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Recombinant adeno-associated virus (rAAV) has emerged as the vector of choice for in vivo gene delivery, with numerous clinical trials underway for the treatment of various human diseases. Utilizing rAAV in gene therapy requires a highly precise quantification method to determine the viral genome titer and further establish the optimal therapeutic dosage for a rAAV product. The conventional single-channel droplet digital PCR (1D ddPCR) method offers only partial information regarding the viral vector genome titer, lacking insights into its integrity. In our pursuit of further advancing rAAV analysis, we have developed a novel 3D ddPCR assay with advanced 3D linkage analysis. We have designed the three amplicon sites targeting both ends of the viral genome, as well as the center of key therapeutic gene of interest (GOI). This study aims to offer a more comprehensive and insightful assessment of rAAV products which includes not only quantity of viral genome titer but also the quality, distinguishing between partial ones and intact full-length viral genomes with the right GOI. Importantly, due to the random partitioning property of a digital PCR system, the 3D linkage analysis of rAAV viral genome requires a proper mathematical model to identify the true linked DNA molecules (full-length/intact DNA) from the population of false/unlinked DNA molecules (fragmented/partial DNA). We therefore have developed an AAV 3D linkage analysis workflow to characterize genomic integrity and intact titer for rAAV gene therapy products. In this study, we focus on evaluating our 3D linkage mathematical model by performing DNA mixing experiments and a case study using multiple rAAV samples. Particularly, we rigorously tested our algorithms by conducting experiments involving the mixing of seven DNA fragments to represent various AAV viral genome populations, including 3 single partials, 3 double partials, and 1 full-length genomes. Across all 37 tested scenarios, we validated the accuracy of our workflow’s output for the percentages of 3D linkage by comparing to the known percentages of input DNA. Consequently, our comprehensive AAV analytical package not only offers insights into viral genome titer but also provides valuable information on its integrity and identity. This cost-effective approach, akin to the setup of traditional 1D or 2D dPCR, holds the potential to advance the application of rAAV in cell and gene therapy for the treatment of human diseases.

Spinal Muscular Atrophy (SMA, MIM#253300) is an autosomal recessive neuromuscular disorder caused by defects in the Survival Motor Neuron ( SMN ) gene. The SMN1 gene, recognized as the primary pathogenic gene for SMA, exhibits a high degree of sequence homology with SMN2 gene. Individuals with the SMN1 2 + 0 genotype represent a unique type of SMA carrier, characterized by two SMN1 copies on one chromosome and zero copies on the other. Accurate identification of this type of carrier is crucial for genetic counseling in families. This study included 28 samples from five SMA families, each with an affected patient carrying a homozygous deletion of the SMN1 gene and a parent suspected to be a SMN1 2 + 0 carrier. Comprehensive Analysis of SMA (CASMA), based on third-generation sequencing technology, was used to detect the SMN1 and SMN2 copy numbers in the samples, and SMN1 2 + 0 carriers were identified through SMN1 haplotypes in parent-child trios (CASMA-trio). The results were compared with those obtained using Multiplex Ligation-dependent Probe Amplification (MLPA) combined with Short Tandem Repeat (STR) linkage analysis. The SMN1 and SMN2 copy numbers detected by MLPA and CASMA were concordant across 25 peripheral blood samples, whereas CASMA failed to accurately determine the copy numbers in the remaining 3 amniotic fluid samples. CASMA-trio identified 5 members from 4 families as SMN1 2 + 0 carriers, which were consistent with the results from STR linkage analysis. However, the two methods yielded inconsistent results for the proband’s father in one family. These findings suggest that CASMA has the potential to detect SMN1 and SMN2 copy numbers. Compared to STR linkage analysis, CASMA-trio only requires a parent-child trio to analyze SMN1 2 + 0 carriers, demonstrating a broader application prospect. Implementing CASMA-trio can facilitate comprehensive screening for SMA carriers.

Understanding knowledge transfer patterns is essential in providing valuable insights for shaping innovations and supporting economic growth. Our study identifies the main contributors and patterns of knowledge transfer within the pharmacology field from 2000 to 2019 by analyzing citation linkage and collaborative information between sector categories, affiliated institutions, and biomedical entities in articles from the Web of Science database. Our main contribution is mapping the knowledge transfer flow and identifying the main contributors to knowledge transfer within the pharmacology domain. We manually categorized affiliated institutions into four sector categories to observe knowledge transfer patterns. Subsequently, we performed a citation linkage analysis at three levels: sector categories, institution names, and biomedical entities. The results show that academic institutions are the most significant contributors to knowledge transfer in the pharmacology field, followed by commercial and government institutions. Although the majority of knowledge transfers originated from academic institutions, our study uncovered notable transfers from commercial to academic sectors and from government to academic sectors. Through named entity analysis on diseases, drugs, and genes, we found that research in the pharmacology field predominantly concentrates on subjects pertaining to cancers, chronic diseases, and neurodegenerative disorders.

Human Cytotoxic-T-lymphocyte-antigen-4 (CTLA-4) insufficiency caused by heterozygous germline mutations in is a complex immune dysregulation and immunodeficiency syndrome presenting with reduced penetrance and variable disease expressivity, suggesting the presence of disease modifiers that trigger the disease onset and severity. Various genetic and non-genetic potential triggers have been analyzed in CTLA-4 insufficiency cohorts, however, none of them have revealed a clear association to the disease. Multiple HLA haplotypes have been positively or negatively associated with various autoimmune diseases and inborn errors of immunity (IEI) due to the relevance of MHC in the strength of the T cell responses. In this exploratory study, we investigated the association of disease onset, severity and clinical manifestations of CTLA-4 insufficiency with specific HLA class I (A, B and C) and class II (DRB1 and DQB1) alleles in forty-three individuals harboring heterozygous mutations in . Twenty-six out of the 43 recruited individuals presented moderate or severe clinical symptoms whereas 17 were completely healthy. HLA frequency analysis, odds ratio analysis and genetic linkage analysis were used. The principal statistical analyses showed no positive association between the HLA genotypes analyzed with the disease onset or the disease severity. We found potential risk associations of and with respiratory tract involvement and with affection of the neurological system in the CTLA-4-insufficient patients. Additionally, we found a potential protective association of with gastrointestinal symptoms. Even though, our findings suggest that HLA-A, -B, -C, DRB1, and DQB1 do not contribute to the onset or severity of disease in CTLA-4 insufficiency, certain HLA-alleles may influence the manifestation of specific symptoms. We advocate for further investigation of specific class I and class II HLA alleles as potential disease modifiers in larger clinical cohorts of CTLA-4 insufficiency.

PurposeThis study aimed to evaluate the value of long-read sequencing for preimplantation haplotype linkage analysis.MethodsThe genetic material of the three β-thalassemia mutation carrier couples was sequenced using single-molecule real-time sequencing in the 7.7-kb region of the HBB gene and a 7.4-kb region that partially overlapped with it to detect the presence of 17 common HBB gene mutations in the Chinese population and the haplotypes formed by the continuous array of single-nucleotide polymorphisms linked to these mutations. By using the same method to analyze multiple displacement amplification products of embryos from three families and comparing the results with those of the parents, it could be revealed whether the embryos carry disease-causing mutations without the need for a proband.ResultsThe HBB gene mutations of the three couples were accurately detected, and the haplotype linked to the pathogenic site was successfully obtained without the need for a proband. A total of 68.75% (22/32) of embryos from the three families successfully underwent haplotype linkage analysis, and the results were consistent with the results of NGS-based mutation site detection.ConclusionThis study supports long-read sequencing as a potential tool for preimplantation haplotype linkage analysis.

Background Transition to flowering at the right time is critical for local adaptation and to maximize grain yield in crops. Canola is an important oilseed crop with extensive variation in flowering time among varieties. However, our understanding of underlying genes and their role in canola productivity is limited. Results We report our analyses of a diverse GWAS panel (300–368 accessions) of canola and identify SNPs that are significantly associated with variation in flowering time and response to photoperiod across multiple locations. We show that several of these associations map in the vicinity of FLOWERING LOCUS T ( FT ) paralogs and its known transcriptional regulators. Complementary QTL and eQTL mapping studies, conducted in an Australian doubled haploid population, also detected consistent genomic regions close to the FT paralogs associated with flowering time and yield-related traits. FT sequences vary between accessions. Expression levels of FT in plants grown in field (or under controlled environment cabinets) correlated with flowering time. We show that markers linked to the FT paralogs display association with variation in multiple traits including flowering time, plant emergence, shoot biomass and grain yield. Conclusions Our findings suggest that FT paralogs not only control flowering time but also modulate yield-related productivity traits in canola.

Background Preimplantation genetic testing (PGT) for monogenic disorders (PGT-M) for germline mosaicism was previously highly dependent on polymerase chain reaction (PCR)-based directed mutation detection combined with linkage analysis of short tandem repeats (STRs). However, the number of STRs is usually limited. In addition, designing suitable probes and optimizing the reaction conditions for multiplex PCR are time-consuming and laborious. Here, we evaluated the effectiveness of next generation sequencing (NGS)-based haplotype linkage analysis in PGT of germline mosaicism. Methods PGT-M with NGS-based haplotype linkage analysis was performed for two families with maternal germline mosaicism for an X-linked Duchenne muscular dystrophy (DMD) mutation (del exon 45–50) or an autosomal TSC1 mutation (c.2074C > T). Trophectoderm biopsy and multiple displacement amplification (MDA) were performed for a total of nine blastocysts. NGS and Sanger sequencing were performed in genomic DNA of family members and embryonic MDA products to detect DMD deletion and TSC1 mutation, respectively. Single nucleotide polymorphism (SNP) sites closely linked to pathogenic mutations were detected with NGS and served in haplotype linkage analysis. NGS-based aneuploidy screening was performed for all embryos to reduce the risk of pregnancy loss. Results All nine blastocytes showed conclusive PGT results. Each family underwent one or two frozen-thawed embryo transfer cycles to obtain a clinical pregnancy, and the prenatal diagnosis showed that the fetus was genotypically normal and euploid for both families. Conclusions NGS-SNP could effectively realize PGT for germline mosaicism. Compared with PCR-based methods, the NGS-SNP method with increased polymorphic informative markers can achieve a greater diagnostic accuracy. Further studies are warranted to verify the effectiveness of NGS-based PGT of germline mosaicism cases in the absence of surviving offsprings.

Polymer‐based dielectrics are extensively applied in various electrical and electronic devices such as capacitors, power transmission cables and microchips, in which a variety of distinct performances such as the dielectric and thermal properties are desired. To fulfil these properties, the emerging machine learning (ML) technique has been used to establish a surrogate model for the structure–property linkage analysis, which provides an effective tool for the rational design of the chemical and morphological structure of polymers/nanocomposites. In this article, the authors reviewed the recent progress in the ML algorithms and their applications in the rational design of polymer‐based dielectrics. The main routes for collecting training data including online libraries, experiments and high‐throughput computations are first summarized. The fingerprints charactering the microstructures of polymers/nanocomposites are presented, followed by the illustration of ML models to establish a mapping between the fingerprinted input and the target properties. Further, inverse design methods such as evolution searching strategies and generative models are described, which are exploited to accelerate the discovery of new polymer‐based dielectrics. Moreover, structure–property linkage analysis techniques such as Pearson correlation calculation, decision‐tree‐based methods and interpretable neural networks are summarized to identify the key features affecting the target properties. The future development prospects of the ML‐driven design method for polymer‐based dielectrics are also presented in this review.

Genetic diseases encompass a spectrum of disorders resulting from DNA variations. Preimplantation genetic testing (PGT) is a critical strategy for preventing recurrent miscarriage, fetal malformations, and the birth of children affected by chromosomal abnormalities and monogenic disorders. Traditional PGT techniques necessitate comprehensive pedigree genetic data for haplotype linkage analysis. In contrast, PGT employing third-generation sequencing (TGS) has distinct advantages, particularly in cases of incomplete pedigree information, de novo mutations, and complex pathogenic variants. Nevertheless, the widespread application of TGS-based PGT in clinical practice encounters hurdles owing to its high costs. Targeted sequencing technologies present a promising solution by selectively enriching regions of interest while disregarding non-targeted regio n s, offering a more cost-effective and flexible alternative. In this proof-of-principle study, we employed low-coverage short-read next-generation sequencing (NGS), microarray analysis and nanopore adaptive sampling to analyze samples from five couples who carried balanced translocations and HBB gene pathogenic mutations, as well as three additional couples with monogenic diseases caused by mutations in PKD1 , ASNS , or ALPL . Nanopore adaptive sampling successfully identified various mutations and facilitated haplotype linkage analysis, confirming its accuracy and reliability. Successful embryo transfer and subsequent prenatal diagnosis in certain families underscore the potential of nanopore adaptive sampling in assisted reproduction. Compared with traditional PGT techniques based on low-coverage short-read NGS combined with microarray analysis, our work highlights that nanopore adaptive sampling is a promising tool for PGT, offering cost-effective solutions, especially for incomplete pedigrees and de novo mutations, and provides preliminary proof-of-principle evidence for its broader clinical application. Graphical Abstract Workflow summarizing embryonic CNV detection, haplotype analysis, targeted variant profiling, and verification by Sanger sequencing, leading to the transfer of euploid embryos with normal haplotypes

PurposeProviding feasible preimplantation genetic testing strategies for monogenic disorders (PGT-M) for prevention and control of genetic cancers.MethodsInclusion of families with a specific pathogenic mutation or a clear family history of genetic cancers. Identification of the distribution of hereditary cancer-related mutations in families through genetic testing. After a series of assisted reproductive measures such as down-regulation, stimulation, egg retrieval, and in vitro fertilization, a biopsy of trophectoderm cells from a blastocyst was performed for single-cell level whole-genome amplification (WGA). Then, the detection of chromosomal aneuploidies was performed by karyomapping. Construction of a haplotype-based linkage analysis to determine whether the embryo carries the mutation. Meanwhile, we performed CNV testing. Finally, embryos can be selected for transfer, and the results will be verified in 18–22 weeks after pregnancy.ResultsSix couples with a total of 7 cycles were included in our study. Except for cycle 1 of case 5 which did not result in a transferable embryo, the remaining 6 cycles produced transferable embryos and had a successful pregnancy. Four couples have had amniotic fluid tests to confirm that the fetus does not carry the mutation, while 1 couple was not tested due to insufficient pregnancy weeks. And the remaining couples had to induce labor due to fetal megacystis during pregnancy.ConclusionOur strategy has been proven to be feasible. It can effectively prevent transmission of hereditary cancer-related mutations to offspring during the prenatal stage.