Establishing national reference materials for genetic testing of cytochrome P450.

OBJECTIVES: Reference materials for in-vitro diagnostic reagents play a critical role in determining the quality of reagents and ensuring the accuracy of clinical test results. This study aimed to establish a national reference material (NRM) for detecting cytochrome P450 (CYP) genes related to drug metabolism by screening databases on the Chinese population to identify CYP gene polymorphism characteristics. METHODS: To prepare the NRM, we used DNA extracted from healthy human immortalized B lymphoblastoid cell lines as the raw material. Samples of these cell lines were obtained from the Chinese Population PGx Gene Polymorphism Biobank. Further, we used Sanger sequencing, next-generation sequencing, and commercial assay kits to validate the polymorphic genotypes. RESULTS: Among the CYP superfamily genes, we confirmed 24 riboswitch loci related to drug metabolism, with evidence levels of 1A, 2A, 3, and 4. We confirmed the polymorphic loci and validated their genotypes using various sequencing techniques. Our results were consistent with the polymorphism information of samples obtained from the biobank, thus demonstrating high precision and stability of the established NRM. CONCLUSION: An NRM (360 056-202 201) for CYP genetic testing covering 24 loci related to drug metabolism was established and approved to assess in-vitro diagnostic reagents containing CYP family gene polymorphisms and perform clinical inter-room quality evaluations.

Nanopore Third-Generation Sequencing for Comprehensive Analysis of Hemoglobinopathy Variants.

BACKGROUND: Oxford Nanopore Technology (ONT) third-generation sequencing (TGS) is a versatile genetic diagnostic platform. However, it is nonetheless challenging to prepare long-template libraries for long-read TGS, particularly the ONT method for analysis of hemoglobinopathy variants involving complex structures and occurring in GC-rich and/or homologous regions. METHODS: A multiplex long PCR was designed to prepare library templates, including the whole-gene amplicons for HBA2/1, HBG2/1, HBD, and HBB, as well as the allelic amplicons for targeted deletions and special structural variations. Library construction was performed using long-PCR products, and sequencing was conducted on an Oxford Nanopore MinION instrument. Genotypes were identified based on integrative genomics viewer (IGV) plots. RESULTS: This novel long-read TGS method distinguished all single nucleotide variants and structural variants within HBA2/1, HBG2/1, HBD, and HBB based on the whole-gene sequence reads. Targeted deletions and special structural variations were also identified according to the specific allelic reads. The result of 158 α-/ß-thalassemia samples showed 100% concordance with previously known genotypes. CONCLUSIONS: This ONT TGS method is high-throughput, which can be used for molecular screening and genetic diagnosis of hemoglobinopathies. The strategy of multiplex long PCR is an efficient strategy for library preparation, providing a practical reference for TGS assay development.

Comprehensive evaluation of BRCA1/2 variant interpretation ability among laboratories in China.

High-quality interpretation of BRCA1/2 variants plays a critical role in the clinical practice of precision medicine. However, a comprehensive system to evaluate the quality and accuracy of variant interpretation has yet to be established. This study investigates the performance of an interpretation system in evaluating the capacities of BRCA1/2 interpretation among distinct laboratories in China. The evaluation system is based on a reference database that contains 750 different variants in BRCA1/2 Evaluation was performed among 41 laboratories in China. We classified their performance into five levels. Only level A was considered qualified. This level allows for a 0.3% error rate for clinical decision-related misinterpretation; 26 of 41 laboratories (63%) met the qualified standard, while 7 laboratories were at levels D and E, which indicated egregious mistakes and systemic problems in variant interpretation. Due to strict quality demands, the interpretation of several variants was amended, which largely influenced the quality rate. The number of qualified laboratories would decrease from 26 to 17 if those incorrect recommended interpretations were not corrected. This evaluation system provides a potential approach for standardisation of variant interpretation and lowers the discordance of variant interpretation between different laboratories. A well-designed interpretation ability evaluation is essential to evaluate the interpretation level of laboratories before they provide service in real-world clinical settings.

Harmonization of distributed multi-center analysis based on dried blood spot reference materials supporting the screening of neonatal inherited metabolic disorders.

BACKGROUND: The standardization of quantification data is critical for ensuring the reliability and measurement traceability in the screening of neonatal inherited metabolic disorders. However, the availability of national certified reference materials is limited in China. METHODS: In this study, we developed a series of dried blood spot (DBS) reference materials containing 9 amino acids (AA) and 10 acylcarnitines (AC) for neonatal screening. Four levels of the reference materials were measured with tandem mass spectrometry (MS/MS) by seven laboratories using different commercial In Vitro Diagnostic Device (IVD) kits. Then, 100 clinical samples were measured using both derivatization and non-derivatization methods by the same laboratory. RESULTS: We found high homogeneity and stability at all levels of the reference materials, with the coefficient of variation (CV) of the analytes less than 15%. These reference materials can be used to assess the testing capabilities of different laboratories. Our test also revealed that the correction factors (CF) calculated by the reference materials, along with clinical samples, could increase the consistency for different kits. CONCLUSION: The DBS reference materials proposed in this study provide reliability for the harmonization in multi-center analysis for the screening of neonatal inherited metabolic disorders. And applying our correction method for the screening could improve the data consistency of the DBS samples prepared by different methods.

Integrative analyses of maternal plasma cell-free DNA nucleosome footprint differences reveal chromosomal aneuploidy fetuses gene expression profile.

BACKGROUND: Chromosomal aneuploidy is the most common birth defect. However, the developmental mechanism and gene expression profile of fetuses with chromosomal aneuploidy are relatively unknown, and the maternal immune changes induced by fetal aneuploidy remain unclear. The inability to obtain the placenta multiple times in real-time is a bottleneck in research on aneuploid pregnancies. Plasma cell-free DNA (cfDNA) carries the gene expression profile information of its source cells and may be used to evaluate the development of fetuses with aneuploidy and the immune changes induced in the mother owing to fetal aneuploidy. METHODS: Here, we carried out whole-genome sequencing of the plasma cfDNA of 101 pregnant women carrying a fetus with trisomy (trisomy 21, n = 42; trisomy 18, n = 28; trisomy 13, n = 31) based on non-invasive prenatal testing (NIPT) screening and 140 normal pregnant women to identify differential genes according to the cfDNA nucleosome profile in the region around the transcription start sites (TSSs). RESULTS: The plasma cfDNA promoter profiles were found to differ between aneuploid and euploid pregnancies. A total of 158 genes with significant differences were identified, of which 43 genes were upregulated and 98 genes were downregulated. Functional enrichment and signaling pathway analysis were performed based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases found that these signal pathways were mainly related to the coordination of developmental signals during embryonic development, the control of cell growth and development, regulation of neuronal survival, and immune regulation, such as the MAPK, Hippo, TGF-ß, and Rap1 signaling pathways, which play important roles in the development of embryonic tissues and organs. Furthermore, based on the results of differential gene analysis, a total of 14 immune-related genes with significant differences from the ImmPort database were collected and analyzed. These significantly different immune genes were mainly associated with the maintenance of embryonic homeostasis and normal development. CONCLUSIONS: These results suggest that the distribution characteristics of cfDNA nucleosomes in maternal plasma can be used to reflect the status of fetal development and changes of the immune responses in trisomic pregnancies. Overall, our findings may provide research ideas for non-invasive detection of the physiological and pathological states of other diseases.

Simulated confined placental mosaicism proportion (SCPMP) based on cell-free fetal DNA fraction enrichment can reduce false-positive results in non-invasive prenatal testing.

OBJECTIVE: To decrease the false-positive rate of NIPT using cell-free fetal DNA (cffDNA) fraction enrichment and the simulated confined placental mosaicism proportion (SCPMP) threshold application via cffDNA quantification. METHOD: Using a cffDNA enrichment method, 303 plasma samples with positive NIPT results (Z-score > 3.0; 200 true-positive and 103 false-positive cases) were re-sequenced. A method to calculate the SCPMP based on the quantified cffDNA fraction was developed; the SCPMP threshold between true- and false-positive NIPT results was determined and used for re-analyses. RESULTS: With enrichment, the fetal fraction of the 303 samples was 26.9 ± 8.4%, compared to 11.0 ± 3.2% without enrichment. The optimized threshold method with double determination using the Z-value-defined SCPMP can reduce the false-positive rates for trisomies 21, 18, and 13 by 87%, 80%, and 88.9%, respectively. CONCLUSION: Our optimized method can decrease the false-positive rate of NIPT results.

The Setup and Application of Reference Material in Sequencing-Based Noninvasive Prenatal Testing.

INTRODUCTION: The sequencing-based noninvasive prenatal testing (NIPT) has been successfully integrated into clinical practice and facilitated the early detection of fetal chromosomal anomalies. However, a comprehensive reference material to evaluate and quality control NIPT services from different NIPT providers remains unavailable. METHODS: In this study, we established a set of NIPT reference material consisting of 192 simulated samples. Most of the potential factors influencing the accuracy of NIPT, such as fetal fraction, mosaicism, and interfering substances, were included in the reference material. We compared the performance of chromosomal abnormalities detection on 3 widely used sequencers (NextSeq 500, BGISEQ-500, and Ion Proton) based on the reference material. RESULTS: All 3 sequencers provided highly accurate and reliable results to samples with ≥3.5% fetal fractions and high percentage of mosaicism. CONCLUSIONS: The established reference material can serve as a universal standard quality control for the current and new-coming NIPT providers based on various sequencers.

[Study on Proficiency Testing of Determination Potassium in Serum].

OBJECTIVE: To evaluate the capacity of laboratories participated in the proficiency testing (PT) of determination potassium in serum and improve the quality of testing, and put forward technical suggestions for unsatisfied laboratories. METHODS: According to the requirements of CNAS related documents, the homogeneity and stability of the real PT sample were evaluated by one-way ANOVA and t test, respectively. The values of real PT samples were assigned by reference method which was used in PT results assay. It is required that the deviation of value of real PT samples (code:2, 3, 5) between the measured value and the assigned value shall be within ±15.0%. The precision of values for all samples should not be greater than 3.0%. RESULTS: All the laboratories submitted valid data according to the requirements. Only one laboratory did not meet the requirements, and the satisfaction rate was 90.9%. CONCLUSIONS: The ability of most of laboratories are accurate and reliable.

[The Quality Analysis of National Supervising Sampling for Rubella Virus IgM Diagnostic Kits in 2014].

OBJECTIVE: To evaluate the quality status of rubella virus IgM diagnostic kits by national supervising sampling. METHODS: Using legal inspection combining with exploratory study, the positive and negative coincidence rate, detection limit and repeatability of kits were verified. RESULTS: The results showed that 15 of 16 batches of kits were qualified using legal inspection, and the passing rate was 93.8%. The unqualified item was negative coincidence rate. In exploratory study, only 11 batches (68.8%) complied with industry standard. The unqualified items were negative coincidence rate, detection limit and repeatability. CONCLUSION: At present, rubella virus IgM diagnostic Kits have some quality problems in the market. It is recommended that we adopt industry standard and national reference panel in the registration inspection for the future, which will prompt enterprises to improve quality.

Development of preimplantation genetic testing for monogenic reference materials using next-generation sequencing.

OBJECTIVE: Preimplantation genetic testing for monogenic disorders (PGT-M) has been used for over 20 years to detect many serious genetic conditions. However, there is still a lack of reference materials (RMs) to validate the test performance during the development and quality control of PGT-M. METHOD: Sixteen thalassemia cell lines from four thalassemia families were selected to establish the RMs. Each family consisted of parents with heterozygous mutations for α- and/or ß-thalassemia and two children, at least one of whom carried a homozygous thalassemia mutation (proband). The RM panel consisted of 12 DNA samples (parents and probands in 4 families) and 4 simulated embryos (cell lines constructed from blood samples from the four nonproband children). Four accredited genetics laboratories that offer verification of thalassemia samples were invited to evaluate the performance of the RM panel. Furthermore, the stability of the RMs was determined by testing after freeze‒thaw cycles and long-term storage. RESULTS: PGT-M reference materials containing 12 genome DNA (gDNA) reference materials and 4 simulated embryo reference materials for thalassemia testing were successfully established. Next-generation sequencing was performed on the samples. The genotypes and haplotypes of all 16 PGT-M reference materials were concordant across the four labs, which used various testing workflows. These well-characterized PGT-M reference materials retained their stability even after 3 years of storage. CONCLUSION: The establishment of PGT-M reference materials for thalassemia will help with the standardization and accuracy of PGT-M in clinical use.

Restriction endonuclease-mediated real-time digestion-PCR for somatic mutation detection.

PCR is a powerful platform for clinical and diagnostic applications, but challenges remain in detecting somatic mutations, as mutant cells are often mixed with more numerous wild-type cells at the tissue-sample sites. Here, we describe a novel method that couples PCR with restriction endonuclease digestion (designated real-time digestion-PCR, or RTD-PCR) in a one-step reaction tube for detecting somatic mutations from a minority of cells. The PCR mixture contains a thermostable restriction enzyme that digests wild-type alleles during the PCR program, allowing selective amplification of the mutant alleles. To validate this method, we used real-time digestion-PCR for the specific detection of the EGFR (epidermal growth factor receptor) treatment resistance-inducing mutation, T790M, combining with three different platforms: Sanger sequencing, TaqMan probe PCR and Sequenom MassArray. From 78 clinical samples, seven T790M mutations were consistently detected on all three platforms, indicating that RTD-PCR may be a useful clinical tool for analyzing the T790M point mutation.

Development and characterization of reference materials for EGFR, KRAS, NRAS, BRAF, PIK3CA, ALK, and MET genetic testing.

BACKGROUND: Along with the dramatic development of molecular diagnostic testing for the detection of oncogene variations, reference materials (RMs) have become increasingly important in performance evaluation of genetic testing. OBJECTIVE: In this study, we built a set of RMs for genetic testing based on next-generation sequencing (NGS). METHOD: Solid tumor tissues were selected as the samples of RMs for preparation. NGS was used to determine and validate the variants and the mutation frequency in DNA samples. Digital PCR was used to determine the copy numbers of RNA samples. The performance of the RMs was validated by six laboratories. RESULTS: Thirty common genetic alterations were designed based on these RMs. RMs consisted of a positive reference, a limit of detection reference, and a negative reference. The validation results confirmed the performance of the RMs. CONCLUSION: These RMs may be an attractive tool for the development, validation, and quality monitoring of molecular genetic testing.

Haplotype-resolved Chinese male genome assembly based on high-fidelity sequencing.

The advantages of both the length and accuracy of high-fidelity (HiFi) reads enable chromosome-scale haplotype-resolved genome assembly. In this study, we sequenced a cell line named HJ, established from a Chinese Han male individual by using HiFi and Hi-C. We assembled two high-quality haplotypes of the HJ genome (haplotype 1 (H1): 3.1 Gb, haplotype 2 (H2): 2.9 Gb). The continuity (H1: contig N50 = 28.2 Mb, H2: contig N50 = 25.9 Mb) and completeness (BUSCO: H1 = 94.9%, H2 = 93.5%) are substantially better than those of other Chinese genomes, for example, HX1, NH1.0, and YH2.0. By comparing HJ genome with GRCh38, we reported the mutation landscape of HJ and found that 176 and 213 N-gaps were filled in H1 and H2, respectively. In addition, we detected 12.9 Mb and 13.4 Mb novel sequences containing 246 and 135 protein-coding genes in H1 and H2, respectively. Our results demonstrate the advantages of HiFi reads in haplotype-resolved genome assembly and provide two high-quality haplotypes of a potential Chinese genome as a reference for the Chinese Han population.

Robust Benchmark Structural Variant Calls of An Asian Using State-of-the-art Long-read Sequencing Technologies.

The importance of structural variants (SVs) for human phenotypes and diseases is now recognized. Although a variety of SV detection platforms and strategies that vary in sensitivity and specificity have been developed, few benchmarking procedures are available to confidently assess their performances in biological and clinical research. To facilitate the validation and application of these SV detection approaches, we established an Asian reference material by characterizing the genome of an Epstein-Barr virus (EBV)-immortalized B lymphocyte line along with identified benchmark regions and high-confidence SV calls. We established a high-confidence SV callset with 8938 SVs by integrating four alignment-based SV callers, including 109× Pacific Biosciences (PacBio) continuous long reads (CLRs), 22× PacBio circular consensus sequencing (CCS) reads, 104× Oxford Nanopore Technologies (ONT) long reads, and 114× Bionano optical mapping platform, and one de novo assembly-based SV caller using CCS reads. A total of 544 randomly selected SVs were validated by PCR amplification and Sanger sequencing, demonstrating the robustness of our SV calls. Combining trio-binning-based haplotype assemblies, we established an SV benchmark for identifying false negatives and false positives by constructing the continuous high-confidence regions (CHCRs), which covered 1.46 gigabase pairs (Gb) and 6882 SVs supported by at least one diploid haplotype assembly. Establishing high-confidence SV calls for a benchmark sample that has been characterized by multiple technologies provides a valuable resource for investigating SVs in human biology, disease, and clinical research.

A comparison of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and surface plasmon resonance for genotyping of high-risk human papillomaviruses.

BACKGROUND: Human papillomavirus (HPV) testing coupled with appropriate clinical management is associated with a significant decline in the rate of advanced cervical cancer and associated death. METHODS: In this present study, we evaluated the performance of 2 new HPV genotyping methods, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and surface plasmon resonance (SPR) in 30 kinds of HPV control materials and in 129 cases of cervical smears including 79 HPV-positive samples screened from 1,600 abnormal clinical samples and 50 cervical cytology samples. RESULTS: The HPV genotyping accuracy of both MALDI-TOFMS and SPR was 100% for the HPV genotyping of control materials. In the analysis of the 79 HPV-positive samples by MALDI-TOFMS, HPV positivity was 88.6% (70/79). Nine samples were non-high-risk HPV (non-HR-HPV), which were not targets of MALDI-TOFMS. In the analysis of the 50 cervical samples, the agreement of both tests was 84% with a κ value of 0.660. By using consensus results that mean agreement between 2 of 3 methods, the HR-HPV genotyping accuracy was 100% (77/77) by MALDI-TOFMS and 94.8% (73/77) by SPR in the 129 cervical samples. The sensitivity (88.2%; 82/93) and specificity (88.9%; 32/36) of MALDI-TOFMS were similar to those of SPR. CONCLUSION: These results support that MALDI-TOFMS is a sensitive, specific and feasible method for HR-HPV detection in clinical application, compared with the SPR method.

BDdb: a comprehensive platform for exploration and utilization of birth defect multi-omics data.

BACKGROUND: Birth defects pose a major challenge to infant health. Thus far, however, the causes of most birth defects remain cryptic. Over the past few decades, considerable effort has been expended on disclosing the underlying mechanisms related to birth defects, yielding myriad treatises and data. To meet the increasing requirements for data resources, we developed a freely accessible birth defect multi-omics database (BDdb, http://t21omics.cngb.org ) consisting of multi-omics data and potential disease biomarkers. RESULTS: In total, omics datasets from 136 Gene Expression Omnibus (GEO) Series records, including 5245 samples, as well as 869 biomarkers of 22 birth defects in six different species, were integrated into the BDdb. The database provides a user-friendly interface for searching, browsing, and downloading data of interest. The BDdb also enables users to explore the correlations among different sequencing methods, such as chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq) from different studies, to obtain the information on gene expression patterns from diverse aspects. CONCLUSION: To the best of our knowledge, the BDdb is the first comprehensive database associated with birth defects, which should benefit the diagnosis and prevention of birth defects.

Construction of a reference material panel for detecting KRAS/NRAS/EGFR/BRAF/MET mutations in plasma ctDNA.

BACKGROUND: The absence of high-quality next-generation sequencing (NGS) reference material (RM) has impeded the clinical use of liquid biopsies with plasma cell-free DNA (cfDNA) in China. OBJECTIVE: This study aimed to develop a national RM panel for external quality assessment and performance evaluation during kit registration of non-small-cell lung cancer (NSCLC)-related Kirsten rat sarcoma viral oncogene (KRAS)/neuroblastoma ras oncogene (NRAS)/epidermal growth factor receptor (EGFR)/B-type Raf kinase (BRAF)/mesenchymal-epithelial transition factor (MET) genetic assays using plasma circulating tumor DNA (ctDNA). METHODS: Mutation cell lines detected by NGS and validated by Sanger sequencing were selected to establish the RM. Cell line genomic DNA was sheared and used to spike basal plasma cfDNA at 10% concentration. Then, the calibration accuracy was determined by four sequencing platforms. Average values were adopted and diluted to 0.1%, 0.3%, 1% and 3% concentrations with basal plasma as the RM panel. Then, five manufacturers were invited to evaluate the performance of the RM panel. RESULTS: 20 cell lines with 23 clinically important mutations were selected, including six mutations in KRAS, two mutations in NRAS, three in BRAF, four in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), six in EGFR, one EGFR Gain (4-5 copy) and one MET Gain (2-5 copy). The RM panel consisted of 87 samples, including these 21 mutations at four concentrations (0.1%, 0.3%, 1% and 3%), one MET gain, one EGFR gain and one wild type. The detection rate was 100% for the 3%, 1% and 0.3% samples at all five companies. For the 0.1% concentration, 15 samples had inconsistent results, but at least three companies had correct results for each mutation. CONCLUSION: RM for a KRAS/NRAS/EGFR/BRAF/MET mutation panel for plasma ctDNA was developed, which will be essential for quality control of the performance of independent laboratories.

A Deep-Learning Pipeline for TSS Coverage Imputation From Shallow Cell-Free DNA Sequencing.

Cell-free DNA (cfDNA) serves as a footprint of the nucleosome occupancy status of transcription start sites (TSSs), and has been subject to wide development for use in noninvasive health monitoring and disease detection. However, the requirement for high sequencing depth limits its clinical use. Here, we introduce a deep-learning pipeline designed for TSS coverage profiles generated from shallow cfDNA sequencing called the Autoencoder of cfDNA TSS (AECT) coverage profile. AECT outperformed existing single-cell sequencing imputation algorithms in terms of improvements to TSS coverage accuracy and the capture of latent biological features that distinguish sex or tumor status. We built classifiers for the detection of breast and rectal cancer using AECT-imputed shallow sequencing data, and their performance was close to that achieved by high-depth sequencing, suggesting that AECT could provide a broadly applicable noninvasive screening approach with high accuracy and at a moderate cost.

Influence of low tumor content on tumor mutational burden estimation by whole-exome sequencing and targeted panel sequencing.

BACKGROUND: Tumor mutational burden (TMB) is a promising biomarker for stratifying patient subpopulation who would benefit from immune checkpoint blockade (ICB) therapies. Although great efforts have been made for standardizing TMB measurement, mutation calling and TMB quantification can be challenging in samples with low tumor content including liquid biopsies. The effect of varying tumor content on TMB estimation by different assay methods has never been systematically investigated. METHOD: We established a series of reference standard DNA samples derived from 11 pairs of tumor-normal matched human cell lines across different cancer types. Each tumor cell line was mixed with its matched normal at 0% (control), 1%, 2%, 5%, and 10% mass-to-mass ratio to mimic the clinical samples with low tumor content. TMB of these reference standards was evaluated by both ∼1000× whole-exome sequencing (wesTMB) and targeted panel sequencing (psTMB) at four different vendors. Both regression and classification analyses of TMB were performed for theoretical investigation and clinical practice purposes. RESULTS: Linear regression model was established that demonstrated in silico psTMB determined by regions of interest (ROI) as a great representative of wesTMB based on TCGA dataset. It was also true in our reference standard samples as the predicted psTMB interval based on the observed wesTMB captured the intended 90% of the in silico psTMB values. Although ∼1000× deep WES was applied, reference standard samples with less than 5% of tumor proportions are below the assay limit of detection (LoD) of wesTMB quantification. However, predicted wesTMB based on observed psTMB accurately classify (>0.97 AUC) for TMB high and low patient stratification even in samples with 2% of tumor content, which is more clinically relevant, as TMB determination should be a qualitative assay for TMB high and low patient classification. One targeted panel sequencing vendor using an optimized blood psTMB pipeline can further classify TMB status accurately (>0.82 AUC) in samples with only 1% of tumor content. CONCLUSIONS: We developed a linear model to establish the quantitative correlation between wesTMB and psTMB. A set of DNA reference standards was produced in aid to standardize TMB measurements in samples with low tumor content across different targeted sequencing panels. This study is a significant contribution aiming to harmonize TMB estimation and extend its future application in clinical samples with low tumor content including liquid biopsy.

Development of a genomic DNA reference material panel for thalassemia genetic testing.

INTRODUCTION: Thalassemia is one of the most common autosomal recessive inherited diseases worldwide, and it is also highly prevalent and variable in southern China. Various types of genetic testing technologies have been developed for diagnosis and screening of thalassemia. Characterized genomic DNA reference materials (RMs) are necessary for assay development, validation, proficiency testing, and quality assurance. However, there are no publicly available RMs for thalassemia genetic testing as yet. METHODS: To address the need for the publicly available DNA RMs for thalassemia genetic testing, the National Institutes for Food and Drug Control and the China National GeneBank established 32 new cell lines with three wild-type genotypes and 29 distinct genotypes of thalassemia which account for approximately 90% thalassemia carriers in China. The genomic DNA of 32 cell lines was characterized by four clinical genetic testing laboratories using different genetic testing methods and technology platforms. RESULTS: The genotyping results are concordant among four laboratories. In addition, the results of stability test demonstrated that the genotypes of these DNA samples are not influenced by preanalytical conditions such as long-term exposure to high-temperature (37°C) environment and repeated freeze-thawing. CONCLUSION: We developed the first national panel of 32 genomic DNA RMs which are renewable and publicly available for the quality assurance of various genetic testing methods and will facilitate research and development in thalassemia genetic testing.