The accurate typing of human leukocyte antigen (HLA) alleles is critical for a variety of medical applications, such as genomic studies of multifactorial diseases, including immune system and ...inflammation‐related disorders, and donor selection in organ transplantation and regenerative medicine. Here, we developed a new algorithm for determining HLA alleles using next‐generation sequencing (NGS) results. The method consists of constructing an extensive dictionary of HLA alleles, precise mapping of the NGS reads, and calculating a score based on weighted read counts to select the most suitable pair of alleles. The developed algorithm compares the score of all allele pairs, taking into account variation not only in the domain for antigen presentation (G‐DOMAIN), but also outside this domain. Using this method, HLA alleles could be determined with 6‐digit precision. We showed that our method was more accurate than other NGS‐based methods and revealed limitations of the conventional HLA typing technologies. Furthermore, we determined the complete genomic sequence of an HLA‐A‐like‐pseudogene when we assembled NGS reads that had caused arguable typing, and found its identity with HLA‐Y*02:01. The accuracy of the HLA‐A allele typing was improved after the HLA‐Y*02:01 sequence was included in the HLA allele dictionary.
HLA‐HD is a new algorithm for determining HLA alleles using next‐generation sequencing (NGS) data. The method consists of constructing an extensive dictionary of HLA alleles, precise mapping of the NGS reads, and calculating a score based on weighted read counts to select the most suitable pair of alleles. HLA‐HD compares the score of all allele pairs, taking into account variation not only in the domain for antigen presentation, but also outside this domain.
Phospholipids are asymmetrically distributed in the plasma membrane. This asymmetrical distribution is disrupted during apoptosis, exposing phosphatidylserine (PtdSer) on the cell surface. Using a ...haploid genetic screen in human cells, we found that ATP11C (adenosine triphosphatase type 11C) and CDC50A (cell division cycle protein 50A) are required for aminophospholipid translocation from the outer to the inner plasma membrane leaflet; that is, they display flippase activity. ATP11C contained caspase recognition sites, and mutations at these sites generated caspase-resistant ATP11C without affecting its flippase activity. Cells expressing caspase-resistant ATP11C did not expose PtdSer during apoptosis and were not engulfed by macrophages, which suggests that inactivation of the flippase activity is required for apoptotic PtdSer exposure. CDC50A-deficient cells displayed PtdSer on their surface and were engulfed by macrophages, indicating that PtdSer is sufficient as an "eat me" signal.
HLA is essential for various medical applications, such as genomic studies of multifactorial diseases, including immune system and inflammation-related disorders. Therefore, an accurate HLA typing ...method that is applicable for any allele registered in HLA allele databases is required to deduce scientific evidence related to disorders. Here, we describe a method for determining HLA alleles from next-generation sequencing (NGS) results by using currently available HLA sequence data in public HLA databases and show its application in association analysis.
Retinitis pigmentosa (RP), a major cause of blindness in developed countries, has multiple causative genes; its prevalence differs by ethnicity. Usher syndrome is the most common form of syndromic RP ...and is accompanied by hearing impairment. Although molecular diagnosis is challenging, recent technological advances such as targeted high-throughput resequencing are efficient screening tools.
We performed comprehensive molecular testing in 329 Japanese RP and Usher syndrome patients by using a custom capture panel that covered the coding exons and exon/intron boundaries of all 193 known inherited eye disease genes combined with Illumina HiSequation 2500. Candidate variants were screened using systematic data analyses, and their potential pathogenicity was assessed according to the frequency of the variants in normal populations, in silico prediction tools, and compatibility with known phenotypes or inheritance patterns.
Molecular diagnoses were made in 115/317 RP patients (36.3%) and 6/12 Usher syndrome patients (50%). We identified 104 distinct mutations, including 66 novel mutations. EYS, USH2A, and RHO were common causative genes. In particular, mutations in EYS accounted for 15.0% of the autosomal recessive/simplex RP patients or 10.7% of the entire RP cohort. Among the 189 previously reported mutations detected in the current study, 55 (29.1%) were found commonly in Japanese or other public databases and were excluded from molecular diagnoses.
By screening a large cohort of patients, this study catalogued the genetic variations involved in RP and Usher syndrome in a Japanese population and highlighted the different distribution of causative genes among populations.
Next-generation sequencing (NGS) has greatly advanced the studies of causative genes and variants of inherited diseases. While it is sometimes challenging to determine the pathogenicity of identified ...variants in NGS, the American College of Medical Genetics and Genomics established the guidelines to help the interpretation. However, as to the genetic screenings for patients with retinitis pigmentosa (RP) in Japan, none of the previous studies utilized the guidelines. Considering that EYS is the major causative gene of RP in Japan, we conducted stepwise genetic screening of 220 Japanese patients with RP utilizing the guidelines. Step 1-4 comprised the following, in order: Sanger sequencing for two major EYS founder mutations; targeted sequencing of all coding regions of EYS; whole genome sequencing; Sanger sequencing for Alu element insertion in RP1, a recently determined founder mutation for RP. Among the detected variants, 2, 19, 173, and 1 variant(s) were considered pathogenic and 8, 41, 44, and 5 patients were genetically solved in step 1, 2, 3, and 4, respectively. Totally, 44.5% (98/220) of the patients were genetically solved, and 50 (51.0%) were EYS-associated and 5 (5.1%) were Alu element-associated. Among the unsolved 122 patients, 22 had at least one possible pathogenic variant.
Central serous chorioretinopathy (CSC) is a common disease affecting younger people and may lead to vision loss. CSC shares phenotypic overlap with age-related macular degeneration (AMD). As recent ...studies have revealed a characteristic increase of choroidal thickness in CSC, we conducted a genome-wide association study on choroidal thickness in 3,418 individuals followed by TaqMan assays in 2,692 subjects, and we identified two susceptibility loci: CFH rs800292, an established AMD susceptibility polymorphism, and VIPR2 rs3793217 (P = 2.05 × 10−10 and 6.75 × 10−8, respectively). Case–control studies using patients with CSC confirmed associations between both polymorphisms and CSC (P = 5.27 × 10−5 and 5.14 × 10−5, respectively). The CFH rs800292 G allele is reportedly a risk allele for AMD, whereas the A allele conferred risk for thicker choroid and CSC development. This study not only shows that susceptibility genes for CSC could be discovered using choroidal thickness as a defining variable but also, deepens the understanding of differences between CSC and AMD pathophysiology.
Spermidine (SPD) delays age-related pathologies in various organisms. SPD supplementation overcame the impaired immunotherapy against tumors in aged mice by increasing mitochondrial function and ...activating CD8
+
T cells. Treatment of naïve CD8
+
T cells with SPD acutely enhanced fatty acid oxidation. SPD conjugated to beads bound to the mitochondrial trifunctional protein (MTP). In the MTP complex, synthesized and purified from
Escherichia coli
, SPD bound to the α and β subunits of MTP with strong affinity and allosterically enhanced their enzymatic activities. T cell–specific deletion of the MTP α subunit abolished enhancement of programmed cell death protein 1 (PD-1) blockade immunotherapy by SPD, indicating that MTP is required for SPD-dependent T cell activation.
Spermidine fights cancer in aging mice
Abundance of the polyamine spermidine decreases in aging mice, and supplementation can have restorative effects and extend life span. Al-Habsi
et al
. explored whether loss of spermidine might contribute to loss of antitumor immunity in aged mice. Restoration of spermidine concentrations enhanced antitumor responses stimulated by programmed death ligand-1 (PD-L1) monoclonal antibody therapy. Spermidine appeared to directly affect T cell function by increasing fatty acid oxidation. Tagged spermidine bound to components of the mitochondrial trifunctional protein complex, thus increasing fatty acid oxidation and the production of ATP. The authors propose that these effects may contribute to the effects of spermidine in promoting longevity. —LBR
Spermidine helps to restore antitumor immunity in older mice.
INTRODUCTION
In mammals, the power of the immune system decreases with age. This is because of multiple factors, including a decrease in the output and diversity of the antigenic repertoire of T cells caused by thymus involution; changes in the cellular metabolism caused by inflammation; and defective proliferative, differentiation, or survival capacities of the immune cells. Aged individuals frequently suffer from severe infections and cancers, and often the therapies applied, including programmed cell death protein 1 (PD-1) blockade in cancer immunotherapy, are ineffective when compared with results in young patients. A biogenic polyamine, spermidine (SPD), decreases with age, and SPD supplementation was shown to improve or delay several age-related pathologies, including those of the immune system. Among the proposed mechanisms responsible for rejuvenation of the immune system by SPD were enhanced autophagy, translational activity, and mitochondrial metabolism. SPD supplementation has previously been shown to enhance the antitumor immunity in animal models. However, it remains largely unknown how SPD deficiency relates to the T cell immune suppression induced by aging.
RATIONALE
Because CD8
+
T cells are key players in tumor immunity, we investigated how aging would affect the metabolic and functional characteristics of CD8
+
T cells. We asked whether SPD insufficiency could be a factor contributing to nonresponsiveness to PD-1 antibody therapy in aged mice. We sought to characterize the CD8
+
T cell population changes induced by SPD supplementation in aged mice and to identify the molecular mechanisms for the SPD action.
RESULTS
We found that the total and free intracellular concentrations of SPD in CD8
+
T cells from aged mice were about half as much as the concentrations found in young mice. Bioenergetically, aged CD8
+
T cells showed impaired mitochondrial activity with lower oxygen consumption rate, adenosine 5′-triphosphate (ATP) production, and fatty acid oxidation (FAO) activity compared with young CD8
+
T cells.
We show that SPD supplementation enhanced the antitumor activity of PD-1 blockade immunotherapy in aged mice. SPD supplementation proved to also be effective in young mice with tumors unresponsive to single anti–programmed death-ligand 1 (PD-L1) antibody therapy. SPD and anti–PD-L1 antibody combination treatment enhanced the proliferation, cytokine production, and mitochondrial ATP production of CD8
+
T cells in vivo. In vitro, SPD effectively enhanced mitochondrial functions and metabolized palmitate into tricarboxylic acid cycle components within 1 hour, which suggests the possibility of direct SPD binding to mitochondria-related proteins. Biochemical analysis identified SPD binding to mitochondrial trifunctional protein (MTP), which is the central enzyme of fatty acid β-oxidation. MTP is composed of α and β subunits, both of which bind SPD. Several assays using the MTP synthesized and purified from
Escherichia coli
revealed that SPD bound with strong affinity binding affinity (dissociation constant,
K
d
) = 0.1 μM and allosterically enhanced their enzymatic FAO activities. Furthermore, we found that spermine, another polyamine derived from SPD with important cellular protective functions, also directly binds to MTP and competitively inhibits FAO activity of SPD, which suggests the importance of SPD and spermine balance for FAO evaluation in aged cells. T cell–specific deletion of the MTP α subunit abolished enhancement of PD-1 blockade immunotherapy by SPD, indicating that MTP is required for SPD-dependent T cell activation.
CONCLUSION
SPD enhances FAO by directly binding and activating the MTP. SPD supplementation enhances the FAO activity and boosts the mitochondrial activities and cytotoxic functions of CD8
+
T cells. We provide new insights into the properties of SPD that may facilitate the development of strategies to prevent and improve outcomes of age-related immune pathologies and combat unresponsiveness to PD-1 blockade therapy in cancers, regardless of age.
SPD binds to MTP and activates FAO in T cells.
SPD directly activates MTP, which plays a central role in FAO. SPD concentration is decreased in aged T cells, leading to low FAO activity and ATP production compared with those in young T cells. SPD supplementation activates FAO in aged and young T cells, which enhances the efficacy of PD-1 blockade cancer immunotherapy. CoA, coenzyme A; TCA, tricarboxylic acid; ETC, electron transport chain. Figure created by Biorender
Purpose
To evaluate the association between central serous chorioretinopathy (CSC) susceptibility genes and choroidal parameters in a large Japanese cohort.
Study design
Retrospective cohort study.
...Methods
Of the 9850 individuals in the Nagahama study whose second visit was between 2013 and 2016, those with optical coherence tomography (OCT) images with enhanced depth imaging (EDI), axial length, and genome-wide single nucleotide polymorphism (SNP) genotyping data were included. We calculated subfoveal choroidal thickness (SFCT), choroidal vascularity index (CVI), normalized choroidal intensity (NCI), and vertical asymmetry of choroidal thickness. Genome-wide quantitative trait locus (QTL) analyses were performed for each parameter. We screened for four CSC susceptibility SNPs:
CFH
rs800292,
TNFRSF10A
rs13278062,
GATA5
rs6061548, and
VIPR2
rs3793217. Whenever an SNP was not included in the genotyping data after quality control, its proxy SNP was selected.
Results
In total, 4586 participants were evaluated.
CFH
rs800292 was significantly associated with SFCT (
P
< 0.001) and CVI (
P
< 0.001).
VIPR2
rs3793217 was significantly associated with SFCT (
P
< 0.001) but not with CVI. Whereas,
TNFRSF10A
rs13254617 and
GATA5
rs6061548 were not significantly associated with SFCT or CVI. None of these SNPs was associated with NCI
EDI
and asymmetry of choroidal thickness.
Conclusion
CFH
,
VIPR2
,
TNFRSF10A
, and
GATA5
showed different association patterns with choroidal parameters. Although the mechanism of CSC pathogenesis by choroidal changes is not fully understood, this finding suggests that each gene may be involved in different mechanisms of CSC development. Our genetic study provides a basis for understanding the role of CSC susceptibility genes.
Profiles of sequence variants that influence gene transcription are very important for understanding mechanisms that affect phenotypic variation and disease susceptibility. Using genotypes at 1.4 ...million SNPs and a comprehensive transcriptional profile of 15,454 coding genes and 6,113 lincRNA genes obtained from peripheral blood cells of 298 Japanese individuals, we mapped expression quantitative trait loci (eQTLs). We identified 3,804 cis-eQTLs (within 500 kb from target genes) and 165 trans-eQTLs (>500 kb away or on different chromosomes). Cis-eQTLs were often located in transcribed or adjacent regions of genes; among these regions, 5' untranslated regions and 5' flanking regions had the largest effects. Epigenetic evidence for regulatory potential accumulated in public databases explained the magnitude of the effects of our eQTLs. Cis-eQTLs were often located near the respective target genes, if not within genes. Large effect sizes were observed with eQTLs near target genes, and effect sizes were obviously attenuated as the eQTL distance from the gene increased. Using a very stringent significance threshold, we identified 165 large-effect trans-eQTLs. We used our eQTL map to assess 8,069 disease-associated SNPs identified in 1,436 genome-wide association studies (GWAS). We identified genes that might be truly causative, but GWAS might have failed to identify for 148 out of the GWAS-identified SNPs; for example, TUFM (P = 3.3E-48) was identified for inflammatory bowel disease (early onset); ZFP90 (P = 4.4E-34) for ulcerative colitis; and IDUA (P = 2.2E-11) for Parkinson's disease. We identified four genes (P<2.0E-14) that might be related to three diseases and two hematological traits; each expression is regulated by trans-eQTLs on a different chromosome than the gene.
Abstract
Circulating levels of inflammatory proteins have to be prognostic markers of all-cause mortality. α1-Antitrypsin (AAT) is a major inflammatory plasma protein, but its association with ...all-cause mortality is unclear. We aimed to evaluate the prognostic significance of AAT levels for all-cause mortality. Study participants comprised 9682 community residents (53.5 ± 13.3 years old). During the 9.8-year follow-up period, 313 participants died from any cause. The mortality rate increased linearly with AAT quintiles (Q1, 18.2; Q2, 24.7; Q3, 23.8; Q4, 31.9; Q5, 64.6 per 10,000 person-years). There were significant correlations between AAT and high-sensitivity C-reactive protein (hsCRP) levels (correlation coefficient, 0.331;
P
< 0.001). However, the Cox model analysis, when adjusted for possible covariates including hsCRP, identified the fifth AAT quintile as a risk factor for all-cause death (hazard ratio, 2.12 95% confidence interval, 1.41–3.18;
P
< 0.001). An analysis of participants older than 50 years (hazard ratio, 1.98,
P
< 0.001) yielded similar results. The hazard ratio increased proportionately in combination with high AAT and high hsCRP levels, and the highest hazard ratio reached 4.51 (95% confidence interval, 3.14–6.54,
P
< 0.001). High AAT levels were determined to be an independent risk factor for mortality in the general population.
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