Objective
Genomics Quality Assessment has provided external quality assessments (EQAs) for preimplantation genetic testing (PGT) for 12 years for eight monogenic diseases to identify sub‐optimal PGT ...strategies, testing and reporting of results, which can be shared with the genomics community to aid optimised standards of PGT services for couples.
Method
The EQAs were provided in two stages to mimic end‐to‐end protocols. Stage 1 involved DNA feasibility testing of a couple undergoing PGT and affected proband. Participants were required to report genotyping results and outline their embryo testing strategy. Lymphoblasts were distributed for mock embryo testing for stage 2. Submitted clinical reports and haplotyping results were assessed against peer‐ratified criteria. Performance was monitored to identify poor performance.
Results
The most common testing methodology was short tandem repeat linkage analysis (59%); however, the adoption of single nucleotide polymorphism‐based platforms was observed and a move from blastomere to trophectoderm testing. There was a variation in testing strategies, assigning marker informativity and understanding test limitations, some clinically unsafe. Critical errors were reported for genotyping and interpretation.
Conclusion
EQA provides an overview of the standard of preimplantation genetic testing‐M clinical testing and identifies areas of improvement for accurate detection of high‐risk embryos.
Key points
External quality assessment (EQA) is required to assess of the standard of testing for monogenic disorders, which incorporates the end‐to‐end process for preimplantation genetic testing (PGT‐M).
12 years of EQA demonstrates the evolution of testing and identifies disease‐specific challenges for delivering appropriate strategies to identify high‐risk/low‐risk haplotypes.
The importance of genotyping accuracy and understanding marker informativity is critical for the assignment of risk haplotypes and this manuscript highlights the variability in clinical practice.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Next‐generation sequencing is radically changing how DNA diagnostic laboratories operate. What started as a single‐gene profession is now developing into gene panel sequencing and whole‐exome and ...whole‐genome sequencing (WES/WGS) analyses. With further advances in sequencing technology and concomitant price reductions, WGS will soon become the standard and be routinely offered. Here, we focus on the critical steps involved in performing WGS, with a particular emphasis on points where WGS differs from WES, the important variables that should be taken into account, and the quality control measures that can be taken to monitor the process. The points discussed here, combined with recent publications on guidelines for reporting variants, will facilitate the routine implementation of WGS into a diagnostic setting.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Summary
β‐thalassaemia is one of the commonest autosomal recessive single‐gene disorders worldwide. Prenatal tests use invasive methods, posing a risk for the pregnancy itself. Development of a ...noninvasive prenatal diagnostic method is, therefore, of paramount importance. The aim of the present study is to identify high‐heterozygote informative single‐nucleotide polymorphisms (SNPs), suitable for the development of noninvasive prenatal diagnosis (NIPD) of β‐thalassaemia. SNP genotyping analysis was performed on 75 random samples from the Cypriot population for 140 SNPs across the β‐globin cluster. Shortlisted, highly heterozygous SNPs were then examined in 101 carrier families for their applicability in the noninvasive detection of paternally inherited alleles. Forty‐nine SNPs displayed more than 6% heterozygosity and were selected for NIPD analysis, revealing 72.28% of the carrier families eligible for qualitative SNP‐based NIPD, and 92% for quantitative detection. Moreover, inference of haplotypes showed predominant haplotypes and many subhaplotypes with sufficient prevalence for diagnostic exploitation. SNP‐based analyses are sensitive and specific for the detection of the paternally inherited allele in maternal plasma. This study provides proof of concept for this approach, highlighting its superiority to NIPD based on single markers and thus providing a blueprint for the general development of noninvasive prenatal diagnostic assays for β‐thalassaemia.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
When the molecular background of couples requesting prevention is unclear, family analysis and tools to define rare mutations are essential. We report two novel deletion defects observed in an ...Italian and in a Turkish couple. The first proband presented with microcytic hypochromic parameters without iron deficiency, a normal HbA(2) and an elevated HbF (10.6%). His father presented with a similar phenotype and his wife was heterozygous for the common Mediterranean codon 39 (HBB:c.118C>T) mutation. Having excluded point mutations and common deletions, Multiplex Ligation-dependent Probe Amplification was performed revealing an unknown Ggamma(Agammadeltabeta)(0)-thalassemia defect spanning from the Agamma gene to downstream of the beta-globin gene provisionally named Leiden 69.5 kb deletion. In the second case, the wife presented with a mild thalassemic picture, normal HbA(2), elevated HbF (18.5%) and a beta/alpha globin chain synthesis ratio of 0.62, without iron deficiency or any known beta-thalassemia defect, while the husband was a simple carrier of the common Mediterranean IVS-I-110 (HBB:c.93-21 G>A) mutation. A new large deletion involving the beta-gene and part of the delta-gene was identified by Multiplex Ligation-dependent Probe Amplification provisionally named "Leiden 7.4 kb".
Objectives: To determine the molecular basis in a Greek child suspected of having HbH disease and β‐thalassemia trait.
Methods: Standard hematology, Hb electrophoresis, and HPLC. Multiplex ...ligation‐dependent probe amplification (MLPA), direct sequencing, and breakpoint characterization by NimbleGen fine‐tiling array analysis.
Results: The index patient showed a moderate microcytic hypochromic anemia with normal ZPP and elevated HbA2, indicative for β‐thalassemia trait. However, the moderate microcytic hypochromic anemia along with the observation of HbH inclusions in occasional red blood cells suggested a coexisting α‐thalassemia. Molecular analysis indicated that the propositus inherited the β+‐thalassemia mutation IVS2‐745 (c>g) and a novel α0‐thalassemia deletion from the mother, and the common non‐deletion α‐thalassemia allele α2(−5nt)α from the father. The α0‐thalassemia deletion, named ‐ ‐BGS, is approximately 131.6 kb in length. It removes the major regulatory elements along with the functional α‐globin genes but leaves the theta‐gene intact.
Conclusions: The compound interaction of a β‐thalassemia defect along with a single functional α‐globin gene is quite rare. Although patients with HbH/β‐thal and simple HbH disease have comparable levels of Hb, the absence of free β‐globin chains and thus detectable non‐functional HbH means that in HbH/β‐thal, the levels of functional Hb are higher, resulting in a better compensated functional anemia. Rare large deletions as the one described here remain undetected by gap‐PCR in routine molecular screening. The introduction of MLPA as a diagnostic screening tool may improve laboratory diagnostics for these defects. The use of NimbleGen fine‐tiling arrays may give additional information about the precise location of breakpoints.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Background: The detection and diagnosis of β-thalassaemia for populations with molecular heterogeneity, or diverse ethnic groups, has increased the need for the development of an array ...high-throughput diagnostic tool that can deliver large scale genetic detection. We report on the update and validation of the ThalassoChip, a β-thalassaemia genetic diagnostic tool which is based on arrayed primer extension (APEX) technology. Methods: ThalassoChip slides with new and redesigned probes were prepared for testing the microarray. Six hundred and sixty DNA samples collected from eight Mediterranean countries were used for standardisation, optimisation and validation of the ThalassoChip. The β-globin gene region was amplified by PCR, the products were hybridised to the probes after fragmentation and the APEX reaction followed. Results: The ThalassoChip was updated with new probes and now has the ability to detect 57 β-globin gene mutations and three single nucleotide polymorphisms (SNPs) in a single test. The ThalassoChip as well as the PCR and APEX reactions were standardised and optimised using 500 DNA samples that were previously genotyped using conventional diagnostic techniques. Some probes were redesigned in order to improve the specificity and sensitivity of the test. Validation of the ThalassoChip performed using 160 samples analysed in blinded fashion showed no error. Conclusions: The updated version of the ThalassoChip is versatile, robust, cost-effective and easily adaptable, but most notably can provide comprehensive genetic diagnosis for β-thalassaemia and other haemoglobinopathies. Clin Chem Lab Med 2010;48:1713–8.
DNA biosensors involve molecular recognition of the target sequence by hybridization with specific probes and detection by electrochemical, optical or gravimetric transduction. Disposable, ...dipstick-type biosensors have been developed recently, which enable visual detection of DNA without using instruments. In this context, we report a multianalyte DNA biosensor for visual genotyping of two single-nucleotide polymorphisms (SNPs). As a model, the biosensor was applied to the simultaneous genotyping of two SNPs, entailing the detection of four alleles. A PCR product that flanks both polymorphic sites is subjected to a single primer extension (PEXT) reaction employing four allele-specific primers, each containing a region complementary to an allele and a characteristic segment that enables subsequent capture on a test zone of the biosensor. The primers are extended with dNTPs and biotin-dUTP only if there is perfect complementarity with the interrogated sequence. The PEXT mixture is applied to the biosensor. As the developing buffer migrates along the strip, all the allele-specific primers are captured by immobilized oligonucleotides at the four test zones of the biosensor and detected by antibiotin-functionalized gold nanoparticles. As a result, the test zones are colored red if extension has occurred denoting the presence of the corresponding allele in the original sample. The excess nanoparticles are captured by immobilized biotinylated albumin at the control zone of the sensor forming another red zone that indicates the proper performance of the system. The assay was applied successfully to the genotyping of twenty clinical samples for two common SNPs of MBL2 gene.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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