Identification of the hybrid gene LILRB5-3 by long-read sequencing and implication of its novel signaling function.

Leukocyte immunoglobulin (Ig)-like receptors (LILRs) on human chromosome 19q13.4 encode 11 immunoglobulin superfamily receptors, exhibiting genetic diversity within and between human populations. Among the LILR genes, the genomic region surrounding LILRB3 and LILRA6 has yet to be fully characterized due to their significant sequence homology, which makes it difficult to differentiate between them. To examine the LILRB3 and LILRA6 genomic region, a tool named JoGo-LILR CN Caller, which can call copy number from short-read whole genome sequencing (srWGS) data, was applied to an extensive international srWGS dataset comprising 2,504 samples. During this process, a previously unreported loss of both LILRB3 and LILRA6 was detected in three samples. Using long-read sequencing of these samples, we have discovered a novel large deletion (33,692 bp) in the LILRB3 and LILRA6 genomic regions in the Japanese population. This deletion spanned three genes, LILRB3, LILRA6, and LILRB5, resulting in LILRB3 exons 12-13 being located immediately downstream of LILRB5 exons 1-12 with the loss of LILRA6, suggesting the potential expression of a hybrid gene between LILRB5 and LILRB3 (LILRB5-3). Transcription and subsequent translation of the LILRB5-3 hybrid gene were also verified. The hybrid junction was located within the intracellular domain, resulting in an LILRB5 extracellular domain fused to a partial LILRB3 intracellular domain with three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), suggesting that LILRB5-3 acquired a novel signaling function. Further application of the JoGo-LILR tool to srWGS samples suggested the presence of the LILRB5-3 hybrid gene in the CEU population. Our findings provide insight into the genetic and functional diversity of the LILR family.

Large-Scale Whole-Genome Analysis of HTLV-1-Associated Myelopathy Identified Hereditary Spastic Paraplegias.

Objectives: Distinguishing human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy from hereditary spastic paraplegia in patients infected with HTLV-1 is challenging due to overlapping clinical symptoms. The aim of this study was to explore the possibility that hereditary spastic paraplegia is inherently present in patients diagnosed with HTLV-1-associated myelopathy. Methods: We performed whole-genome sequencing on 315 unrelated patients registered in the HTLV-1-Associated Myelopathy patient registry "HAM-net," from 2013 to 2022 in Japan. CSF inflammatory biomarkers, including CXCL10, were measured. Results: We identified 5 patients with pathogenic variants in the genes RTN2, SPAST, VCP, and UBAP1, which are the known causes of hereditary spastic paraplegia. These patients had no family history of hereditary spastic paraplegia. The levels of CSF inflammatory biomarkers were lower than expected in these patients, compared with disease severity. Discussion: Genetic analysis is useful for the differentiation of hereditary spastic paraplegia patients from HTLV-1-associated myelopathy patients, especially for the patients with low levels of CSF inflammatory markers. Here we report the presence of hereditary spinal cord diseases in patients diagnosed with HTLV-1-associated myelopathy and provides evidence that genetic analysis would be helpful in the diagnostic workflow.

A novel NODAL variant in a young embolic stroke patient with visceral heterotaxy.

BACKGROUND: Ischemic stroke in young adults can be caused by a variety of etiologies including the monogenic disorders. Visceral heterotaxy is a condition caused by abnormal left-right determinations during embryonic development. We aimed to determine the cause of a young ischemic stroke patient with visceral heterotaxy. CASE PRESENTATION: We performed neurological, radiological, and genetic evaluations in a 17-year-old male patient presenting ischemic stroke and visceral heterotaxy to determine the underlying cause of this rare disease combination. Brain magnetic resonance imaging (MRI) showed evidence of embolic stroke, abdominal computed tomography (CT) showed visceral heterotaxy, and echocardiogram showed cardiac anomaly with right-to-left-shunt (RLS). Whole genome sequencing (WGS) revealed a heterozygous missense variant (NM_018055.5: c.1016 T > C, p.(Met339Val)) in the NODAL gene, which is essential to the determination of the left-right body axis. CONCLUSIONS: Our study highlights the importance of evaluating genetic etiology in young ischemic stroke and the need for stroke risk management in visceral heterotaxy patients with RLS. To the best of our knowledge, we report the first genetically-confirmed case of visceral heterotaxy with young embolic stroke reported to date.

Identification of region-specific gene isoforms in the human brain using long-read transcriptome sequencing.

In neurological and neuropsychiatric diseases, different brain regions are affected, and differences in gene expression patterns could potentially explain this mechanism. However, limited studies have precisely explored gene expression in different regions of the human brain. In this study, we performed long-read RNA sequencing on three different brain regions of the same individuals: the cerebellum, hypothalamus, and temporal cortex. Despite stringent filtering criteria excluding isoforms predicted to be artifacts, over half of the isoforms expressed in multiple samples across multiple regions were found to be unregistered in the GENCODE reference. We then especially focused on genes with different major isoforms in each brain region, even with similar overall expression levels, and identified that many of such genes including GAS7 might have distinct roles in dendritic spine and neuronal formation in each region. We also found that DNA methylation might, in part, drive different isoform expressions in different regions. These findings highlight the significance of analyzing isoforms expressed in disease-relevant sites.

Cost-Effective Cas9-Mediated Targeted Sequencing of Spinocerebellar Ataxia Repeat Expansions.

Hereditary repeat diseases are caused by an abnormal expansion of short tandem repeats in the genome. Among them, spinocerebellar ataxia (SCA) is a heterogeneous disease, and currently, 16 responsible repeats are known. Genetic diagnosis is obtained by analyzing the number of repeats through separate testing of each repeat. Although simultaneous detection of candidate repeats using current massively parallel sequencing technologies has been developed to avoid complicated multiple experiments, these methods are generally expensive. This study developed a cost-effective SCA repeat panel [Flongle SCA repeat panel sequencing (FLO-SCAp)] using Cas9-mediated targeted long-read sequencing and the smallest long-read sequencing apparatus, Flongle. This panel enabled the detection of repeat copy number changes, internal repeat sequences, and DNA methylation in seven patients with different repeat expansion diseases. The median (interquartile range) values of coverage and on-target rate were 39.5 (12 to 72) and 11.6% (7.5% to 16.5%), respectively. This approach was validated by comparing repeat copy number changes measured by FLO-SCAp and short-read whole-genome sequencing. A high correlation was observed between FLO-SCAp and short-read whole-genome sequencing when the repeat length was ≤250 bp (r = 0.98; P < 0.001). Thus, FLO-SCAp represents the most cost-effective method for conducting multiplex testing of repeats and can serve as the first-line diagnostic tool for SCA.

Exploring the genetic diversity of the Japanese population: Insights from a large-scale whole genome sequencing analysis.

The Japanese archipelago is a terminal location for human migration, and the contemporary Japanese people represent a unique population whose genomic diversity has been shaped by multiple migrations from Eurasia. We analyzed the genomic characteristics that define the genetic makeup of the modern Japanese population from a population genetics perspective from the genomic data of 9,287 samples obtained by high-coverage whole-genome sequencing (WGS) by the National Center Biobank Network. The dataset comprised populations from the Ryukyu Islands and other parts of the Japanese archipelago (Hondo). The Hondo population underwent two episodes of population decline during the Jomon period, corresponding to the Late Neolithic, and the Edo period, corresponding to the Early Modern era, while the Ryukyu population experienced a population decline during the shell midden period of the Late Neolithic in this region. Haplotype analysis suggested increased allele frequencies for genes related to alcohol and fatty acid metabolism, which were reported as loci that had experienced positive natural selection. Two genes related to alcohol metabolism were found to be 12,500 years out of phase with the time when they began to increase in the allele frequency; this finding indicates that the genomic diversity of Japanese people has been shaped by events closely related to agriculture and food production.

A case of hyperphosphatemic familial tumoral calcinosis due to maternal uniparental disomy of a GALNT3 variant.

Hyperphosphatemic familial tumoral calcinosis (HFTC) is a rare, inherited autosomal recessive disorder caused by fibroblast growth factor-23 (FGF23), N-acetylgalactosaminyltransferase 3 (GALNT3), or Klotho (KL) gene variants. Here, we report the case of a Japanese boy who presented with a mass in his left elbow at the age of three. Laboratory test results of the patient revealed normocalcemia (10.3 mg/dL) and hyperphosphatemia (8.7 mg/dL); however, despite hyperphosphatemia, serum intact FGF23 level was low, renal tubular reabsorption of phosphate (TRP) level was inappropriately increased, and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) level was inappropriately normal. Genetic analysis revealed maternal uniparental disomy (UPD) of chromosome 2, which included a novel GALNT3 variant (c.1780-1G>C). Reverse transcription-polymerase chain reaction (RT-PCR) analysis of GALNT3 mRNA confirmed that this variant resulted in the destruction of exon 11. We resected the mass when the patient was five years old, owing to its gradual enlargement. No relapse or new pathological lesions were observed four years after tumor resection. This is the first case report of a Japanese patient with HFTC associated with a novel GALNT3 variant, as well as the first case of HFTC caused by maternal UPD of chromosome 2 that includes the GALNT3 variant.

Genome-wide host-pathogen analyses reveal genetic interaction points in tuberculosis disease.

The genetics underlying tuberculosis (TB) pathophysiology are poorly understood. Human genome-wide association studies have failed so far to reveal reproducible susceptibility loci, attributed in part to the influence of the underlying Mycobacterium tuberculosis (Mtb) bacterial genotype on the outcome of the infection. Several studies have found associations of human genetic polymorphisms with Mtb phylo-lineages, but studies analysing genome-genome interactions are needed. By implementing a phylogenetic tree-based Mtb-to-human analysis for 714 TB patients from Thailand, we identify eight putative genetic interaction points (P < 5 × 10-8) including human loci DAP and RIMS3, both linked to the IFNγ cytokine and host immune system, as well as FSTL5, previously associated with susceptibility to TB. Many of the corresponding Mtb markers are lineage specific. The genome-to-genome analysis reveals a complex interactome picture, supports host-pathogen adaptation and co-evolution in TB, and has potential applications to large-scale studies across many TB endemic populations matched for host-pathogen genomic diversity.

Intraocular human cytomegaloviruses of ocular diseases are distinct from those of viremia and are capable of escaping from innate and adaptive immunity by exploiting HLA-E-mediated peripheral and central tolerance.

Human cytomegalovirus (HCMV) infections develop into CMV diseases that result in various forms of manifestations in local organs. CMV-retinitis is a form of CMV disease that develops in immunocompromised hosts with CMV-viremia after viruses in the peripheral circulation have entered the eye. In the HCMV genome, extensive diversification of the UL40 gene has produced peptide sequences that modulate NK cell effector functions when loaded onto HLA-E and are subsequently recognized by the NKG2A and NKG2C receptors. Notably, some HCMV strains carry UL40 genes that encode peptide sequences identical to the signal peptide sequences of specific HLA-A and HLA-C allotypes, which enables these CMV strains to escape HLA-E-restricted CD8+T cell responses. Variations in UL40 sequences have been studied mainly in the peripheral blood of CMV-viremia cases. In this study, we sought to investigate how ocular CMV disease develops from CMV infections. CMV gene sequences were compared between the intraocular fluids and peripheral blood of 77 clinical cases. UL40 signal peptide sequences were more diverse, and multiple sequences were typically present in CMV-viremia blood compared to intraocular fluid. Significantly stronger NK cell suppression was induced by UL40-derived peptides from intraocular HCMV compared to those identified only in peripheral blood. HCMV present in intraocular fluids were limited to those carrying a UL40 peptide sequence corresponding to the leader peptide sequence of the host's HLA class I, while UL40-derived peptides from HCMV found only in the peripheral blood were disparate from any HLA class I allotype. Overall, our analyses of CMV-retinitis inferred that specific HCMV strains with UL40 signal sequences matching the host's HLA signal peptide sequences were those that crossed the blood-ocular barrier to enter the intraocular space. UL40 peptide repertoires were the same in the intraocular fluids of all ocular CMV diseases, regardless of host immune status, implying that virus type is likely to be a common determinant in ocular CMV disease development. We thus propose a mechanism for ocular CMV disease development, in which particular HCMV types in the blood exploit peripheral and central HLA-E-mediated tolerance mechanisms and, thus, escape the antivirus responses of both innate and adaptive immunity.

National Center Biobank Network.

There are six national centers (6NCs) for advanced and specialized medicine in Japan that conduct basic and clinical research on major diseases that have a substantial impact on national health. Disease-specific bioresources and information collected by each NC are stored in a separate biobank. The National Center Biobank Network (NCBN) was established in 2011 and coordinates the biobanks and researchers of the 6NCs via an open-access database (Catalogue Database: http://www2.ncbiobank.org/Index_en ) as an efficient means of providing registered biological resources and data for use in research communities. The NCBN resources are characterized by their high-quality and rich medical information and are available for life science research and for the development of novel testing methodologies (biomarkers), new treatments, and drugs for future health care in the scope of personalized medicine through a deeper understanding of disease pathogenesis. Here, we explain the activities of the NCBN and the characteristics of the NCBN Catalogue Database.

Detection of the HLA-B*15:01:74 allele, an HLA-B*15 variant discovered in a Japanese individual.

HLA-B*15:01:74 differs from B*15:01:01:01 by one nucleotide in exon 2 at position 470.

Detection of the novel HLA-C*06:02:01:62 allele by next-generation sequencing in a Japanese individual.

HLA-C*06:02:01:62 differs from HLA-C*06:02:01:01 by one nucleotide in intron 3 at position 1413.

Genomic sequencing of the novel HLA-C*07:02:01:107 allele by next-generation sequencing in two Japanese individuals.

HLA-C*07:02:01:107 differs from HLA-C*07:02:01:01 by two nucleotides in 3' UTR at position 3789 and 3806.

Identification of the novel HLA-B*40:01:02:40 and HLA-B*40:01:02:41 alleles in a Japanese individual.

HLA-B*40:01:02:40 and -B*40:01:02:41 differ from B*40:01:02:04 by one and two nucleotides in the 3'UTR, respectively.

The HLA-B*15:02:01:05 allele identified by two next-generation sequencing methods in a Japanese individual.

HLA-B*15:02:01:05 differs from HLA-B*15:02:01:01 by four nucleotides in 3'UTR at positions 3435, 3457, 3472, and 3511.

Discovery of a novel HLA-B*40 allele, HLA-B*40:02:01:30 in a Japanese individual.

HLA-B*40:02:01:30 differs from HLA-B*40:02:01:01 by one nucleotide in 3'UTR at position 2869.

Full genomic sequence of the novel HLA-C*14:132 allele identified using next-generation sequencing.

HLA-C*14:132 differs from HLA-C*14:02:01:01 by one nucleotide in exon 3 at positions 829.

Full-length sequence of a novel HLA-C*03:03:01 allele, HLA-C*03:03:01:54 identified in a Japanese individual.

HLA-C*03:03:01:54 differs from HLA-C*03:03:01:01 by one nucleotide in intron 2 at position 531.

HLA-B*13:01:01:09, a variant of HLA-B*13:01:01:01, detected in a Japanese individual.

HLA-B*13:01:01:09 differs from HLA-B*13:01:01:01 by one nucleotide in intron 4 at positions 1913.

An Association Study of HLA with the Kinetics of SARS-CoV-2 Spike Specific IgG Antibody Responses to BNT162b2 mRNA Vaccine.

BNT162b2, an mRNA-based SARS-CoV-2 vaccine (Pfizer-BioNTech, New York, NY, USA), is one of the most effective COVID-19 vaccines and has been approved by more than 130 countries worldwide. However, several studies have reported that the COVID-19 vaccine shows high interpersonal variability in terms of humoral and cellular responses, such as those with respect to SARS-CoV-2 spike protein immunoglobulin (Ig)G, IgA, IgM, neutralizing antibodies, and CD4+ and CD8+ T cells. The objective of this study is to investigate the kinetic changes in anti-SARS-CoV-2 spike IgG (IgG-S) profiles and adverse reactions and their associations with HLA profiles (HLA-A, -C, -B, -DRB1, -DQA1, -DQB1, -DPA1 and -DPB1) among 100 hospital workers from the Center Hospital of the National Center for Global Health and Medicine (NCGM), Tokyo, Japan. DQA1*03:03:01 (p = 0.017; Odd ratio (OR) 2.80, 95%confidence interval (CI) 1.05-7.25) was significantly associated with higher IgG-S production after two doses of BNT162b2, while DQB1*06:01:01:01 (p = 0.028, OR 0.27, 95%CI 0.05-0.94) was significantly associated with IgG-S declines after two doses of BNT162b2. No HLA alleles were significantly associated with either local symptoms or fever. However, C*12:02:02 (p = 0.058; OR 0.42, 95%CI 0.15-1.16), B*52:01:01 (p = 0.031; OR 0.38, 95%CI 0.14-1.03), DQA1*03:02:01 (p = 0.028; OR 0.39, 95%CI 0.15-1.00) and DPB1*02:01:02 (p = 0.024; OR 0.45, 95%CI 0.21-0.97) appeared significantly associated with protection against systemic symptoms after two doses of BNT162b2 vaccination. Further studies with larger sample sizes are clearly warranted to determine HLA allele associations with the production and long-term sustainability of IgG-S after COVID-19 vaccination.