Mutations In Structural Genes Research Articles

Genetic characteristics associated with the virulence of porcine epidemic diarrhea virus (PEDV) with a naturally occurring truncated ORF3 gene

Porcine epidemic diarrhea virus (PEDV) has emerged in American countries, and it has reemerged in Asia and Europe, causing significant economic losses to the pig industry worldwide. In the present study, the 17GXCZ-1ORF3d strain, which has a naturally large deletion at the 172–554 bp position of the ORF3 gene, together with the 17GXCZ-1ORF3c strain, was serially propagated in Vero cells for up to 120 passages. The adaptability of the two strains gradually increased through serial passages in vitro. Genetic variation analysis of the variants of the two strains from different generations revealed that the naturally truncated ORF3 gene in the 17GXCZ-1ORF3d variants was stably inherited. Furthermore, the survival, viral shedding and histopathological lesions following inoculation of piglets demonstrated that the virulence of 17GXCZ-1ORF3d-P120 was significantly attenuated. These results indicate that the naturally truncated ORF3 gene may accelerate the attenuation of virulence and is involved in PEDV virulence together with mutations in other structural genes. Importantly, immunization of sows with G2b 17GXCZ-1ORF3d-P120 increased PEDV-specific IgG and IgA antibody levels in piglets and conferred partial passive protection against heterologous G2a PEDV strains. Our findings suggest that an attenuated strain with a truncated ORF3 gene may be a promising candidate for protection against PEDV.

Analysis of the role of Na<sub>v</sub>1.5 slow inactivation in the development of inherited cardiac pathology

Voltage-gated cardiac sodium channels Nav1.5 are responsible for the initiation and propagation of action potentials in cardiomyocytes. Dysfunction of Nav1.5 can be caused both by pathogenic variants in the SCN5A gene itself, which encodes Nav1.5, and by genetic variants in the genes of other proteins, regulating channel activity and trafficking. The change of different phases of the action potential is determined by the strict temporal organization of activation and inactivation of various ion channels. Transitions between channel functional states (for example, to slow inactivated state) can be influenced by various factors and proteins interacting with the channel. Despite the fact that the process of slow inactivation of the channel has been known for several decades, its role in the mechanism of development of hereditary heart pathology remains unclear. In this work, using the patch clamp method in whole-cell leads, we studied changes in the process of slow Nav1.5 inactivation under the influence of various mutations in structural genes (DSP-H1684R, LMNA-R249Q, FLNC-R1267Q, FLNC-V2264M) associated with a genetically determined myocardial pathology leading to dysfunction of cardiomyocytes. The study used a model of cardiomyocytes differentiated from induced pluripotent stem cells (СM-iPSCs). We have demonstrated an increase in slow inactivation in the model of CM-iPSCs obtained from patients with a phenotype of cardiomyopathy combined with ventricular arrhythmias. Thus, this work contributes to understanding the role of the slow inactivation process in the mechanism of the development of heart pathology.

Mutations in Structural Genes of the Mitochondrial Complex IV May Influence Breast Cancer.

Although it has gained more attention in recent years, the relationship between breast cancer (BC) and mitochondrial oxidative phosphorylation (OXPHOS) is still not well understood. Importantly, Complex IV or Cytochrome C Oxidase (COX) of OXPHOS is one of the key players in mitochondrial balance. An in silico investigation of mutations in structural genes of Complex IV was conducted in BC, comprising 2107 samples. Our findings show four variants (rs267606614, rs753969142, rs199476128 and rs267606884) with significant pathogenic potential. Moreover, we highlight nine genes (MT-CO1, MT-CO2, MT-CO3, CO4I2, COX5A, COX5B, COX6A2, COX6C and COX7B2) with a potential impact on BC.

ВОЗМОЖНОСТИ МУЛЬТИЛОКУСНОГО АНАЛИЗА В ОЦЕНКЕ ГЕНЕТИЧЕСКОГО ПОТЕНЦИАЛА ПРОДУКТИВНЫХ КАЧЕСТВ КУР ГЕНОФОНДНЫХ ПОРОД

The article discusses the use of genome-wide analysis of associations of single nucleotide polymorphisms (SNPs) in DNA with growth rate in chickens and other productive traits. The chip technology is considered, which makes it possible to detect SNPs localized in multiple places in the genome. Some examples of the use of mutations in structural genes that determine traits and various aspects of the application of genomic selection are also given.

Comprehensive Analysis Reveals USP45 as a Novel Putative Oncogene in Pan-Cancer

Background: Deubiquitinating enzymes specifically removes ubiquitin molecules from ubiquitin-tagged target proteins, thereby inhibiting the degradation of target proteins and playing an important role in tumor. However, the mechanism of deubiquitinating enzyme USP45 in tumors remains unclear. Methods: Based on the RNA-seq data of tissues and cell lines in The Cancer Genome Atlas (TCGA) database, GTEx and CCLE database, the pan-cancer analysis of USP45 expression and survival outcome were performed using R software and Kaplan-Meier Plotter. The structural variants, gene mutations and gene copy number alteration of USP45 were analyzed using the TCGA Pan-Cancer Atlas Studies dataset in the cBioPortal database. The relationships between USP45 and mRNA methylation, tumor heterogeneity, tumor stemness, and tumor immunity were performed by Sangerbox platform and TIMER2.0 using Pearson correlation analysis. Through the ENCORI database and string database, we constructed the ceRNA regulatory mechanism and protein-protein interaction network for USP45. Based on the RNA-seq data in TCGA and GTEx databases, we also constructed the downstream regulatory network for USP45 using the Limma and ClusterProfiler packages of R software. At last, the protein expression levels of USP45 were detected by immunohistochemistry in tumor tissue microarrays. Results: USP45 is upregulated in most types of tumors and negatively correlated with the overall survival and recurrence-free survival of patient. Furthermore, the structural variation, gene mutations and gene copy number variation of USP45 were identified in different types of tumors. The pan-cancer analysis showed that USP45 was closely related to mRNA methylation, tumor heterogeneity and tumor stemness. In most types of tumors, the expression of USP45 was positively correlated with many immune checkpoint molecules and immune regulators such as PD-L1, while negatively correlated with the infiltration levels of NK cells, Th1 cells, macrophages, and dendritic cells in the tumor microenvironment. Finally, we constructed the ceRNA regulatory network, protein-protein interaction network and downstream regulatory network for USP45 in different types of tumors. Conclusion: Our study firstly explored the putative oncogenic role of USP45 in pan-cancer, and provided insights for further investigation of USP45.

Mutations in PmUFGT3 contribute to color variation of fruit skin in Japanese apricot (Prunus mume Sieb. et Zucc.)

BackgroundJapanese apricot (Prunus mume Sieb. et Zucc.) is popular for both ornamental and processing value, fruit color affects the processing quality, and red pigmentation is the most obvious phenotype associated with fruit color variation in Japanese apricot, mutations in structural genes in the anthocyanin pathway can disrupt the red pigmentation, while the formation mechanism of the red color trait in Japanese apricot is still unclear. ResultsOne SNP marker (PmuSNP_27) located within PmUFGT3 gene coding region was found highly polymorphic among 44 different fruit skin color cultivars and relative to anthocyanin biosynthesis in Japanese apricot. Meantime, critical mutations were identified in two alleles of PmUFGT3 in the green-skinned type is inactivated by seven nonsense mutations in the coding region, which leads to seven amino acid substitution, resulting in an inactive UFGT enzyme. Overexpression of the PmUFGT3 allele from red-skinned Japanese apricot in green-skinned fruit lines resulted in greater anthocyanin accumulation in fruit skin. Expression of same allele in an Arabidopsis T-DNA mutant deficient in anthocyanidin activity the accumulation of anthocyanins. In addition, using site-directed mutagenesis, we created a single-base substitution mutation (G to T) of PmUFGT3 isolated from green-skinned cultivar, which caused an E to D amino acid substitution and restored the function of the inactive allele of PmUFGT3 from a green-skinned individual.ConclusionThis study confirms the function of PmUFGT3, and provides insight into the mechanism underlying fruit color determination in Japanese apricot, and possible approaches towards genetic engineering of fruit color.

Abstract 6171: A single-cell solution for solid tumors to detect mutations and quantify copy number variations

Abstract Cancer is a highly heterogeneous disease characterized by the presence of chromosomal structural copy number variation (CNV) and driver gene mutation events, which appear somatically at the early stages of oncogenesis and can drive tumor progression. Previously, we have developed a high throughput single cell DNA analysis platform that leverages droplet microfluidics and a multiplex-PCR based targeted DNA sequencing approach. It demonstrates high sensitivity detection of SNVs and indels in the same cells and generation of high resolution maps of clonal architecture based on mutational profiling. Here we expand upon the same approach and create an algorithm to accurately quantify CNVs along with genetic mutations from the same single cells. To estimate the CNV the amplicon read counts were corrected for artefacts related to cell to cell variability due sequencing efficiencies along with amplicon to amplicon variability that occur due to amplification efficiency differences. We used a spiked in diploid cell line as a reference to normalize all the cells. The spiked cell line was identified using its mutational profile. We validated the system using cell lines with known CNVs and found that we could at least accurately estimate copy numbers ranging from 0 through 5. Some of the regions were also validated orthogonally using droplet digital PCR. Another set of validation was performed using well characterized cell lines from NIST. We enable multiple visualizations of the copy number estimates in karyotype plots and line plots projected on SNV clones. With improved biochemistry, panel design and novel data analysis algorithms we develop a complete solution to detect amplification or loss of function in oncogenes and tumor suppressors reliably. Integration of CNVs and SNVs facilitates more accurate reconstruction of tumor evolution to better understand cancer progression mechanisms as well for quality control of gene edited cells, to further advance cancer research and therapy. Citation Format: Saurabh Parikh, Khushali Patel, Alex Li, Anup Parikh. A single-cell solution for solid tumors to detect mutations and quantify copy number variations [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 6171.

Abstract 13546: Putative Titin Mutations Associated With Both Long QT Syndrome and Left Ventricular Noncompaction

Introduction: Inherited long QT Syndrome (LQTS) is characterized by prolonged QTc intervals on electrocardiogram (EKG), ventricular arrhythmias and sudden death (SCD), and is caused by mutations in ion channel or ion-channel related genes. Inherited forms of dilated cardiomyopathy (DCM) and left ventricular noncompaction (LVNC), on the other hand, are caused by mutations in sarcomeric and structural genes including titin. Methods: Two unrelated families with histories of SCD presented to us for clinical evaluation of LQTS. Clinical genetic testing covering arrhythmia and cardiomyopathy genes was performed on each proband, with carrier testing on other family members. Results (Figure): The proband of family 1 had a strong family history of SCD, carried the clinical diagnosis of LQTS treated with propranolol, and had normal cardiac function by echocardiography. Following chemotherapy with adriamycin for breast cancer, evaluation showed a QTc of 514 ms on EKG, non-sustained VT on Holter, and LVNC with an LVEF=43% on cardiac MRI (cMRI). Genetic testing identified a likely pathogenic nonsense TTN variant (p.W18202*) along with a benign TTN polymorphism (p.E689K). Her clinically normal daughter did not carry the p.W18202* variant. The proband of family 2 had a strong family history of LQTS and SCD and was diagnosed with LQTS in her 20s after her mother suffered a cardiac arrest. Evaluation showed a QTc of 500 ms on EKG and biventricular LVNC with an LVEF=48% on cMRI. Genetic testing identified a likely pathogenic frameshift TTN variant (p.E18038Rfs*47). Three of her children carried the variant and had borderline or prolonged QTc intervals; one had hypertrabeculation on cMRI. Conclusion: We have identified two truncating TTN variants in close proximity to each other in unrelated families with overlapping LQTS/LVNC/DCM syndromes. Mutations in TTN may play a previously unappreciated role in the pathogenesis of inherited arrhythmia syndromes.

Sensing of intracellular Hcp levels controls T6SS expression in Vibrio cholerae

The type 6 secretion system (T6SS) is a bacterial weapon broadly distributed in gram-negative bacteria and used to kill competitors and predators. Featuring a long and double-tubular structure, this molecular machine is energetically costly to produce and thus is likely subject to diverse regulation strategies that are largely ill defined. In this study, we report a quantity-sensing control of the T6SS that down-regulates the expression of secreted components when they accumulate in the cytosol due to T6SS inactivation. Using Vibrio cholerae strains that constitutively express an active T6SS, we demonstrate that mRNA levels of secreted components, including the inner-tube protein component Hcp, were down-regulated in T6SS structural gene mutants while expression of the main structural genes remained unchanged. Deletion of both hcp gene copies restored expression from their promoters, while Hcp overexpression negatively impacted expression. We show that Hcp directly interacts with the RpoN-dependent T6SS regulator VasH, and deleting the N-terminal regulator domain of VasH abolishes this interaction as well as the expression difference of hcp operons between T6SS-active and inactive strains. We find that negative regulation of hcp also occurs in other V. cholerae strains and the pathogens Aeromonas dhakensis and Pseudomonas aeruginosa This Hcp-dependent sensing control is likely an important energy-conserving mechanism that enables T6SS-encoding organisms to quickly adjust T6SS expression and prevent wasteful build-up of its major secreted components in the absence of their efficient export out of the bacterial cell.

Histone Lysine Methyltransferase SETD2 Regulates Coronary Vascular Development in Embryonic Mouse Hearts.

Congenital heart defects are the most common birth defect and have a clear genetic component, yet genomic structural variations or gene mutations account for only a third of the cases. Epigenomic dynamics during human heart organogenesis thus may play a critical role in regulating heart development. However, it is unclear how histone mark H3K36me3 acts on heart development. Here we report that histone-lysine N-methyltransferase SETD2, an H3K36me3 methyltransferase, is a crucial regulator of the mouse heart epigenome. Setd2 is highly expressed in embryonic stages and accounts for a predominate role of H3K36me3 in the heart. Loss of Setd2 in cardiac progenitors results in obvious coronary vascular defects and ventricular non-compaction, leading to fetus lethality in mid-gestation, without affecting peripheral blood vessel, yolk sac, and placenta formation. Furthermore, deletion of Setd2 dramatically decreased H3K36me3 level and impacted the transcriptional landscape of key cardiac-related genes, including Rspo3 and Flrt2. Taken together, our results strongly suggest that SETD2 plays a primary role in H3K36me3 and is critical for coronary vascular formation and heart development in mice.

Adaptation of a live-attenuated genotype I Japanese encephalitis virus to vero cells is associated with mutations in structural protein genes

The SD12-F120 is a live-attenuated genotype I strain of Japanese encephalitis virus (JEV) and was obtained by serial passage of wild-type strain SD12 on BHK-21 cells combined with multiple plaque purification and virulence selection in mice. The large scale production and vast clinical trials always demand ideal safety and efficacy profile of live-attenuated vaccines. In the present study, SD12-F120VC has undergone serial passaging of P1-P30 in WHO qualified Vero cells to assess the potential effect of adaptation to growth on Vero cells. The series of experiments showed that vaccine SD12-F120VC (Vero cell adapted) variants have consistently increased in peak virus titer compared to early passages and have good adaptation to growth in Vero cells. The animal experiments showed that Vero cell adapted SD12-F120VC variants have attenuation phenotype in suckling mice and the plaque morphology for all SD12-F120VC variants was small. Vaccination of mice with SD12-F120VC vaccine produced complete protection for homologous SD12 genotype I strain, but failed to give the complete protection of vaccinated mice against the challenge of heterologous N28 genotype III strain. In response to immunization of SD12-F120VC in mice, the neutralizing antibodies titer against homologous SD12-F120VC and SD12 (GI) was higher than heterologous N28 (GIII) strain. The prM protein has 6 amino acid substitutions, of which 5 amino acid changes were confined at the start of the pr domain in the ∼40 amino acids, and some mutations in the pr domain of prM might contribute to Vero cell adaptation. Our findings in this study are important for validation, evaluation and quality control study of live attenuated flaviviruses vaccines and show that Vero cells are a suitable substrate for the production of a safe and stable live-attenuated JEV vaccine.

Genetic characterization of fowl aviadenovirus 4 isolates from Guangxi, China, during 2017–2019

Hepatitis-hydropericardium syndrome (HHS) is a severe disease that causes 20 to 80% mortality in chickens aged 3 to 6 wk. Fowl aviadenovirus serotype 4 (FAdV-4) plays an important role in the etiology of HHS. Since 2015, outbreaks of HHS have been reported in several provinces of China; however, details regarding the FAdV-4 genome properties are lacking. In the present study, the complete genomes of 9 isolates responsible for these outbreaks in Guangxi Province, China, were sequenced. To investigate the molecular characteristics of these FAdV-4 isolates, we compared their genomes with those of other reported pathogenic and nonpathogenic FAdV-4 isolates. A variable number of GA repeats were observed in the isolates of this study. Each of the isolates GX2017-01, GX2017-02, GX2018-08, and GX2019-09 had 11 GA repeats; GX2017-03, GX2017-04, and GX2017-05 each had 10 GA repeats, while GX2017-06 and GX2018-07 each had 8 GA repeats. We observed several deletions and distinct amino acid mutations in the major structural genes of these isolates when compared with non-Chinese isolates. We found 2 novel putative genetic markers in the hexon protein, one present in GX2017-02, in which aspartic acid (D) was changed to tyrosine (Y), and another present in each of isolates GX2018-08 and GX2019-09, in which serine (S) was changed to arginine (R), when compared with selected Chinese and some non-Chinese isolates. Moreover, the phylogenetic analysis revealed that all the isolates of this study were clustered within FAdV-C. We found that these isolates were closely related to other recently isolated Chinese strains. The data presented in this study will not only increase the understanding of the molecular epidemiology and genetic diversity of FAdV-4 isolates in China but also has an important reference value of the major factors that determine the virulence of FAdV-4 strains.

Insight into the Genome of Diverse Penicillium chrysogenum Strains: Specific Genes, Cluster Duplications and DNA Fragment Translocations.

Background: There are eighteen species within the Penicillium genus section chrysogena, including the original penicillin producers Penicillium notatum (Fleming strain) and Penicillium chrysogenum NRRL 1951. Other wild type isolates of the Penicillium genus are relevant for the production of useful proteins and primary or secondary metabolites. The aim of this article is to characterize strain specific genes and those genes which are involved in secondary metabolite biosynthesis, particularly the mutations that have been introduced during the β-lactams strain improvement programs. Results: The available genomes of several classical and novel P. chrysogenum strains have been compared. The first genome sequenced was that of the reference strain P. chrysogenum Wis54-1255, which derives from the wild type P. chrysogenum NRRL 1951; its genome size is 32.19 Mb and it encodes 12,943 proteins. Four chromosomes were resolved in P. chrysogenum and P. notatum by pulse field gel electrophoresis. The genomes of three industrial strains have a similar size but contain gene duplications and truncations; the penicillin gene cluster copy number ranges from one in the wild type to twelve in the P. chrysogenum ASP-E1 industrial strain and is organized in head to tail tandem repeats. The genomes of two new strains, P. chrysogenum KF-25, a producer of antifungal proteins isolated from a soil sample, and P. chrysogenum HKF2, a strain with carbohydrate-converting activities isolated from a sludge treatment plant, showed strain specific genes. Conclusions: The overall comparison of all available P. chrysogenum genomes indicates that there are a significant number of strain-specific genes, mutations of structural and regulatory genes, gene cluster duplications and DNA fragment translocations. This information provides important leads to improve the biosynthesis of enzymes, antifungal agents, prebiotics or different types of secondary metabolites.

Nutritional intervention with cyanidin hinders the progression of muscular dystrophy

Muscular Dystrophies are severe genetic diseases due to mutations in structural genes, characterized by progressive muscle wasting that compromises patients’ mobility and respiratory functions. Literature underlined oxidative stress and inflammation as key drivers of these pathologies. Interestingly among different myofiber classes, type I fibers display a milder dystrophic phenotype showing increased oxidative metabolism. This work shows the benefits of a cyanidin-enriched diet, that promotes muscle fiber-type switch and reduced inflammation in dystrophic alpha-sarcoglyan (Sgca) null mice having, as a net outcome, morphological and functional rescue. Notably, this benefit is achieved also when the diet is administered in dystrophic animals when the signs of the disease are seriously evident. Our work provides compelling evidence that a cyanidin-rich diet strongly delays the progression of muscular dystrophies, paving the way for a combinatorial approach where nutritional-based reduction of muscle inflammation and oxidative stress facilitate the successful perspectives of definitive treatments.

A Deleted Deletion Site in a New Vector Strain and Exceptional Genomic Stability of Plaque-Purified Modified Vaccinia Ankara (MVA)

Vectored vaccines based on highly attenuated modified vaccinia Ankara (MVA) are reported to be immunogenic, tolerant to pre-existing immunity, and able to accommodate and stably maintain very large transgenes. MVA is usually produced on primary chicken embryo fibroblasts, but production processes based on continuous cell lines emerge as increasingly robust and cost-effective alternatives. An isolate of a hitherto undescribed genotype was recovered by passage of a non-plaque-purified preparation of MVA in a continuous anatine suspension cell line (CR.pIX) in chemically defined medium. The novel isolate (MVA-CR19) replicated to higher infectious titers in the extracellular volume of suspension cultures and induced fewer syncytia in adherent cultures. We now extend previous studies with the investigation of the point mutations in structural genes of MVA-CR19 and describe an additional point mutation in a regulatory gene. We furthermore map and discuss an extensive rearrangement of the left telomer of MVA-CR19 that appears to have occurred by duplication of the right telomer. This event caused deletions and duplications of genes that may modulate immunologic properties of MVA-CR19 as a vaccine vector. Our characterizations also highlight the exceptional genetic stability of plaque-purified MVA: although the phenotype of MVA-CR19 appears to be advantageous for replication, we found that all genetic markers that differentiate wildtype and MVA-CR19 are stably maintained in passages of recombinant viruses based on either wildtype or MVA-CR.

Molecular mechanisms underlying intraspecific variation in snake venom

Elucidating the molecular mechanisms underlying snake venom variability provides important clues for understanding how the biological functions of this powerful toxic arsenal evolve. We analyzed in detail individual transcripts and venom protein isoforms produced by five specimens of a venomous snake (Bothrops atrox) from two nearby but genetically distinct populations from the Brazilian Amazon rainforest which show functional similarities in venom properties. Individual variation was observed among the venoms of these specimens, but the overall abundance of each general toxin family was conserved both in transcript and in venom protein levels. However, when expression of independent paralogues was analyzed, remarkable differences were observed within and among each toxin group, both between individuals and between populations. Transcripts for functionally essential venom proteins (“core function” proteins) were highly expressed in all specimens and showed similar transcription/translation rates. In contrast, other paralogues (“adaptive” proteins) showed lower expression levels and the toxins they coded for varied among different individuals. These results provide support for the inferences that (a) expression and translational differences play a greater role in defining adaptive variation in venom phenotypes than does sequence variation in protein coding genes and (b) convergent adaptive venom phenotypes can be generated through different molecular mechanisms. SignificanceAnalysis of individual transcripts and venom protein isoforms produced by specimens of a venomous snake (Bothrops atrox), from the Brazilian Amazon rainforest, revealed that transcriptional and translational mechanisms contribute to venom phenotypic variation. Our finding of evidence for high expression of toxin proteins with conserved function supports the hypothesis that the venom phenotype consists of two kinds of proteins: conserved “core function” proteins that provide essential functional activities with broader relevance and less conserved “adaptive” proteins that vary in expression and may permit customization of protein function. These observations allowed us to suggest that genetic mechanisms controlling venom variability are not restricted to selection of gene copies or mutations in structural genes but also to selection of the mechanisms controlling gene expression, contributing to the plasticity of this important phenotype for venomous snakes.

Silencing Nfix rescues muscular dystrophy by delaying muscle regeneration

Muscular dystrophies are severe disorders due to mutations in structural genes, and are characterized by skeletal muscle wasting, compromised patient mobility, and respiratory functions. Although previous works suggested enhancing regeneration and muscle mass as therapeutic strategies, these led to no long-term benefits in humans. Mice lacking the transcription factor Nfix have delayed regeneration and a shift toward an oxidative fiber type. Here, we show that ablating or silencing the transcription factor Nfix ameliorates pathology in several forms of muscular dystrophy. Silencing Nfix in postnatal dystrophic mice, when the first signs of the disease already occurred, rescues the pathology and, conversely, Nfix overexpression in dystrophic muscles increases regeneration and markedly exacerbates the pathology. We therefore offer a proof of principle for a novel therapeutic approach for muscular dystrophies based on delaying muscle regeneration.

Identification of an HLA class I allele closely involved in the autoantigen presentation in acquired aplastic anemia

AbstractTo identify HLA alleles closely involved in the autoantigen presentation in acquired aplastic anemia (AA), we studied the HLA allelic loss frequencies of 312 AA patients, including 43 patients with loss of heterozygosity of 6p chromosome (6pLOH). An analysis of the HLA alleles contained in the lost haplotype revealed HLA-B*40:02 to be the most frequently lost allele. When we examined 28 AA (12 6pLOH[+] and 16 6pLOH[−]) patients with HLA-B*40:02 for the presence of leukocytes lacking HLA-B4002 (B4002−) using a new monoclonal antibody specific to this allele, B4002− granulocytes were detected not only in all 6pLOH(+) patients but also in 9 (56%) of the 16 6pLOH(−) patients. Furthermore, 10 (83%) of the 12 6pLOH(+) patients possessed 1.0% to 78% B4002− granulocytes that retained the HLA-A allele on the same haplotype (B4002−A+), suggesting the frequent coexistence of granulocytes that underwent mutations restricted to HLA-B*40:02 with 6pLOH(+) (B4002−A−) granulocytes. Deep sequencing of the HLA-B*40:02 of sorted B4002−A+ granulocytes revealed various somatic mutations, such as frameshift, nonsense, and splice site mutations, in all 15 patients studied. Surprisingly, missense mutations in the α-3 domain of HLA-B*40:02 that are not involved in the antigen presentation were detected exclusively in the B4002+ granulocytes of 3 patients possessing B4002− granulocytes. The markedly high prevalence of leukocytes lacking HLA-B4002 as a result of either 6pLOH or structural gene mutations, or both, suggests that antigen presentation by hematopoietic stem/progenitor cells to cytotoxic T cells via the HLA-B allele plays a critical role in the pathogenesis of AA.

Association between mutation status and left ventricular reverse remodelling in dilated cardiomyopathy

ObjectiveTo explore the genetic landscape of a well selected dilated cardiomyopathy (DCM) cohort, assessing the possible relation between different genotypes and left ventricular reverse remodelling (LVRR).MethodsA cohort of 152 patients...

Identification of an HLA Class I Allele Closely Involved in the Pathogenesis of Acquired Aplastic Anemia

[Background] The frequent loss of heterozygosity of the HLA haplotype in the short arm of chromosome 6 (6pLOH) in leukocytes is thought to offer compelling evidence of cytotoxic T cell (CTL) involvement in the development of acquired aplastic anemia (AA) because it represents the escape of hematopoietic stem/progenitor cells (HSPCs) with 6pLOH from the attack of CTLs that are specific to autoantigens presented by the lacked HLA class I allele. Although our previous study suggested that HLA-B*40:02 is the major allele involved in this phenomenon, the exact role of B*40:02 remained unclear because 6pLOH involving this allele is always associated with a lack of HLA-A and C alleles in the haplotype, and the presence of B*40:02-missing leukocytes were unable to be shown due to the lack of monoclonal antibodies (mAbs) specific to B61, the HLA-B antigen that corresponds to B*40:02. We recently succeeded in generating a mAb specific for HLA-B61 that enabled us to explore the role of B*40:02 in the development of AA.[Methods] Using the new mAb, we examined peripheral blood samples of 28 AA (12 with 6pLOH and 16 without 6pLOH) patients carrying this allele for the presence of B61(-) leukocytes using flow cytometry. HLA genes were enriched by sequence capture, a hybridization-based gene enrichment method, from genomic DNA of sorted B61(-) granulocytes, and were subjected to deep sequencing using an NGS (MiSeq). B61(+) granulocytes or T cells were used as controls. Potential mutations responsible for the B61-missing were identified when 10 or more variant reads were found only in B61(-) granulocytes. Thereafter, HLA-B alleles carrying those mutations were determined taking advantage of the nearest allele-specific SNPs.[Results] Among the 12 6pLOH(+) patients, 10 (83%) possessed 0.5%-60% B61-missing granulocytes that were not lacking HLA-A, in addition to 12% to 99% 6pLOH(+) granulocytes that lacked both B61 and an HLA-A allele on the same haplotype (Figure 1). B61(-) granulocytes that accounted for 0.5%-99% of the total granulocytes were detected in 9 (56%) of the 16 6pLOH(-) patients. The prevalence of missing B61 in the 28 AA patients was 21/28 (75%), much more frequent than those of the 3 other major alleles (A*02:01, 32%; A*02:06, 30%; A*24:02, 6%). B61(-) granulocytes were available for mutation analyses of HLA-B alleles in 15 of the 19 patients who possessed B61(-) granulocytes. The mean coverage of HLA-B gene was 426x. In total, 43 somatic mutations of HLA-B were identified in B61(-) granulocytes, all of which were present in B*40:02 but not in any of the other HLA-B alleles. Median variant allele frequency was 4.8% (range, 1.0% - 43%) and the number of mutations in each patient was 1 to 6 (Figure 2). Thirty-nine mutations were exonic while 4 were intronic. Exonic mutations included frameshift insertions (n=12), frameshift deletions (n=16), non-frameshift deletions (n=2), nonsense mutations (n=7), a missense mutation (n=1) and a start codon mutation (n=1). All four intronic mutations were considered to be a splice site mutation; two mutations deactivated 5’ and 3’ splice sites, whereas the other two were single base substitutions within intron 3, making alternative 5’ splicing site with strong consensus sequence: GGC [A>G] TGAGT and TTC [C>G] TGAGT. Surprisingly, missense mutations in the alpha-2 and alpha-3 chain-coding region of HLA-B*40:02 were detected exclusively in the B61(+) granulocytes of two patients possessing B61(-) granulocytes, suggesting the inability of the mutant HSPCs to interact with CTLs. Variant allele frequencies of the two missense mutation were 40% and 45%, respectively. As a result of the mutation, virtually all granulocytes of the two patients were affected by B*40:02 mutations that allowed the HSPCs to escape the T cell attack.[Conclusions] The markedly high prevalence of leukocytes lacking HLA-B*40:02 as a result of either or both 6pLOH or structural gene mutations clearly indicates that antigen presentation by HSPCs to CTLs via the HLA-B allele plays a critical role in the pathogenesis of AA. [Display omitted] [Display omitted] DisclosuresTakamatsu:Celgene: Honoraria; Janssen Pharmaceuticals: Honoraria. Nakao:Alexion Pharmaceuticals: Honoraria, Research Funding.