. A YAC clone corresponding to one of the two isolated DNA fragments was used for fluorescence in situ hybridization on normal human lymphocyte metaphases and tumor-derived nuclei. This resulted in the localization of this YAC to 19q12-13.1 and confirmed the amplification status of the isolated fragment in the tumors. The availability of such RDA-isolated sequences may be instrumental in the search for genes relevant for tumor development.

Detection of chromosomal DNA gains and losses in testicular germ cell tumors by comparative genomic hybridization.

To extend the results of conventional cytogenetic analysis of testicular germ cell tumors (TGCTs), we applied the new molecular cytogenetic method of comparative genomic hybridization (CGH), which enables the detection of chromosomal imbalances without the need for dividing cells. DNA from II TGCTs was studied by CGH. In all tumors examined, gain of 12p, mostly of the whole p arm, could be demonstrated. However, in three tumors, an amplification of 12p material restricted to the chromosomal bands 12p11.2-p12.1 was found. Further fluorescence in situ hybridization (FISH) analysis using a yeast artificial chromosome (YAC) that was previously mapped to that region revealed multiple copies of that chromosomal segment in interphase nuclei of these tumors. This finding is an important clue to the localization of candidate protooncogenes at 12p involved in TGCTs. Gains of small chromosomal regions at 2p, 4q, 6p, and 19p were also detected recurrently. Furthermore, gains of chromosomes 8, 14, 21, and X as well as loss of chromosome 13 were frequent findings. In conclusion, CGH provides new insights into genetic alterations of TGCTs. By using CGH, chromosomal subregions could be identified that may harbor genes involved in the pathogenesis of this malignancy.

Comparative genomic hybridization of germ cell tumors of the adult testis: confirmation of karyotypic findings and identification of a 12p-amplicon.

Comparative genomic hybridization (CGH) was carried out on 15 primary testicular germ cell tumors (TGCT) of adolescents and adults and two metastatic residual tumors after chemotherapeutic treatment. The results were compared with karyotypic data obtained form the same tumor specimens after direct harvesting of metaphases or short-term in vitro culture. Both techniques revealed that the most consistent abnormality in primary TGCT is gain of 12p-sequences. Although in most cases over-representation of the complete short arm was observed, CGH revealed a specific amplification of 12p11.1-p12.1 region in two independent primary tumors. In addition, loss of (parts of) chromosome 13 (always involving q31-qter), and gain of (parts of) chromosome 7 (mostly involving q11), (parts of) chromosome 8, and the X chromosome were detected in more than 25% of the tumors by this latter technique. Loss of 6q15-q21 in both residual tumors analyzed may suggest a role for this anomaly in acquired resistance to chemotherapeutic treatment. Overall, the CGH analyses confirmed gains and losses of certain chromosomal regions in TGCT as observed by karyotyping, and thus support their role in the development of these neoplasms. The amplification of a restricted region of 12p in primary TGCT confirms and extends our previous observations and, as such, represents an important step forward in the identification of gene(s) on 12p relevant for the pathogenesis of these tumors.

Cloning and characterization of DXS6673E, a candidate gene for X-linked mental retardation in Xq13.1.

In several families with non-specific X-linked mental retardation (XLMR) linkage analyses have assigned the underlying gene defect to the pericentromeric region of the X chromosome, but none of these genes have been isolated so far. Here, we report on the cloning and characterization of a novel gene, DXS6673E, that maps to Xq13.1, is subject to X-inactivation and is disrupted in the 5' untranslated region by a balanced X;13 translocation in a mentally retarded female. The DXS6673E gene is highly conserved among vertebrates and its expression is most abundant in brain. It encodes a hydrophilic protein of 1358 amino acids (aa) that does not show sequence homology to other known proteins. A segment of this protein consisting of neutral and hydrophobic aa with a proline residue in every second position may represent a transmembrane domain. Almost complete sequence identity was found between the 3' end of the DXS6673E gene and two expressed sequence tags (ESTs) and between the 5' end of the DXS6673E gene and a third EST. Moreover, weaker sequence similarity was observed between coding regions and two other ESTs.

Reviews of chromosome studies in urological tumors. III. Cytogenetics and genes in testicular tumors.

PURPOSE: We reviewed available cytogenetic and molecular findings in testicular germ cell tumors, and their possible application to clinical, pathological and basic parameters. MATERIALS AND METHODS: Findings in the literature on testicular germ cell tumors as well as those from our laboratory were summarized, including a listing of the cytogenetic findings on testicular germ cell tumors to date with some illustrations. RESULTS: Testicular germ cell tumors are characterized in most cases by the presence of an i(12p) isochromosome. In tumors without such an abnormal chromosome studies using fluorescence in situ hybridization and molecular approaches have demonstrated either masking of the i(12p) or the presence of extra 12p sequences in the karyotype. Although testicular germ cell tumors are often associated with chromosome changes in addition to the i(12p), no other specifically recurrent structural chromosome changes have been found. Based on the cytogenetic and molecular findings in testicular germ cell tumors, a hypothetical scheme for the genetic events leading to these tumors is presented. CONCLUSIONS: The genetic events leading to genesis of testicular germ cell tumors in men appear to be related to aneuploidization followed by the formation of an i(12p) isochromosome, the latter characterizing the preponderant number of testicular germ cell tumors. The exact role of the i(12p) isochromosome in testicular germ cell tumor pathogenesis remains to be determined, as is true of the genes involved in or affected by these tumors. Based on presently available information, a hypothetical pathogenetic and oncogenetic model for the development of testicular germ cell tumors is presented.

Detection of numerical alterations for chromosomes 7 and 12 in benign thyroid lesions by in situ hybridization. Histological implications.

Polysomies of chromosomes 7 and 12 have been frequently observed by conventional cytogenetics in a subgroup of thyroid follicular adenomas and in some cases of thyroid goiters. To further study possible cytogenetic similarities between these two types of thyroid lesions, we have used fluorescence in situ hybridization (FISH) to detect polysomies of chromosomes 7 and/or 12 in isolated nuclei from frozen and paraffin-embedded material of goiters and thyroid follicular adenomas and compared results with previous ones obtained by flow cytometry and conventional cytogenetics. With a set of two alpha-satellite DNA probes specific for the centromeric regions of chromosomes 7 and 12, used either separately (single-target fluorescence in situ hybridization) or simultaneously (double-target fluorescence in situ hybridization), we detected polysomies of chromosome 7 in 35.7% of the thyroid follicular adenomas and in 10.7% of the goiters. Polysomies of chromosome 12 were detected in 29.6% of the thyroid follicular adenomas and 6.7% of the goiters. The significantly higher frequency of adenomas with numerical alterations for chromosomes 7 and/or 12 supports the idea of a biological continuum and karyotypic evolution between both lesions. It is also noteworthy that polysomies of chromosomes 7 and/or 12 were observed only in lesions with an exclusive (or predominant) microfollicular histological component, as detected by enzymatic in situ hybridization on frozen sections.

Increasing levels of MYC and MET co-amplification during tumor progression of a case of gastric cancer.

The cytogenetic study of a nodal metastasis from a gastric carcinoma, after two passages in nude mice, revealed a large number of double minutes. Comparative genomic in situ hybridization (CGH) analysis using DNA extracted from this xenograft revealed the existence of three clear amplification units that originated from the chromosomal subregions 6q24-25, 7q31-32, and 8q24 in the xenograft DNA. Similar, though less prominent, CGH results were found with DNAs extracted from the primary tumor and its metastasis, implying that the same amplicons were also present, albeit less abundantly, in the DNAs of these neoplastic tissues. Southern analysis of the second-passage xenograft detected 18- and 10-fold amplification of MET (located at 7q31) and MYC (located at 8q24), respectively. The retrospective study of the first passage of the xenograft, as well as of the metastatic and primary tumors before xenografting, showed amplification levels of MET of, respectively, 12-, 9-, and 5-fold and MYC of, respectively, 8-, 7-, and 5-fold. Our results suggest that increased levels of co-amplification of MYC and MET correlate with enhanced growth potential in this case of gastric carcinoma.

Amplification of chromosome subregion 12p11.2-p12.1 in a metastasis of an i(12p)-negative seminoma: relationship to tumor progression?

Cytogenetic analysis of a metastasis of a human testicular germ cell tumor (seminoma) revealed multiple numerical and structural anomalies, including an abnormally banding region (ABR) present on the short arm of one of the chromosome 12 homologs. Fluorescence in situ- and comparative genomic hybridization experiments revealed that the ABR results from the amplification of 12p11.2-p12.1 derived sequences. We speculate that this particular region may harbor gene(s) relevant for testicular germ cell tumor progression.

Complex composition and co-amplification of SAS and MDM2 in ring and giant rod marker chromosomes in well-differentiated liposarcoma.

Extra abnormal chromosomes (rings and giant rods) containing chromosome 12 sequences are characteristic of well-differentiated liposarcoma (WDLPS). By whole chromosome painting we found in 6 WDLPS that minimally 5 chromosomes had contributed to the formation of the extra abnormal chromosomes. To the constant chromosome 12 contribution, sequences were variably added from chromosomes 1, 4, and 16. Material from chromosomes 1, 4, and 12 was identified by painting in interphase nuclear projections ("blebs") and in micronuclei consistent with the concept that blebs are precursors to micronuclei. The complexity of the mechanisms generating the extra abnormal chromosomes in WDLPS was also attested to by the diversity and, in some cases, intricacy of the patterns of fluorescence. To begin to fathom the function of the extra abnormal chromosomes we examined the amplification of genes, including SAS, MDM2, and GADD153/CHOP, known to be in the region 12q13-14. SAS and MDM2 demonstrated constant co-amplification. GADD153/CHOP, which is critically rearranged in myxoid liposarcoma, was not amplified in WDLPS.

Localization of the gene encoding the alpha 2/delta-subunits of the L-type voltage-dependent calcium channel to chromosome 7q and analysis of the segregation of flanking markers in malignant hyperthermia susceptible families.

Malignant hyperthermia susceptibility (MHS) is an autosomal dominant disorder of skeletal muscle which manifests as a potentially fatal hypermetabolic crisis triggered by commonly used anaesthetic agents. The demonstration of genetic heterogeneity in MHS prompted the investigation of the roles played by calcium regulatory proteins other than the ryanodine receptor (RYR1), which is known to be linked to MHS in fewer than half of the European MHS families studied to date. Previously, we have excluded the genes encoding the skeletal muscle L-type voltage-dependent calcium channel alpha 1-, beta 1- and gamma-subunits as candidates for MHS. In this report, we describe the cloning and partial DNA sequence analysis of the gene encoding the alpha 2/delta-subunits, CACNL2A, and its localization on the proximal long arm of chromosome 7q. A new dinucleotide repeat marker close to CACNL2A was identified at the D7S849 locus and tested for linkage in six MHS families. D7S849 and flanking genetic markers were found to co-segregate with the MHS locus through 11 meioses in one, three-generation family. These results suggest that mutations in or near CACNL2A may be involved in some forms of this heterogeneous disorder.