Prenatal diagnosis of BACs-on-Beads assay in 1520 cases from Fujian Province, China.

BACKGROUND: The aim of this study was to evaluate the application of BACs-on-Beads (BoBs™) assay for rapid detection of chromosomal abnormalities for prenatal diagnosis (PND). METHODS: A total of 1520 samples, including seven chorionic villi biopsy samples, 1328 amniotic fluid samples, and 185 umbilical cord samples from pregnant women were collected to detect the chromosomal abnormalities using BoBs™ assay and karyotyping. Furthermore, abnormal specimens were verified by chromosome microarray analysis (CMA) and fluorescence in situ hybridization (FISH). RESULTS: The results demonstrated that the success rate of karyotyping and BoBs™ assay in PND was 98.09% and 100%, respectively. BoBs™ assay was concordant with karyotyping for Trisomy 21, Trisomy 18, and Trisomy 13, sex chromosomal aneuploidy, Wolf-Hirschhorn syndrome, and mosaicism. BoBs™ assay also detected Smith-Magenis syndrome, Williams-Beuren syndrome, DiGeorge syndrome, Miller-Dieker syndrome, Prader-Willi syndrome, Xp22.31 microdeletions, 22q11.2, and 17p11.2 microduplications. However, karyotyping failed to show these chromosomal abnormalities. A case of 8q21.2q23.3 duplication which was found by karyotyping was not detected by BoBs™ assay. Furthermore, all these chromosomal abnormalities were consistent with CMA and FISH verifications. According to the reports, we estimated that the detection rates of karyotyping, BoBs™, and CMA in the present study were 4.28%, 4.93%, and 5%, respectively, which is consistent with the results of a previous study. The respective costs for the three methods were about $135-145, $270-290, and $540-580. CONCLUSION: BoBs™ assay is considered a reliable, rapid test for use in PND. A variety of comprehensive technological applications can complement each other in PND, in order to maximize the diagnosis rate and reduce the occurrence of birth defects.

Cost-effectiveness analysis of chromosomal microarray as a primary test for prenatal diagnosis in Hong Kong.

BACKGROUND: Chromosomal microarray (CMA) has been shown to be cost-effective over karyotyping in invasive prenatal diagnosis for pregnancies with fetal ultrasound anomalies. Yet, information regarding preceding and subsequent tests must be considered as a whole before the true cost-effectiveness can emerge. Currently in Hong Kong, karyotyping is offered free as the standard prenatal test while genome-wide array comparative genome hybridization (aCGH), a form of CMA, is self-financed. A new algorithm was proposed to use aCGH following quantitative fluorescent polymerase chain reaction (QF-PCR) as primary test instead of karyotyping. This study aims to evaluate the cost-effectiveness of the proposed algorithm versus the current algorithm for prenatal diagnosis in Hong Kong. METHODS: Between November 2014 and February 2016, 129 pregnant women who required invasive prenatal diagnosis at two public hospitals in Hong Kong were prospectively recruited. The proposed algorithm was performed for all participants in this demonstration study. For the cost-effectiveness analysis, cost and outcome (diagnostic rate) data were compared with that of a hypothetical scenario representing the current algorithm. Further analysis was performed to incorporate women's willingness-to-pay for the aCGH test. Impact of government subsidies on the aCGH test was explored as a sensitivity analysis. RESULTS: The proposed algorithm dominated the current algorithm for prenatal diagnosis. Both algorithms were equally effective but the proposed algorithm was significantly cheaper (p ≤ 0.05). Taking into account women's willingness-to-pay for an aCGH test, the proposed algorithm was more effective and less costly than the current algorithm. When the government subsidy reaches 100%, the maximum number of diagnoses could be made. CONCLUSION: By switching to the proposed algorithm, cost saving can be achieved whilst maximizing the diagnostic rate for invasive prenatal diagnosis. It is recommended to implement aCGH as a primary test following QF-PCR to replace the majority of karyotyping for prenatal diagnosis in Hong Kong.

An efficient genetic test flow for multiple congenital anomalies and intellectual disability.

BACKGROUND: Genetic testing has enabled the diagnosis of multiple congenital anomalies and/or intellectual disabilities. However, because of the phenotypic variability in these disorders, selection of an appropriate genetic test can be difficult and complex. For clinical examination, particularly in clinical facilities, a simple and standardized system is needed. METHODS: We compared microarray comparative genomic hybridization and clinical exome sequencing with regard to diagnostic yield, cost, and time required to reach a definitive diagnosis. After first performing G-banding for 200 patients with multiple congenital anomalies and/or intellectual disability, as a subsequent genetic test, microarray and clinical exome sequencing were compared with regard to diagnostic yield, cost, and time required. RESULTS: There was no obvious difference in the diagnostic rate between the two methods; however, clinical exome sequencing was superior in terms of cost and time. In addition, clinical exome sequencing could sufficiently identify copy number variants, and even smaller copy number variants could be identified. CONCLUSIONS: Clinical exome sequencing should be implemented earlier as a genetic test for undiagnosed patients with multiple congenital anomalies and/or intellectual disabilities. Our results can be used to establish inspection methods in clinical facilities.

[Comparative genomic hybridisation as a first option in genetic diagnosis: 1,000 cases and a cost-benefit analysis]. / Array CGH como primera opción en el diagnóstico genético: 1.000 casos y análisis de coste-beneficio.

BACKGROUND AND OBJECTIVE: Conventional cytogenetics diagnoses 3-5% of patients with unexplained developmental delay/intellectual disability and/or multiple congenital anomalies. The Multiplex Ligation-dependent Probe Amplification increases diagnostic rates from between 2.4 to 5.8%. Currently the comparative genomic hybridisation array or aCGH is the highest performing diagnostic tool in patients with developmental delay/intellectual disability, congenital anomalies and autism spectrum disorders. Our aim is to evaluate the efficiency of the use of aCGH as first-line test in these and other indications (epilepsy, short stature). PATIENTS AND METHOD: A total of 1000 patients referred due to one or more of the abovementioned disorders were analysed by aCGH. RESULTS: Pathogenic genomic imbalances were detected in 14% of the cases, with a variable distribution of diagnosis according to the phenotypes: 18.9% of patients with developmental delay/intellectual disability; 13.7% of multiple congenital anomalies, 9.76% of psychiatric pathologies, 7.02% of patients with epilepsy, and 13.3% of patients with short stature. Within the multiple congenital anomalies, central nervous system abnormalities and congenital heart diseases accounted for 14.9% and 10.6% of diagnoses, respectively. Among the psychiatric disorders, patients with autism spectrum disorders accounted for 8.9% of the diagnoses. CONCLUSIONS: Our results demonstrate the effectiveness and efficiency of the use of aCGH as the first line test in genetic diagnosis of patients suspected of genomic imbalances, supporting its inclusion within the National Health System.

Cost Effectiveness of Using Array-CGH for Diagnosing Learning Disability.

OBJECTIVE: To undertake a cost-effectiveness analysis of using microarray comparative genomic hybridisation (array-CGH) as a first-line test versus as a second-line test for the diagnosis of causal chromosomal abnormalities in patients referred to a NHS clinical genetics service in the U.K. with idiopathic learning disability, developmental delay and/or congenital anomalies. METHODS: A cost-effectiveness study was conducted. The perspective is that of a U.K. NHS clinical genetics service provider (with respect to both costs and outcomes). A cohort of patients (n = 1590) referred for array-CGH testing of undiagnosed learning disability and developmental delay by a single NHS regional clinical genetics service (South East Thames Regional Genetics Service), were split into a before-and-after design where 742 patients had array-CGH as a second-line test (before group-comparator intervention) and 848 patients had array-CGH as a first-line test (after group-evaluated intervention). The mean costs were calculated from the clinical genetics testing pathway constructed for each patient including the costs of genetic testing undertaken and clinical appointments scheduled. The outcome was the number of diagnoses each intervention produced so that a mean cost-per-diagnosis could be calculated. The cost effectiveness of the two interventions was calculated as an incremental cost-effectiveness ratio to produce an incremental cost-per-diagnosis (in 2013 GBP). Sensitivity analyses were conducted by altering both costs and effects to check the validity of the outcome. RESULTS: The incremental mean cost of testing patients using the first-line testing strategy was -GBP241.56 (95% CIs -GBP256.93 to -GBP226.19) and the incremental mean gain in the percentage diagnoses was 0.39% (95% CIs -2.73 to 3.51%), which equates to an additional 1 diagnosis per 256 patients tested. This cost-effectiveness study comparing these two strategies estimates that array-CGH first-line testing dominates second-line testing because it was both less costly and as effective. The sensitivity analyses conducted (adjusting both costs and effects) supported the dominance of the first-line testing strategy (i.e. lower cost and as effective). CONCLUSIONS: The first-line testing strategy was estimated to dominate the second-line testing strategy because it was both less costly and as effective. These findings are relevant to the wider UK NHS clinical genetics service, with two key strengths of this study being the appropriateness of the comparator interventions and the direct applicability of the patient cohort within this study and the wider UK patient population.

[Comparative cost analysis of molecular biology methods in the diagnosis of sarcomas]. / Analyse comparative des coûts de techniques de biologie moléculaire dans le diagnostic de sarcomes.

Sarcomas represent a complex and heterogeneous group of rare malignant tumors and their correct diagnosis is often difficult. Recent molecular biological techniques have been of great diagnostic use and there is a need to assess the cost of these procedures in routine clinical practice. Using prospective and observational data from eight molecular biology laboratories in France, we used "microcosting" method to assess the cost of molecular biological techniques in the diagnosis of five types of sarcoma. The mean cost of fluorescence in situ hybridization (FISH) was 318 € (273-393) per sample; mean reverse transcription polymerase chain reaction (RT-PCR) cost ranged from 300 € (229-481) per formalin-fixed, paraffin-embedded specimen to 258 € (213-339) per frozen specimen; mean quantitative polymerase chain reaction (Q-PCR) cost was 184 € (112-229) and mean CGH-array cost was 332 € (329-335). The cost of these recently implemented techniques varied according to the type of sarcoma; the method of tissue collection and local organizational factors including the level of local expertise and investment. The cost of molecular diagnostic techniques needs to be balanced against their respective performance.

A robust method to analyze copy number alterations of less than 100 kb in single cells using oligonucleotide array CGH.

Comprehensive genome wide analyses of single cells became increasingly important in cancer research, but remain to be a technically challenging task. Here, we provide a protocol for array comparative genomic hybridization (aCGH) of single cells. The protocol is based on an established adapter-linker PCR (WGAM) and allowed us to detect copy number alterations as small as 56 kb in single cells. In addition we report on factors influencing the success of single cell aCGH downstream of the amplification method, including the characteristics of the reference DNA, the labeling technique, the amount of input DNA, reamplification, the aCGH resolution, and data analysis. In comparison with two other commercially available non-linear single cell amplification methods, WGAM showed a very good performance in aCGH experiments. Finally, we demonstrate that cancer cells that were processed and identified by the CellSearch® System and that were subsequently isolated from the CellSearch® cartridge as single cells by fluorescence activated cell sorting (FACS) could be successfully analyzed using our WGAM-aCGH protocol. We believe that even in the era of next-generation sequencing, our single cell aCGH protocol will be a useful and (cost-) effective approach to study copy number alterations in single cells at resolution comparable to those reported currently for single cell digital karyotyping based on next generation sequencing data.

High quality genomic copy number data from archival formalin-fixed paraffin-embedded leiomyosarcoma: optimisation of universal linkage system labelling.

Most soft tissue sarcomas are characterized by genetic instability and frequent genomic copy number aberrations that are not subtype-specific. Oligonucleotide microarray-based Comparative Genomic Hybridisation (array CGH) is an important technique used to map genome-wide copy number aberrations, but the traditional requirement for high-quality DNA typically obtained from fresh tissue has limited its use in sarcomas. Although large archives of Formalin-fixed Paraffin-embedded (FFPE) tumour samples are available for research, the degradative effects of formalin on DNA from these tissues has made labelling and analysis by array CGH technically challenging. The Universal Linkage System (ULS) may be used for a one-step chemical labelling of such degraded DNA. We have optimised the ULS labelling protocol to perform aCGH on archived FFPE leiomyosarcoma tissues using the 180k Agilent platform. Preservation age of samples ranged from a few months to seventeen years and the DNA showed a wide range of degradation (when visualised on agarose gels). Consistently high DNA labelling efficiency and low microarray probe-to-probe variation (as measured by the derivative log ratio spread) was seen. Comparison of paired fresh and FFPE samples from identical tumours showed good correlation of CNAs detected. Furthermore, the ability to macro-dissect FFPE samples permitted the detection of CNAs that were masked in fresh tissue. Aberrations were visually confirmed using Fluorescence in situ Hybridisation. These results suggest that archival FFPE tissue, with its relative abundance and attendant clinical data may be used for effective mapping for genomic copy number aberrations in such rare tumours as leiomyosarcoma and potentially unravel clues to tumour origins, progression and ultimately, targeted treatment.

A clinical algorithm for efficient, high-resolution cytogenomic analysis of uncultured perinatal tissue samples.

PURPOSE: Cytogenetic analysis of solid tissue is indispensable in perinatal care, reproductive planning, and detection of gestational trophoblastic disease. Unfortunately, methods in common use suffer from drawbacks including culture artifact, low resolution, and high cost. We propose a new diagnostic algorithm based on direct genetic analysis of tissues (without cell culture) using QF-PCR and array CGH. METHODS: Study samples consisted of specimens submitted to the cytogenetics laboratory between January and June of 2011 that were split and analyzed in parallel by our traditional algorithm (culture and G-banding, plus an interphase FISH aneuploidy panel for culture failures) and the proposed "no-culture" algorithm (first line QF-PCR, plus array CGH on normal QF-PCRs). Data on clinical impact, cost, and turnaround time were collected. RESULTS: Forty specimens were included. The algorithms produced results that were fully concordant in 22 cases, partially concordant in 9 cases, and discordant in 9 cases. The no-culture algorithm detected new, clinically-significant abnormalities in 8 of 40 cases (20%), corrected the sex chromosome assortment in 1 case, reduced the analysis failure rate from 10% to 0%, and provided at least one of these three important benefits in 12 of 40 cases (30%). The algorithm also demonstrated a reduced cost per-specimen and per diagnosis, as well as improved turnaround time, with virtually all cases reported per guidelines. CONCLUSION: These striking results favor the "no-culture" algorithm, which may have the potential to replace standard cytogenetic methods in the clinical laboratory.

Evaluation of one- and two-color gene expression arrays for microbial comparative genome hybridization analyses in routine applications.

DNA microarray technology has already revolutionized basic research in infectious diseases, and whole-genome sequencing efforts have allowed for the fabrication of tailor-made spotted microarrays for an increasing number of bacterial pathogens. However, the application of microarrays in diagnostic microbiology is currently hampered by the high costs associated with microarray experiments and the specialized equipment needed. Here, we show that a thorough bioinformatic postprocessing of the microarray design to reduce the amount of unspecific noise also allows the reliable use of spotted gene expression microarrays for gene content analyses. We further demonstrate that the use of only single-color labeling to halve the costs for dye-labeled nucleotides results in only a moderate decrease in overall specificity and sensitivity. Therefore, gene expression microarrays using only single-color labeling can also reliably be used for gene content analyses, thus reducing the costs for potential routine applications such as genome-based pathogen detection or strain typing.

Improved detection of aneuploid blastocysts using a new 12-chromosome FISH test.

Fluorescence in-situ hybridization (FISH) has been the principal method used for the identification and preferential transfer of chromosomally normal embryos, in the context of both preimplantation genetic diagnosis (PGD) and screening (PGS). Generally, the probe combinations used during PGS have focused on chromosomes frequently identified as abnormal in prenatal samples or material derived from first-trimester spontaneous abortions. Recent data, however, obtained with the use of comparative genomic hybridization (CGH), have suggested that commonly used PGS strategies may fail to detect a large number of aneuploidies affecting preimplantation embryos. Some chromosomes, which have been relatively neglected in PGS protocols thus far, display a disproportionate contribution to embryo aneuploidy and should be prioritized for screening. Using CGH data, it is possible to design new probe combinations that examine between 10 and 12 chromosomes and are capable of accurately diagnosing 89-91% of anomalies seen in embryos. At present, 24-chromosome tests, such as CGH, array CGH or single nucleotide polymorphism arrays, remain relatively costly and, in some cases, are yet to be fully validated. For these reasons, a cost-effective method, capable of accurately detecting almost all aneuploid embryos, represents an attractive alternative to comprehensive chromosome screening approaches.

Valuing the benefit of diagnostic testing for genetic causes of idiopathic developmental disability: willingness to pay from families of affected children.

Idiopathic developmental disability (DD) has been found to put significant psychological distress on families of children with DD. The cause of the disability, however, is unknown for up to one-half of the affected children. Chromosomal abnormalities identified by cytogenetic analysis are the most frequently recognized cause of DD, although they account for less than 10% of cases. Array genomic hybridization (AGH) is a new diagnostic tool that provides a much higher detection rate for chromosomal imbalance than conventional cytogenetic analysis. This increase in diagnostic capability comes at greater monetary costs, which provides an impetus for understanding how individuals value genetic testing for DD. This study estimated the willingness to pay (WTP) for diagnostic testing to find a genetic cause of DD from families of children with DD. A discrete choice experiment was used to obtain WTP values. When it was assumed that AGH resulted in twice as many diagnoses and a 1-week reduction in waiting time compared with conventional cytogenetic analysis, this study found that families were willing to pay up to CDN$1118 (95% confidence interval, $498-1788) for the expected benefit. These results support the conclusion that the introduction of AGH into the Canadian health care system may increase the perceived welfare of society, but future studies should examine the cost-benefit of AGH vs cytogenetic testing.