Infant case of severe immune thrombocytopenia caused by COVID-19 infection.

Immune thrombocytopenia (ITP) is a common childhood acute autoimmune bleeding disorder caused by numerous viruses and characterized by isolated thrombocytopenia. Although cases of ITP caused by coronavirus disease 2019 (COVID-19) infection have been reported in adults, pediatric reports are limited. We present the case of a 1-year-old girl who developed COVID-19-infection-related ITP with a very low platelet count (0.0 × 104/µL). We searched for COVID-19-related pediatric ITP cases and found 10 other cases, with the majority having platelet counts of <1.0 × 104/µL. Although pediatric ITP cases caused by COVID-19 infection may be severe, further studies are needed.

Sarcomere maturation: function acquisition, molecular mechanism, and interplay with other organelles.

During postnatal cardiac development, cardiomyocytes mature and turn into adult ones. Hence, all cellular properties, including morphology, structure, physiology and metabolism, are changed. One of the most important aspects is the contractile apparatus, of which the minimum unit is known as a sarcomere. Sarcomere maturation is evident by enhanced sarcomere alignment, ultrastructural organization and myofibrillar isoform switching. Any maturation process failure may result in cardiomyopathy. Sarcomere function is intricately related to other organelles, and the growing evidence suggests reciprocal regulation of sarcomere and mitochondria on their maturation. Herein, we summarize the molecular mechanism that regulates sarcomere maturation and the interplay between sarcomere and other organelles in cardiomyocyte maturation. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.

A case of congenital fiber-type disproportion syndrome presenting dilated cardiomyopathy with ACTA1 mutation.

BACKGROUND: Actin, alpha, skeletal muscle 1 (ACTA1) is one of the causative genes of nemaline myopathy (NM) and congenital fiber-type disproportion (CFTD). CFTD is characterized by type 1 fiber atrophy and distinguished from NM in the absence of rods. Eight patients with CFTD, including one patient with dilated cardiomyopathy (DCM), have previously been reported. Herein, we report the case of a 10-year-old boy presenting with CFTD and DCM. METHODS: We performed exome sequencing and analyzed the effect of Met327Lys mutations on cultured C2C12 muscle cells compared with that seen in the wild type (WT, ACTA1) and previously identified Asp294Val mutations associated with a severe phenotype of CFTD without cardiomyopathy. RESULTS: Exome sequencing revealed a de novo mutation, c.980 T > A, p.(Met327Lys), in ACTA1 (NM_001100.4). C2C12 cells transfected with the WT plasmid expressed ACTA1 in the nucleus and cytoplasm. Cells with the Asp294Val mutant showed needle-like structures in the cytoplasm, whereas the expression of the Met327Lys mutant resulted in few aggregations but many apoptotic cells. CONCLUSION: Apoptosis induced in Met327Lys-transfected muscle cells supports the pathogenicity of the mutation and can be implicated as one of the histopathological features associated with CFTD, as in NM.

Disease Modeling of Mitochondrial Cardiomyopathy Using Patient-Specific Induced Pluripotent Stem Cells.

Mitochondrial cardiomyopathy (MCM) is characterized as an oxidative phosphorylation disorder of the heart. More than 100 genetic variants in nuclear or mitochondrial DNA have been associated with MCM. However, the underlying molecular mechanisms linking genetic variants to MCM are not fully understood due to the lack of appropriate cellular and animal models. Patient-specific induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) provide an attractive experimental platform for modeling cardiovascular diseases and predicting drug efficacy to such diseases. Here we introduce the pathological and therapeutic studies of MCM using iPSC-CMs and discuss the questions and latest strategies for research using iPSC-CMs.

Generation of Efficient Knock-in Mouse and Human Pluripotent Stem Cells Using CRISPR-Cas9.

A knock-in can generate fluorescent or Cre-reporter under the control of an endogenous promoter. It also generates knock-out or tagged-protein with fluorescent protein and short tags for tracking and purification. Recent advances in genome editing with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) significantly increased the efficiencies of making knock-in cells. Here we describe the detailed protocols of generating knock-in mouse and human pluripotent stem cells (PSCs) by electroporation and lipofection, respectively.

Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells provide promising sources for cardiac disease modeling. For cardiomyopathies, sarcomere shortening is one of the standard physiological assessments that are used with adult cardiomyocytes to examine their disease phenotypes. However, the available methods are not appropriate to assess the contractility of PSC-CMs, as these cells have underdeveloped sarcomeres that are invisible under phase-contrast microscopy. To address this issue and to perform sarcomere shortening with PSC-CMs, fluorescent-tagged sarcomere proteins and fluorescent live-imaging were used. Thin Z-lines and an M-line reside at both ends and the center of a sarcomere, respectively. Z-line proteins - α-Actinin (ACTN2), Telethonin (TCAP), and actin-associated LIM protein (PDLIM3) - and one M-line protein - Myomesin-2 (Myom2) - were tagged with fluorescent proteins. These tagged proteins can be expressed from endogenous alleles as knock-ins or from adeno-associated viruses (AAVs). Here, we introduce the methods to differentiate mouse and human pluripotent stem cells to cardiomyocytes, to produce AAVs, and to perform and analyze live-imaging. We also describe the methods for producing polydimethylsiloxane (PDMS) stamps for a patterned culture of PSC-CMs, which facilitates the analysis of sarcomere shortening with fluorescent-tagged proteins. To assess sarcomere shortening, time-lapse images of the beating cells were recorded at a high framerate (50-100 frames per second) under electrical stimulation (0.5-1 Hz). To analyze sarcomere length over the course of cell contraction, the recorded time-lapse images were subjected to SarcOptiM, a plug-in for ImageJ/Fiji. Our strategy provides a simple platform for investigating cardiac disease phenotypes in PSC-CMs.

Comprehensive Genetic Analysis of Non-syndromic Autism Spectrum Disorder in Clinical Settings.

Although genetic factors are involved in the etiology of autism spectrum disorder (ASD), the significance of genetic analysis in clinical settings is unclear. Forty-nine subjects diagnosed with non-syndromic ASD were analyzed by microarray comparative genomic hybridization (CGH) analysis, whole-exome sequencing (WES) analysis, and panel sequencing analysis for 52 common causative genes of ASD to detect inherited rare variants. Genetic analysis by microarray CGH and WES analyses showed conclusive results in about 10% of patients, however, many inherited variants detected by panel sequencing analysis were difficult to interpret and apply in clinical practice in the majority of patients. Further improvement of interpretation of many variants detected would be necessary for combined genetic tests to be used in clinical settings.

Comparative Transcriptome Landscape of Mouse and Human Hearts.

Transcriptome landscape of organs from mice and humans offers perspectives on the process of how organs develop and the similarity and diversity in each organ between the species. Among multi-species and multi-organ dataset, which was previously generated, we focused on the mouse and human dataset and performed a reanalysis to provide a more specific perspective on the maturation of human cardiomyocytes. First, we examined how organs diversify their transcriptome during development across and within two species. We unexpectedly identified that ribosomal genes were differentially expressed between mice and humans. Second, we examined the corresponding ages of organs in mice and humans and found that the corresponding developmental ages did not match throughout organs. Mouse hearts at P0-3 and human hearts at 18-19 wpc showed the most proximity in the regard of the transcriptome. Third, we identified a novel set of maturation marker genes that are more consistent between mice and humans. In contrast, conventionally used maturation marker genes only work well with mouse hearts. Finally, we compared human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) in maturation-enhanced conditions to human fetal and adult hearts and revealed that human PSC-CMs only expressed low levels of the potential maturation marker genes. Our findings provide a novel foundation to study cardiomyocyte maturation and highlight the importance of studying human samples rather than relying on a mouse time-series dataset.

A Brief Review of Current Maturation Methods for Human Induced Pluripotent Stem Cells-Derived Cardiomyocytes.

Cardiovascular diseases are the leading cause of death worldwide. Therefore, the discovery of induced pluripotent stem cells (iPSCs) and the subsequent generation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) was a pivotal point in regenerative medicine and cardiovascular research. They constituted an appealing tool for replacing dead and dysfunctional cardiac tissue, screening cardiac drugs and toxins, and studying inherited cardiac diseases. The problem is that these cells remain largely immature, and in order to utilize them, they must reach a functional degree of maturity. To attempt to mimic in vivo environment, various methods including prolonging culture time, co-culture and modulations of chemical, electrical, mechanical culture conditions have been tried. In addition to that, changing the topology of the culture made huge progress with the introduction of the 3D culture that closely resembles the in vivo cardiac topology and overcomes many of the limitations of the conventionally used 2D models. Nonetheless, 3D culture alone is not enough, and using a combination of these methods is being explored. In this review, we summarize the main differences between immature, fetal-like hiPSC-CMs and adult cardiomyocytes, then glance at the current approaches used to promote hiPSC-CMs maturation. In the second part, we focus on the evolving 3D culture model - it's structure, the effect on hiPSC-CMs maturation, incorporation with different maturation methods, limitations and future prospects.

Treatment of a patient with Kawasaki disease associated with selective IgA deficiency by continuous infusion of cyclosporine A without intravenous immunoglobulin.

Intravenous immunoglobulin therapy is standard for Kawasaki disease (KD) treatment; however, anaphylactic reactions to immunoglobulins are a risk in KD patients with selective IgA deficiency (sIgAD). The therapy for KD associated with sIgAD has not been established. The IgA immune response is believed to play an important role in KD vasculitis. We report the case of a 5-year-old boy with KD and sIgAD treated with intravenous cyclosporine A (CsA, 3.0 mg/kg/day) instead of intravenous immunoglobulin (IVIG). The fever and inflammation immediately resolved without a coronary artery lesion. In KD patients with sIgAD, we believe that an IgA immune response is lacking, which is the reason for milder KD symptoms than in those without sIgAD. This case report aids in clarifying the role of IgA antibodies in KD and provides evidence that CsA is a potential candidate for first-line therapy for patients with KD with contraindications to IVIG.

Rapid evolution of major histocompatibility complex class I genes in primates generates new disease alleles in humans via hitchhiking diversity.

A plausible explanation for many MHC-linked diseases is lacking. Sequencing of the MHC class I region (coding units or full contigs) in several human and nonhuman primate haplotypes allowed an analysis of single nucleotide variations (SNV) across this entire segment. This diversity was not evenly distributed. It was rather concentrated within two gene-rich clusters. These were each centered, but importantly not limited to, the antigen-presenting HLA-A and HLA-B/-C loci. Rapid evolution of MHC-I alleles, as evidenced by an unusually high number of haplotype-specific (hs) and hypervariable (hv) (which could not be traced to a single species or haplotype) SNVs within the classical MHC-I, seems to have not only hitchhiked alleles within nearby genes, but also hitchhiked deleterious mutations in these same unrelated loci. The overrepresentation of a fraction of these hvSNV (hv1SNV) along with hsSNV, as compared to those that appear to have been maintained throughout primate evolution (trans-species diversity; tsSNV; included within hv2SNV) tends to establish that the majority of the MHC polymorphism is de novo (species specific). This is most likely reminiscent of the fact that these hsSNV and hv1SNV have been selected in adaptation to the constantly evolving microbial antigenic repertoire.

Genomic evolution of MHC class I region in primates.

To elucidate the origins of the MHC-B-MHC-C pair and the MHC class I chain-related molecule (MIC)A-MICB pair, we sequenced an MHC class I genomic region of humans, chimpanzees, and rhesus monkeys and analyzed the regions from an evolutionary stand-point, focusing first on LINE sequences that are paralogous within each of the first two species and orthologous between them. Because all the long interspersed nuclear element (LINE) sequences were fragmented and nonfunctional, they were suitable for conducting phylogenetic study and, in particular, for estimating evolutionary time. Our study has revealed that MHC-B and MHC-C duplicated 22.3 million years (Myr) ago, and the ape MICA and MICB duplicated 14.1 Myr ago. We then estimated the divergence time of the rhesus monkey by using other orthologous LINE sequences in the class I regions of the three primate species. The result indicates that rhesus monkeys, and possibly the Old World monkeys in general, diverged from humans 27-30 Myr ago. Interestingly, rhesus monkeys were found to have not the pair of MHC-B and MHC-C but many repeated genes similar to MHC-B. These results support our inference that MHC-B and MHC-C duplicated after the divergence between apes and Old World monkeys.

Rapid assignment of the swine major histocompatibility complex (SLA) class I and II genotypes in Clawn miniature swine using PCR-SSP and PCR-RFLP methods.

BACKGROUND: Inbred miniature swine with defined novel SLA haplotypes will be useful in allo- and xeno-transplantation studies, which can be carried out representing variable combinations of SLA haplotypes. METHODS: In Clawn miniature swine, two haplotypes (c1 and c2) and one crossover haplotype (c3) have been assigned by nucleotide sequence determination of RT-PCR products of the three SLA classical class I genes and two SLA class II genes. To select SLA class I and II homozygotes in Clawn miniature swine individuals, we developed a rapid and simple SLA-class I- and II-DNA typing method by a combination of polymerase chain reaction-sequence specific primer (PCR-SSP) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. RESULTS: Seven allele specific primer pairs were designed for amplification of the second exons of three SLA class I genes, SLA-1, SLA-2, and SLA-3, and one SLA class II gene, DRB1. Furthermore, based on PCR-RFLP patterns in the SLA-DQB1 gene, two allelic variants were recognized in the second exon in the Clawn miniature swine. Three haplotypes, c1, c2 and c3, were simply identified by the combination of PCR-SSP and PCR-RFLP methods in 22 samples from five families. A single allele at each of the class I and II genes was also observed in seven samples as SLA class I and II homozygotes with either the c1 or c2 haplotype. CONCLUSIONS: The combination of PCR-SSP and PCR-RFLP methods facilitate the rapid identification of the three haplotypes and SLA class I and II homozygotes in individual Clawn miniature swine.

Rhesus macaque class I duplicon structures, organization, and evolution within the alpha block of the major histocompatibility complex.

The alpha block of the human and chimpanzee major histocompatibility complex (MHC) class I genomic region contains 10 to 11 duplicated MHC class I genes, including the HLA/Patr-A, -G, and -F genes. In comparison, the alpha block of the rhesus macaque (Macaca mulatta, Mamu) has an additional 20 MHC class I genes within this orthologous region. The present study describes the identification and analysis of the duplicated segmental genomic structures (duplicons) and genomic markers within the alpha block of the rhesus macaque and their use to reconstruct the duplication history of the genes within this region. A variety of MHC class I genes, pseudogenes, transposons, and retrotransposons, such as Alu and ERV16, were used to categorize the 28 duplicons into four distinct structural categories. The phylogenetic relationship of MHC class I genes, Alu, and LTR16B sequences within the duplicons was examined by use of the Neighbor-Joining (NJ) method. Two single-duplicon tandem duplications, two polyduplicon tandem duplications with an accompanying inversion product per duplication, eight polyduplicon tandem duplications steps, 12 deletions, and at least two recombinations were reconstructed to explain the highly complex organization and evolution of the 28 duplicons (nine inversions) within the Mamu alpha block. On the basis of the phylogenetic evidence and the reconstructed tandem duplication history of the 28 duplicons, the Mamu/Patr/HLA-F ortholog was the first MHC class I gene to have been fixed without further duplication within the alpha block of primates. Assuming that the rhesus macaque and the chimpanzee/human lineages had started with the same number of MHC class I duplicons at the time of their divergence approximately 24 to 31 MYA, then the number of genes within the alpha block have been duplicated at an approximately three times greater rate in the rhesus macaque than in either the human or chimpanzee.

Genetic polymorphism of the swine major histocompatibility complex ( SLA) class I genes, SLA-1, -2 and -3.

In order to identify and characterize genetic polymorphism of the swine major histocompatibility complex ( Mhc: SLA) class I genes, RT-PCR products of the second and third exons of the three SLA classical class I genes, SLA-1, SLA-2 and SLA-3 were subjected to nucleotide determination. These analyses allowed the identification of four, eight and seven alleles at the SLA-1, SLA-2 and SLA-3 loci, respectively, from three different breeds of miniature swine and one mixed breed. Among them, 12 alleles were novel. Construction of a phylogenetic tree using the nucleotide sequences of those 19 alleles indicated that the SLA-1 and -2 genes are more closely related to each other than to SLA-3. Selective forces operating at single amino acid sites of the SLA class I molecules were analyzed by the Adaptsite Package program. Ten positive selection sites were found at the putative antigen recognition sites (ARSs). Among the 14 positively selected sites observed in the human MHC ( HLA) classical class I molecules, eight corresponding positions in the SLA class I molecules were inferred as positively selected. On the other hand, four amino acids at the putative ARSs were identified as negatively selected in the SLA class I molecules. These results suggest that selective forces operating in the SLA class I molecules are almost similar to those of the HLA class I molecules, although several functional sites for antigen and cytotoxic T-lymphocyte recognition by the SLA class I molecules may be different from those of the HLA class I molecules.

Comparative sequencing of human and chimpanzee MHC class I regions unveils insertions/deletions as the major path to genomic divergence.

Despite their high degree of genomic similarity, reminiscent of their relatively recent separation from each other ( approximately 6 million years ago), the molecular basis of traits unique to humans vs. their closest relative, the chimpanzee, is largely unknown. This report describes a large-scale single-contig comparison between human and chimpanzee genomes via the sequence analysis of almost one-half of the immunologically critical MHC. This 1,750,601-bp stretch of DNA, which encompasses the entire class I along with the telomeric part of the MHC class III regions, corresponds to an orthologous 1,870,955 bp of the human HLA region. Sequence analysis confirms the existence of a high degree of sequence similarity between the two species. However, and importantly, this 98.6% sequence identity drops to only 86.7% taking into account the multiple insertions/deletions (indels) dispersed throughout the region. This is functionally exemplified by a large deletion of 95 kb between the virtual locations of human MICA and MICB genes, which results in a single hybrid chimpanzee MIC gene, in a segment of the MHC genetically linked to species-specific handling of several viral infections (HIV/SIV, hepatitis B and C) as well as susceptibility to various autoimmune diseases. Finally, if generalized, these data suggest that evolution may have used the mechanistically more drastic indels instead of the more subtle single-nucleotide substitutions for shaping the recently emerged primate species.

Comparative genomic analysis of the MHC: the evolution of class I duplication blocks, diversity and complexity from shark to man.

The major histocompatibility complex (MHC) genomic region is composed of a group of linked genes involved functionally with the adaptive and innate immune systems. The class I and class II genes are intrinsic features of the MHC and have been found in all the jawed vertebrates studied so far. The MHC genomic regions of the human and the chicken (B locus) have been fully sequenced and mapped, and the mouse MHC sequence is almost finished. Information on the MHC genomic structures (size, complexity, genic and intergenic composition and organization, gene order and number) of other vertebrates is largely limited or nonexistent. Therefore, we are mapping, sequencing and analyzing the MHC genomic regions of different human haplotypes and at least eight nonhuman species. Here, we review our progress with these sequences and compare the human MHC structure with that of the nonhuman primates (chimpanzee and rhesus macaque), other mammals (pigs, mice and rats) and nonmammalian vertebrates such as birds (chicken and quail), bony fish (medaka, pufferfish and zebrafish) and cartilaginous fish (nurse shark). This comparison reveals a complex MHC structure for mammals and a relatively simpler design for nonmammalian animals with a hypothetical prototypic structure for the shark. In the mammalian MHC, there are two to five different class I duplication blocks embedded within a framework of conserved nonclass I and/or nonclass II genes. With a few exceptions, the class I framework genes are absent from the MHC of birds, bony fish and sharks. Comparative genomics of the MHC reveal a highly plastic region with major structural differences between the mammalian and nonmammalian vertebrates. Additional genomic data are needed on animals of the reptilia, crocodilia and marsupial classes to find the origins of the class I framework genes and examples of structures that may be intermediate between the simple and complex MHC organizations of birds and mammals, respectively.