Comprehensive Genetic Analysis Research Articles

Similarities of P1-Like Phage Plasmids and Their Role in the Dissemination of blaCTX-M-55.

ABSTRACTThe P1-like phage plasmid (PP) has been widely used as a molecular biology tool, but its role as an active accessory cargo element is not fully understood. In this study, we provide insights into the structural features and gene content similarities of 77 P1-like PPs in the RefSeq database. We also describe a P1-like PP carrying a blaCTX-M-55 gene, JL22, which was isolated from a clinical strain of Escherichia coli from a duck farm. P1-like PPs were very similar and conserved based on gene content similarities, with only eight highly variable regions. Importantly, two kinds of replicon types, namely, IncY and p0111, were identified and can be used to specifically identify the P1-like phage. JL22 is similar to P1, acquiring an important foreign DNA fragment with two obvious features, namely, the plasmid replication gene repA′ (p0111) replacing the gene repA (IncY) and a 4,200-bp fragment mobilized by IS1380 and IS5 and containing a blaCTX-M-55 gene and a trpB gene encoding tryptophan synthase (indole salvaging). The JL22 phage could be induced but had no lytic capacities. However, a lysogenic recipient and intact structure of JL22 virions were observed, showing that the extended-spectrum β-lactamase blaCTX-M-55 gene was successfully transferred. Overall, conserved genes can be a good complement to improve the identification efficiency and accuracy in future screening for P1-like PPs. Moreover, the highly conserved structures may be important for their prevalence and dissemination.IMPORTANCE As a PP, P1 DNA exists as a low-copy-number plasmid and replicates autonomously with a lysogenization style. This unique mode of P1-like elements probably indicates a stable contribution to antibiotic resistance. After analyzing these elements, we show that P1-like PPs are very similar and conserved, with only eight highly variable regions. Moreover, we observed the occurrence of replicon IncY and p0111 only in the P1-like PP community, implying that these conserved regions, coupled with IncY and p0111, can be an important complement in future screening of P1-like PPs. Identification and characterization of JL22 confirmed our findings that major changes were located in variable regions, including the first detection of blaCTX-M-55 in such a mobile genetic element. This suggests that these variable regions may facilitate foreign DNA mobilization. This study features a comprehensive genetic analysis of P1-like PPs, providing new insights into the dissemination mechanisms of antibiotic resistance through P1 PPs.

Cryptic genetic divergence of the red dwarf rasbora, Microrasbora rubescens, in and around Inle Lake: implications for the origin of endemicity in the ancient lake in Myanmar.

Inle Lake, an ancient lake located in the Shan Plateau of Myanmar, is a biogeographically attractive region with high fish endemism. Some endemic species inhabit the lake as well as the surrounding areas. The genetic and ecological relationships between populations in the lake and surrounding areas provide important insights into the process underlying ichthyofaunal formation in Inle Lake. In this study, the authors focused on red dwarf rasbora Microrasbora rubescens, an endemic genus and species in this region, and estimated its population structure and evolutionary scenario based on genome-wide polymorphism, mtDNA and geometric morphometric analyses using samples from Inle Lake and three areas surrounding the lake. The results showed that M. rubescens comprises at least three genetically divergent lineages (Inle, Heho and Hopong) with distinct geographic structures consistent with nuclear and mtDNA data. In contrast, there was no clear regional differentiation in morphology. The divergence time estimation based on mtDNA suggests that the Hopong lineage diverged at 2.7 Ma and the Inle and Heho lineages diverged at 1.9Ma - consistent with the nuclear DNA results. The deep divergence observed in the endemic species supports the ancient history of ichthyofaunal development in this region. The distinct regional differentiation and morphological conservatism of this species might have been shaped by niche conservatism in stagnant water environments that limit dispersal and morphological diversification. Future comprehensive genetic and morphological analyses and comparisons for other native species should reveal the geographic and ecological processes that shaped the ichthyofauna in this region.

Targeted next-generation sequencing for comprehensive genetic analysis of external apical root resorption during orthodontic treatment with premolar extraction in the Korean population

External apical root resorption (EARR) is one of the most common unfavorable consequences of orthodontic treatment and causes loss of tooth structure. The present study aimed to investigate the genetics of EARR using next-generation sequencing comprehensively. Targeted next-generation sequencing was performed for comprehensive genetic analysis of 118 Korean orthodontic patients. The patients were divided into 2 groups on the basis of their EARR value. The association of clinical and genetic parameters with EARR was assessed using the χ2 test or t test for matched pairs, followed by Bonferroni correction and linear regression analysis. In addition, haplotype analysis and in silico prediction were conducted to evaluate functional effects. No statistically significant difference was observed between clinical and treatment-related parameters and EARR. The single nucleotide polymorphisms SPP1 rs9138 (P= 0.001) and SFRP2 rs3810765 (P= 0.04) showed only nominal significance between EARR groups. However, these 2 SNPs were not significant after Bonferroni correction for multiple testing (cutoff P= 0.05/142= 3.52 × 10-4). Variations in SPP1 rs9138 and SFRP2 rs3810765 may be related to EARR during orthodontic treatment. In summary, not only genes related to inflammatory reactions but also those related to Wnt signaling to affect the degree of EARR during orthodontic teeth movement.

Impact of comprehensive family history and genetic analysis in the multidisciplinary pancreatic tumor clinic setting.

Genetic testing is recommended for all pancreatic ductal adenocarcinoma (PDAC) patients. Prior research demonstrates that multidisciplinary pancreatic cancer clinics (MDPCs) improve treatment- and survival-related outcomes for PDAC patients. However, limited information exists regarding the utility of integrated genetics in the MDPC setting. We hypothesized that incorporating genetics in an MDPC serving both PDAC patients and high-risk individuals (HRI) could: (1) improve compliance with guideline-based genetic testing for PDAC patients, and (2) optimize HRI identification and PDAC surveillance participation to improve early detection and survival. Demographics, genetic testing results, and pedigrees were reviewed for PDAC patients and HRI at one institution over 45 months. Genetic testing analyzed 16 PDAC-associated genes at minimum. Overall, 969 MDPC subjects were evaluated during the study period; another 56 PDAC patients were seen outside the MDPC. Among 425 MDPC PDAC patients, 333 (78.4%) completed genetic testing; 29 (8.7%) carried a PDAC-related pathogenic germline variant (PGV). Additionally, 32 (9.6%) met familial pancreatic cancer (FPC) criteria. These PDAC patients had 191 relatives eligible for surveillance or genetic testing. Only 2/56 (3.6%) non-MDPC PDAC patients completed genetic testing (p < 0.01). Among 544 HRI, 253 (46.5%) had a known PGV or a designation of FPC, and were eligible for surveillance at baseline; of the remainder, 15/291 (5.2%) were eligible following genetic testing and PGV identification. Integrating genetics into the multidisciplinary setting significantly improved genetic testing compliance by reducing logistical barriers for PDAC patients, and clarified cancer risks for their relatives while conserving clinical resources. Overall, we identified 206 individuals newly eligible for surveillance or genetic testing (191 relatives of MDPC PDAC patients, and 15 HRI from this cohort), enabling continuity of care for PDAC patients and at-risk relatives in one clinic.

Two large novel alpha-globin gene cluster deletions causing alpha(0)-thalassemia in two Chinese families

IntroductionMonosomy of terminal 16p13.3 is a relatively common subtelomeric abnormality, most affected individuals presented α-thalassemia, some also have mental retardation, developmental abnormalities and/or speech delay and facial dysmorphism, which is termed ATR-16 syndrome. Here, we reported two novel 16p13.3 deletions involving the α-globin gene cluster and multispecies conserved sequences (MCSs), causing only a phenotype of α-thalassemia. MethodsSamples were collected from members of the two families and were subjected to haematological and comprehensive genetic analysis. ResultsThe novel 108 Kb deletion in family A extends from the non-protein coding RNA gene (WASIR2) to the NPRL3 gene, removing MCS-R1 to R3. This deletion should arise de novo because it wasn’t detected in both parents. The novel 336 Kb deletion in family B should extend from telomere to ∼ chr16:336000, removing the entire α-globin gene cluster. Carriers of these two deletions presented with microcytosis and hypochromic red cells, in accordance with a phenotype of α0-thalassemia carrier. ConclusionOur study increases the mutation spectrum of α-thalassemia. MCSs deletion should be considered in clinical practice of thalassemia screening and diagnosis.

Comprehensive genetic analysis of histological components of combined small cell carcinoma.

BackgroundCombined small‐cell lung cancer (cSCLC) is a rare type of small‐cell lung cancer (SCLC) that includes both SCLC and non‐small‐cell lung cancer (NSCLC). The molecular biological mechanisms underlying the heterogeneity of histological types in combined or metachronously transformed SCLC (mtSCLC) remain unclear. This study aimed to investigate the relationship between genetic alterations and each histological component heterogeneously detected in cSCLC and mtSCLC.MethodsThis study included four cSCLC cases and one mtSCLC case. Formalin‐fixed and paraffin‐embedded sections of each histological component of these tumors were subjected to next‐generation sequencing (NGS) and quantitative reverse transcription‐polymerase chain reaction to investigate the genetic mutations and expression levels of neuroendocrine cell‐specific transcription factors (achaete‐scute homolog‐1 [ASCL1], brain‐2 [BRN2] also known as POU domain class 3 transcription factor 2, nuclear factor 1 B [NF1B], insulinoma‐associated protein 1 [INSM1], and thyroid transcription factor‐1 [TTF‐1]).ResultsNGS analysis revealed that SCLC and NSCLC components share the same somatic mutations detected most frequently in TP53, and also in RB1 and EGFR. Gene expression analysis showed ASCL1 expression was significantly lower in the NSCLC component than in the SCLC component.ConclusionWe conclude that the morphological evolution of heterogeneous histological components in cSCLC may be associated with differences in ASCL1 expression levels, but not in acquired somatic gene mutations.

Intra-host genetic population diversity: Role in emergence and persistence of drug resistance among Mycobacterium tuberculosis complex minor variants.

Drug resistant tuberculosis (DR-TB) is an important public health issue in different parts of the world. Mycobacterium tuberculosis complex variants (MTBC vars) preferentially infect certain hosts, limiting their distribution to different ecosystems. However, MTBC vars can infect other hosts beyond their preferred target potentially contributing to persistence of drug resistance (DR) in other niches. Here, we performed a comprehensive intra-host genetic analysis for the identification of DR-related mutations among all MTBC minor vars whole genome sequences (8,095 strains) publicly available worldwide. High confidence drug-resistance mutations in katG (isoniazid), rpsL (streptomycin), pncA (pyrazinamide), rpoB (rifampicin) and gyrA (fluoroquinolones) genes were identified among intrahost minor sub-populations in 197 different strains (2.43%) belonging to vars africanum, bovis, caprae, microti, orygis and pinnipedii. In addition, a three-dimensional structure modeling analysis to assess the role of novel mutations was also performed. Our findings highlight the importance of detecting discrete intra-host populations carrying DR mutations.

Comprehensive Genetic Analyses of Inherited Peripheral Neuropathies in Japan: Making Early Diagnosis Possible.

Various genomic variants were linked to inherited peripheral neuropathies (IPNs), including large duplication/deletion and repeat expansion, making genetic diagnosis challenging. This large case series aimed to identify the genetic characteristics of Japanese patients with IPNs. We collected data on 2695 IPN cases throughout Japan, in which PMP22 copy number variation (CNV) was pre-excluded. Genetic analyses were performed using DNA microarrays, next-generation sequencing-based gene panel sequencing, whole-exome sequencing, CNV analysis, and RFC1 repeat expansion analysis. The overall diagnostic rate and the genetic spectrum of patients were summarized. We identified 909 cases with suspected IPNs, pathogenic or likely pathogenic variants. The most common causative genes were MFN2, GJB1, MPZ, and MME. MFN2 was the most common cause for early-onset patients, whereas GJB1 and MPZ were the leading causes of middle-onset and late-onset patients, respectively. Meanwhile, GJB1 and MFN2 were leading causes for demyelinating and axonal subtypes, respectively. Additionally, we identified CNVs in MPZ and GJB1 genes and RFC1 repeat expansions. Comprehensive genetic analyses explicitly demonstrated the genetic basis of our IPN case series. A further understanding of the clinical characteristics of IPN and genetic spectrum would assist in developing efficient genetic testing strategies and facilitate early diagnosis.

O-210 Cluster analysis reveals two hidden subgroups among patients with cryptozoospermia

Abstract Study question Can cryptozoospermic patients be sub classified based on clinical parameters? Summary answer Cryptozoospermic patients can be subdivided in two subgroups based on the histological phenotype of their testicular tissues, testicular volume and FSH levels. What is known already Cryptozoospermia is a severe form of oligozoospermia in which patients present with a sperm concentration of ≤ 0.1 million per milliliter. Due to such low sperm concentration, cryptozoospermic patients depend on the surgical procedure of testicular sperm extraction (TESE) to retrieve sperm for intracytoplasmic sperm injection (ICSI). The general etiology underlying cryptozoospermia remains hitherto unknown, likely due to the high heterogeneity within this patient cohort regarding histological and endocrine parameters. Study design, size, duration We retrospectively selected 132 cryptozoospermic patients (Crypto) who underwent TESE during their fertility treatment. Exclusion parameters were genetic diseases, a history of cryptorchidism or tumors and hormonal treatment. As controls, we selected 160 patients with obstructive azoospermia and normal spermatogenesis. All 292 patients were used for principal component analysis (PCA) followed by hierarchical clustering on principal components (HCPC). Participants/materials, setting, methods PCA and hierarchical clustering were performed considering age, testicular volume, hormonal values (FSH, LH, testosterone and free testosterone), ejaculate parameters (Ejaculate pH and volume, sperm concentration and total sperm count) and histological information. Histological analyses were performed on two PAS-stained sections from two independent biopsies per testis and patient. The percentage of seminiferous tubules containing elongated spermatids, round spermatids, spermatocytes or spermatogonia, as well as Sertoli cell only and hyalinized tubules was assessed. Main results and the role of chance The PCA and hierarchical clustering subdivided the patient cohort into 3 different clusters. Cluster 1 included all the controls (n = 160) and 2 Crypto patients. Remaining Crypto patients were equally subdivided between cluster 2 (n = 65) and cluster 3 (n = 65). The control patients in cluster 1 were characterized by the highest percentages of tubules with elongated spermatids (&amp;gt;80%), normal testicular volume and hormonal values. Characteristic of Crypto patients in cluster 2 was the arrest of germ cell differentiation at the level of spermatocytes in the majority of seminiferous tubules. In contrast, the majority of seminiferous tubules in Crypto patients in cluster 3 showed a Sertoli cell only phenotype or even tubular shadows. Interestingly, the more severe histological phenotype of the cluster 3 patients was accompanied by higher FSH and LH levels as well as lower testicular volume compared to cluster 2 patients. Despite this, the percentage of tubules with full spermatogenesis was similar in the two Crypto groups and no difference was found in the sperm retrieval rate after TESE in the two patient groups. Limitations, reasons for caution Uncovering the existence of subgroups within the cohort of Crypto-patients is the prerequisite for unveiling potentially distinct etiologies. Comprehensive genetic and scRNA-seq analyses of these Crypto-subgroups remain to be performed to validate the findings. Wider implications of the findings Overall, this study offers novel approaches towards the understanding of the etiology underlying the reduced sperm formation in Crypto patients and the division into two subgroups will facilitate the strategic search for underlying causes. Moreover, this approach could be applied to any infertility type in order to identify hidden subgroups. Trial registration number not applicable

Abstract 2945: Comprehensive analysis of tissue and plasma-related genetic alterations in Hungarian colorectal cancer patients

Abstract Background: Analysis of circulating cell-free DNA (cfDNA) of colorectal cancer (CRC) patients provides an ideal approach to explore genetic alterations in a minimally invasive way. Aims: We aimed to perform a comprehensive analysis of tissue-originated genomic DNA and plasma-derived cfDNA from CRC patients by whole-exome and targeted panel sequencing. Materials &amp; methods: DNA was isolated from tissue and plasma samples of 55 [7 healthy (N), 16 adenomas (AD), and 32 CRC] patients using the High Pure PCR Template Kit (Roche) and the Quick cfDNA Serum &amp; Plasma Kit (Zymo). cfDNA quality was assessed with the High Sensitivity DNA kit on BioAnalyzer 2100 and quantified by the Qubit dsDNA HS assay. Exome libraries from tissue samples were constructed by the Nextera DNA Exome Kit (Illumina). For the cfDNA samples, we used the QIAseq cfDNA All-in-One kit combined with the QIASeq Human Exome Kit, and in the case of 12 patients, CRC-specific oncogenes were further analyzed with a solution developed by QIAGEN for reliable calling of low frequency variants in cfDNA samples. Exome libraries were quantified with the KAPA Library Quantification Kit and the QIAseq™ Library Quant Assay Kit (Qiagen) and were sequenced using the NextSeq 500/550 High Output v2 kit on a NextSeq 500 Instrument (Illumina). Raw data analysis and demultiplexing were completed on the BaseSpace Sequence Hub. Variants of the tissue and cfDNA samples were determined by the Mutect2 and Haplotype caller algorithms of GATK 4.1.4.1, respectively. Clinical significance was evaluated according to the OncoKB database. Results: The most frequently mutated genes were APC, KRAS, DYNC1H1, KCNO5, and MARCH6 in the colorectal adenoma tissue samples, while those in CRC samples were APC, TP53, TTN, KRAS, and DYNCC2H1. CfDNA quantity was significantly higher in the CRC group compared to the AD (p&amp;lt;0.02) and N (p&amp;lt;0.005) patients. Based on the plasma exome results, 4.4-59.4% of the tumor somatic variants could be found in 12.5% of the patients. These patients were above 60 years of age and had Dukes D stage CRC. Panel sequencing of the circulating DNA samples detected tumor somatic variants in 8 out of the 12 enrolled patients. This method could identify 60% of all tumor somatic variants falling on its targeted regions, while whole-exome sequencing recovered only 20% of tumor somatic variants in the respective regions in cfDNA of the same patients. Conclusion: We have performed a comprehensive genetic analysis on CRC tissue and cfDNA samples in the Hungarian population. Exome sequencing offers a broad overview of the coding regions, however, targeted panel sequencing with a higher coverage depth can detect tumor somatic variants more reliably in cfDNA, therefore, it can hold a relevant clinical potential. Citation Format: Alexandra Kalmar, Gitta Szabo, Orsolya Galamb, Barbara Kinga Bartak, Zsofia Brigitta Nagy, Sara Zsigrai, Krisztina Andrea Szigeti, William Kothalawala, Peter Igaz, Istvan Takacs, Bela Molnar. Comprehensive analysis of tissue and plasma-related genetic alterations in Hungarian colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2945.

Establishment and characterization of a novel human induced pluripotent stem cell line stably expressing the iRFP720 reporter

Stem cell therapy has great potential for replacing beta-cell loss in diabetic patients. However, a key obstacle to cell therapy’s success is to preserve viability and function of the engrafted cells. While several strategies have been developed to improve engrafted beta-cell survival, tools to evaluate the efficacy within the body by imaging are limited. Traditional labeling tools, such as GFP-like fluorescent proteins, have limited penetration depths in vivo due to tissue scattering and absorption. To circumvent this limitation, a near-infrared fluorescent mutant version of the DrBphP bacteriophytochrome, iRFP720, has been developed for in vivo imaging and stem/progenitor cell tracking. Here, we present the generation and characterization of an iRFP720 expressing human induced pluripotent stem cell (iPSC) line, which can be used for real-time imaging in various biological applications. To generate the transgenic cells, the CRISPR/Cas9 technology was applied. A puromycin resistance gene was inserted into the AAVS1 locus, driven by the endogenous PPP1R12C promoter, along with the CAG-iRFP720 reporter cassette, which was flanked by insulator elements. Proper integration of the transgene into the targeted genomic region was assessed by comprehensive genetic analysis, verifying precise genome editing. Stable expression of iRFP720 in the cells was confirmed and imaged by their near-infrared fluorescence. We demonstrated that the reporter iPSCs exhibit normal stem cell characteristics and can be efficiently differentiated towards the pancreatic lineage. As the genetically modified reporter cells show retained pluripotency and multilineage differentiation potential, they hold great potential as a cellular model in a variety of biological and pharmacological applications.

Comprehensive genetic analysis in first or second trimester pregnancy loss using chromosomal microarray with single nucleotide polymorphism probes

To explore possible genetic causes associated with early pregnancy loss using chromosomal microarray analysis (CMA) with single nucleotide polymorphism (SNP) probes. A retrospective review was performed by the CMA of samples from 961 patients who spontaneously aborted in our hospital before the 20th week of pregnancy. (1) The total chromosome abnormality rate in miscarriage samples was 54.44% (515/946), including single chromosome abnormality (39.53%), two chromosome abnormality (2.22%), multi-chromosome abnormality (0.42%), triploidy or hypertriploidy (4.86%), copy number variants (CNVs) in 41 cases (4.33%), regions of homozygosity (ROH, 0.74%), mosaic (2.22%) and chimera (0.11%). (2) CNV analysis of 41 cases showed that 85.36% were pathogenic and likely pathogenic, 12.20% were classified as clinical significance unknown and 2.44% were interpreted as likely benign; (3) Among the cases of ROH, 2 cases shown whole-genome homozygosity and 1 case had completely homozygous at chromosome 21. The homozygous regions in 2 cases were located at the end of the short arm of chromosome 16, suggesting the mechanism of ROH in such cases could be the result of isodisomy. Chromosome abnormality is an important genetic factor causing pregnancy loss. The application of CMA with SNP probes can indeed improve the detection rate of chromosome abnormalities and evaluate the risk of reproductive fertility in patients with pregnancy loss.

A genome-wide atlas of antibiotic susceptibility targets and pathways to tolerance

Detailed knowledge on how bacteria evade antibiotics and eventually develop resistance could open avenues for novel therapeutics and diagnostics. It is thereby key to develop a comprehensive genome-wide understanding of how bacteria process antibiotic stress, and how modulation of the involved processes affects their ability to overcome said stress. Here we undertake a comprehensive genetic analysis of how the human pathogen Streptococcus pneumoniae responds to 20 antibiotics. We build a genome-wide atlas of drug susceptibility determinants and generated a genetic interaction network that connects cellular processes and genes of unknown function, which we show can be used as therapeutic targets. Pathway analysis reveals a genome-wide atlas of cellular processes that can make a bacterium less susceptible, and often tolerant, in an antibiotic specific manner. Importantly, modulation of these processes confers fitness benefits during active infections under antibiotic selection. Moreover, screening of sequenced clinical isolates demonstrates that mutations in genes that decrease antibiotic sensitivity and increase tolerance readily evolve and are frequently associated with resistant strains, indicating such mutations could be harbingers for the emergence of antibiotic resistance.

Comprehensive genetic analysis of the human lipidome identifies loci associated with lipid homeostasis with links to coronary artery disease

We integrated lipidomics and genomics to unravel the genetic architecture of lipid metabolism and identify genetic variants associated with lipid species putatively in the mechanistic pathway for coronary artery disease (CAD). We quantified 596 lipid species in serum from 4,492 individuals from the Busselton Health Study. The discovery GWAS identified 3,361 independent lipid-loci associations, involving 667 genomic regions (479 previously unreported), with validation in two independent cohorts. A meta-analysis revealed an additional 70 independent genomic regions associated with lipid species. We identified 134 lipid endophenotypes for CAD associated with 186 genomic loci. Associations between independent lipid-loci with coronary atherosclerosis were assessed in ∼456,000 individuals from the UK Biobank. Of the 53 lipid-loci that showed evidence of association (P < 1 × 10−3), 43 loci were associated with at least one lipid endophenotype. These findings illustrate the value of integrative biology to investigate the aetiology of atherosclerosis and CAD, with implications for other complex diseases.

Generation of hypoimmunogenic induced pluripotent stem cells by CRISPR-Cas9 system and detailed evaluation for clinical application

In order to expand the promise of regenerative medicine using allogeneic induced pluripotent stem cells (iPSCs), precise and efficient genome editing of human leukocyte antigen (HLA) genes would be advantageous to minimize the immune rejection caused by mismatches of HLA type. However, clinical-grade genome editing of multiple HLA genes in human iPSC lines remains unexplored. Here, we optimized the protocol for good manufacturing practice (GMP)-compatible CRISPR-Cas9 genome editing to deplete the three gene locus (HLA-A, HLA-B, and CIITA genes) simultaneously in HLA homozygous iPSCs. The use of HLA homozygous iPSCs has one main advantage over heterozygous iPSCs for inducing biallelic knockout by a single gRNA. RNA-seq and flow cytometry analyses confirmed the successful depletion of HLAs, and lineage-specific differentiation into cardiomyocytes was verified. We also confirmed that the pluripotency of genome-edited iPSCs was successfully maintained by the three germ layers of differentiation. Moreover, whole-genome sequencing, karyotyping, and optical genome mapping analyses revealed no evident genomic abnormalities detected in some clones, whereas unexpected copy number losses, chromosomal translocations, and complex genomic rearrangements were observed in other clones. Our results indicate the importance of multidimensional analyses to ensure the safety and quality of the genome-edited cells. The manufacturing and assessment pipelines presented here will be the basis for clinical-grade genome editing of iPSCs.

Recent advances in hematopoietic cell transplantation for inherited bone marrow failure syndromes.

Inherited bone marrow failure syndromes (IBMFSs) are a group of rare genetic disorders characterized by bone marrow failure with unique phenotypes and predisposition to cancer. Classical IBMFSs primarily include Fanconi anemia with impaired DNA damage repair, dyskeratosis congenita with telomere maintenance dysfunction, and Diamond-Blackfan anemia with aberrant ribosomal protein biosynthesis. Recently, comprehensive genetic analyses have been implemented for the definitive diagnosis of classic IBMFSs, and advances in molecular genetics have led to the identification of novel disorders such as AMeD and MIRAGE syndromes. Allogeneic hematopoietic cell transplantation (HCT), a promising option to overcome impaired hematopoiesis in patients with IBMFSs, does not correct nonhematological defects and may enhance the risk of secondary malignancies. Disease-specific management is necessary because IBMFSs differ in underlying defects and are associated with varying degrees of risk for clonal evolution and early or late complications after HCT. In addition, long-term follow-up is essential to detect complications related to the IBMFS or HCT. This review provides a summary of current clinical practices along with the latest data on HCT in IBMFSs.

Forensic Efficiency Estimation of a Homemade Six-Color Fluorescence Multiplex Panel and In-Depth Anatomy of the Population Genetic Architecture in Two Tibetan Groups.

The genetic information of the Chinese Tibetan group has been a long-standing research hotspot among population geneticists and archaeologists. Herein, 309 unrelated individuals from two Tibetan groups living in Qinghai Province, China (CTQ), and Tibet Autonomous Region, China (CTT), were successfully genotyped using a new homemade six-color fluorescence multiplex panel, which contained 59 autosomal deletion/insertion polymorphisms (au-DIPs), two mini short tandem repeats (miniSTRs), two Y-chromosomal DIPs, and one Amelogenin. The cumulative probability of matching and combined power of exclusion values for this new panel in CTQ and CTT groups were 1.9253E-27 and 0.99999729, as well as 1.5061E-26 and 0.99999895, respectively. Subsequently, comprehensive population genetic analyses of Tibetan groups and reference populations were carried out based on the 59 au-DIPs. The multitudinous statistical analysis results supported that Tibetan groups have close genetic affinities with East Asian populations. These findings showed that this homemade system would be a powerful tool for forensic individual identification and paternity testing in Chinese Tibetan groups and give us an important insight for further perfecting the genetic landscape of Tibetan groups.

Transcription factor Acj6 controls dendrite targeting via a combinatorial cell-surface code

Transcription factors specify the fate and connectivity of developing neurons. We investigate how a lineage-specific transcription factor, Acj6, controls the precise dendrite targeting of Drosophila olfactory projection neurons (PNs) by regulating the expression of cell-surface proteins. Quantitative cell-surface proteomic profiling of wild-type and acj6 mutant PNs in intact developing brains, and a proteome-informed genetic screen identified PN surface proteins that execute Acj6-regulated wiring decisions. These include canonical cell adhesion molecules and proteins previously not associated with wiring, such as Piezo, whose mechanosensitive ion channel activity is dispensable for its function in PN dendrite targeting. Comprehensive genetic analyses revealed that Acj6 employs unique sets of cell-surface proteins in different PN types for dendrite targeting. Combined expression of Acj6 wiring executors rescued acj6 mutant phenotypes with higher efficacy and breadth than expression of individual executors. Thus, Acj6 controls wiring specificity of different neuron types by specifying distinct combinatorial expression of cell-surface executors.

A molecular framework of ethylene-mediated fruit growth and ripening processes in tomato.

Although the role of ethylene in tomato (Solanum lycopersicum) fruit ripening has been intensively studied, its role in tomato fruit growth remains poorly understood. In addition, the relationship between ethylene and the developmental factors NON-RIPENING (NOR) and RIPENING INHIBITOR (RIN) during ripening is under debate. Here, we carried out comprehensive genetic analyses of genome-edited mutants of tomato ETHYLENE INSENSITIVE 2 (SlEIN2), four EIN3-like genes (SlEIL1-4), and three EIN3 BINDING F-box protein genes (SlEBF1-3). Both slein2-1 and the high-order sleil mutant (sleil1 sleil2 sleil3/SlEIL3 sleil4) showed reduced fruit size, mainly due to decreased auxin biosynthesis. During fruit maturation, slein2 mutants displayed the complete cessation of ripening, which was partially rescued by slebf1 but not slebf2 or slebf3. We also discovered that ethylene directly activates the expression of the developmental genes NOR, RIN, and FRUITFULL1 (FUL1) via SlEIL proteins. Indeed, overexpressing these genes partially rescued the ripening defects of slein2-1. Finally, the signal intensity of the ethylene burst during fruit maturation was intimately connected with the progression of full ripeness. Collectively, our work uncovers a critical role of ethylene in fruit growth and supports a molecular framework of ripening control in which the developmental factors NOR, RIN, and FUL1 act downstream of ethylene signaling.

Analysis of complex chromosomal rearrangements using a combination of current molecular cytogenetic techniques

BackgroundUsing combined fluorescence in situ hybridization (FISH) and high-throughput whole-genome sequencing (WGS) molecular cytogenetic technology, we aim to analyze the junction breakpoints of complex chromosome rearrangements (CCR) that were difficult to identify by conventional karyotyping analysis and further characterize the genetic causes of recurrent spontaneous abortion.ResultsBy leveraging a combination of current molecular techniques, including chromosome karyotype analysis, FISH, and WGS, we comprehensively characterized the extremely complex chromosomal abnormalities in this patient with recurrent spontaneous abortions. Here, we demonstrated that combining these current established molecular techniques is an effective and efficient workflow to identify the structural abnormalities of complex chromosomes and locate the rearrangement of DNA fragments.ConclusionsIn conclusion, leveraging results from multiple molecular and cytogenetic techniques can provide the most comprehensive genetic analysis for genetic etiology research, diagnosis, and genetic counseling for patients with recurrent spontaneous abortion and embryonic abortion.