Clinical and analytical validation of an 82-gene comprehensive genome-profiling panel for identifying and interpreting variants responsible for inherited retinal dystrophies.

Inherited retinal dystrophies comprise a clinically complex and heterogenous group of diseases characterized by visual impairment due to pathogenic variants of over 300 different genes. Accurately identifying the causative gene and associated variant is crucial for the definitive diagnosis and subsequent selection of precise treatments. Consequently, well-validated genetic tests are required in the clinical practice. Here, we report the analytical and clinical validation of a next-generation sequencing targeted gene panel, the PrismGuide IRD Panel System. This system enables comprehensive genome profiling of 82 genes related to inherited retinal dystrophies. The PrismGuide IRD Panel System demonstrated 100% (n = 43) concordance with Sanger sequencing in detecting single-nucleotide variants, small insertions, and small deletions in the target genes and also in assessing their zygosity. It also identified copy-number loss in four out of five cases. When assessing precision, we evaluated the reproducibility of variant detection with 2,160 variants in 144 replicates and found 100% agreement in terms of single-nucleotide variants (n = 1,584) and small insertions and deletions (n = 576). Furthermore, the PrismGuide IRD Panel System generated sufficient read depth for variant calls across the purine-rich and highly repetitive open-reading frame 15 region of RPGR and detected all five variants tested. These results show that the PrismGuide IRD Panel System can accurately and consistently detect single-nucleotide variants and small insertions and deletions. Thus, the PrismGuide IRD Panel System could serve as useful tool that is applicable in clinical practice for identifying the causative genes based on the detection and interpretation of variants in patients with inherited retinal dystrophies and can contribute to a precise molecular diagnosis and targeted treatments.

Performance and usefulness of a novel automated immunoassay HISCL SARS-CoV-2 Antigen assay kit for the diagnosis of COVID-19.

Here, we aimed to evaluate the clinical performance of a novel automated immunoassay HISCL SARS-CoV-2 Antigen assay kit designed to detect the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This kit comprises automated chemiluminescence detection systems. Western blot analysis confirmed that anti-SARS-CoV antibodies detected SARS-CoV-2N proteins. The best cut-off index was determined, and clinical performance was tested using 115 serum samples obtained from 46 patients with coronavirus disease 2019 (COVID-19) and 69 individuals who tested negative for COVID-19 through reverse transcription quantitative polymerase chain reaction (RT-qPCR). The HISCL Antigen assay kit showed a sensitivity of 95.4% and 16.6% in samples with copy numbers > 100 and < 99, respectively. The kit did not cross-react with human coronaviruses causing seasonal common cold and influenza, and none of the 69 individuals without COVID-19 were diagnosed with positive results. Importantly, 81.8% of the samples with low virus load (< 50 copy numbers) were diagnosed as negative. Thus, using HISCL antigen assay kits may reduce overdiagnosis compared with RT-qPCR tests. The rapid and high-throughput HISCL SARS-CoV-2 Antigen assay kit developed here proved suitable for screening infectious COVID-19 and may help control the pandemic.

Clinical Validation of Newly Developed Multiplex Kit Using Luminex xMAP Technology for Detecting Simultaneous RAS and BRAF Mutations in Colorectal Cancer: Results of the RASKET-B Study.

Detection of RAS and BRAF mutations is essential to determine the optimal treatment strategy for metastatic colorectal cancer (CRC). We prospectively evaluated the MEBGEN RASKET-B KIT (RASKET-B), a novel multiplex kit, simultaneously detecting 48 types of RAS mutations and the BRAF V600E mutation using Luminex xMAP technology. The aim was to obtain market approval for RASKET-B as an in vitro diagnostic (IVD) option in Japan. Genomic DNA was extracted from 302 formalin-fixed paraffin-embedded tissues obtained from CRC patients. The primary endpoints were the concordance rate (CR) between the results from RASKET-B and the previously approved IVD kit (RASKET) for RAS mutations, and CR between the results from RASKET-B and direct sequencing (DS) for BRAF mutations. The secondary endpoints included the CR between RASKET-B and DS for RAS mutations and between RASKET-B and the pyrosequencing (PYRO) for the BRAF V600E mutation. Among the 302 samples, 142 RAS mutations (47%) and 18 BRAF V600E mutations (6.0%) were detected by RASKET-B. All mutations detected in the recruited patients were mutually exclusive. Both RAS and BRAF mutation rates were statistically higher in right-sided than left-sided CRC. The CR between RASKET-B and RASKET for RAS gene and RASKET-B and DS for BRAF V600E mutation was 100% for both (95% CI: 99%-100%). The results from RASKET-B were also highly concordant with DS for RAS (97.4%) and with PYRO for the BRAF (V600E) gene (99.7%). RASKET-B thus provides rapid, precise, and simultaneous detection of RAS and BRAF mutations in CRC.

[High-throughput screening method of KRAS mutations at codons 12 and 13 in formalin-fixed paraffin-embedded tissue specimens of metastatic colorectal cancer].

Clinical studies overseas using the therapeutic anti-EGFR monoclonal antibodies, cetuximab or panitumumab against metastatic colorectal cancer(mCRC), have revealed KRAS mutations as a negative predictive marker of response. Accordingly, the Ministry of Health, Labour and Welfare in Japan approved medical reimbursement of the KRAS mutation test in April 2010. Anti-EGFR monoclonal antibody therapies are now used as first-line treatment for patients with mCRC. To advance the simple high-throughput KRAS mutation test, we established a high-throughput screening system for detecting KRAS mutations utilizing Luminex(xMAP)technology(the fluorescent bead-based multiplex analyte profiling method), in combination with the polymerase chain reaction-reverse sequence-specific oligonucleotide method. Here we evaluated the basic performance of our system and confirmed its high specificity and reproducibility in detecting KRAS mutations at codons 12 and 13 in both plasmid DNAs carrying mutant KRAS genes and formalin-fixed paraffin-embedded tissues from mCRC patients. We demonstrated the KRAS mutation status in paraffin-embedded tissues of mCRC and confirmed that the results were comparable to those of the direct sequencing method. Our high-throughput method has an advantage in simultaneous analysis of multiple mutations in one well of 96-well PCR plates, and will advance the KRAS mutation test in clinical laboratories.

Cell cycle-dependent accumulation of histone H3.3 and euchromatic histone modifications in pericentromeric heterochromatin in response to a decrease in DNA methylation levels.

In mammals, DNA methylation is an important epigenetic mark that is associated with gene silencing, particularly in constitutive heterochromatin. However, the effect of DNA methylation on other epigenetic properties of chromatin is controversial. In this study, we show that inhibition of DNA methylation in mouse fibroblast cells affects histone modification and the subnuclear localization of histone H3.3 in a cell cycle-dependent manner. Using a DNA methyltransferase (Dnmt) inhibitor 5-aza-2'-deoxycytidine (5-aza-dC), we found that reduced levels of DNA methylation were associated with the activation of transcription from centromeric and pericentromeric satellite repeats. The de-repressed pericentromeric chromatin was enriched in euchromatic histone modifications such as acetylation of histone H4, and di- and tri-methylation of lysine 4 on histone H3. Spatio-temporal analysis showed that the accumulation of these euchromatic histone modifications occurred during the second S phase following 5-aza-dC treatment, corresponding precisely with a shift in replication timing of the pericentromeric satellite repeats from middle/late S phase to early S phase. Moreover, we found that histone H3.3 was deposited on the pericentromeric heterochromatin prior to the accumulation of the euchromatic histone modifications. These results suggest that DNA CpG methylation is essential for the proper organization of pericentromeric heterochromatin in differentiated mouse cells.

Regulation of replication at the R/G chromosomal band boundary and pericentromeric heterochromatin of mammalian cells.

Mammalian chromosomes consist of multiple replicons; however, in contrast to yeast, the details of this replication process (origin firing, fork progression and termination) relative to specific chromosomal domains remain unclear. Using direct visualization of DNA fibers, here we show that the rate of replication fork movement typically decreases in the early-mid S phase when the replication fork proceeds through the R/G chromosomal band boundary and pericentromeric heterochromatin. To support this, fluorescence in situ hybridization (FISH)-based replication profiles at the human 1q31.1 (R-band)-32.1 (G-band) regions revealed that replication timing switched around at the putative R/G chromosomal band boundary predicted by marked changes in GC content at the sequence level. Thus, the slowdown of replication fork movement is thought to be the general property of the band boundaries separating the functionally different chromosomal domains. By simultaneous visualization of replication fork movement and pericentromeric heterochromatin sequences on DNA fibers, we observed that this region is duplicated by many replication forks, some of which proceed unidirectionally, that originate from clustered replication origins. We showed that histone hyperacetylation is tightly associated with changes in the replication timing of pericentromeric heterochromatin induced by 5-aza-2'-deoxycytidine treatment. These results suggest that, similar to the yeast system, histone modification is involved in controlling the timing of origin firing in mammals.