Alleles In Japanese Patients Research Articles

HLA Class I Genotypes Predict the Survival after Hematopoietic Stem Cell Transplantation in Immune Aplastic Anemia

Introduction Immune aplastic anemia (AA) is a bone marrow failure syndrome mediated by cytotoxic T cells, leading to the loss of HLA class I allele expression in hematopoietic stem and progenitor cells. In unrelated hematopoietic stem cell transplantation (HSCT), we recently reported an association between HLA loss and a short time to transplantation (TTT), consistent with an abrupt onset of immune AA. In addition, patients who lost HLA-A*02:06 or HLA-B*40:02 showed better survival outcomes after HSCT than those with other HLA allele losses. To clarify the clinical significance of the loss of specific HLA class I alleles in AA, we correlated loss-prone HLA class I allele genotypes with HSCT outcomes in patients with a short TTT, assuming the frequent absence of these alleles in this subgroup. Methods We conducted a nationwide retrospective analysis of patients with acquired AA ≥16 years old who underwent their first allogeneic HSCT in Japan between 2006 and 2020. Patient data were obtained from the Transplant Registry Unified Management Program (TRUMP ®), a comprehensive Japanese HSCT registry. Results From 1025 patients with acquired AA, we excluded patients with drug-induced AA and those missing 2-field HLA allele information and TTT information, leaving 874 patients for the analysis. The median patient age at HSCT was 34 years old, and the median TTT was 14 months. When focusing on the most frequently absent HLA class I alleles in Japanese patients with AA, including HLA-B*40:02, HLA-A*02:06, HLA-A*02:01, HLA-B*54:01, and HLA-A*31:01, all 5 alleles were associated with higher survival rates than in 220 patients without these alleles (Figure 1A). As expected, this association was more pronounced with a shorter TTT. For instance, among the 424 patients with a TTT <14 months, the 5-year survival rates were 79%, 78%, 85%, 78%, and 81% in patient groups with the respective HLA class I alleles above, while it was 55% in the other 101 patients (Figure 1B). Of note, having any of the 5 alleles (5-allele +) proved to be the best predictor of a survival out of 242792 combinations of 2 to 5 HLA-A, HLA-B, and HLA-DRB1 alleles. To adjust for potential confounders, such as age and graft sources, we performed propensity score matching and confirmed that the overall survival was significantly lower in patients without any of the 5 alleles (5-allele -) than in 5-allele + patients ( P = .00018). A multivariable Cox regression analysis including age, AA severity, prior IST, graft sources, HLA disparity, and HLA-B leader peptide dimorphism as covariates further demonstrated that the 5-allele genotype was an independent predictor of the survival. The consistent effect of the 5 alleles on the survival has been internally validated across various subgroups. In contrast, the 5 HLA class I alleles did not affect the survival in patients with hematological malignancies, including acute myeloid leukemia, acute lymphoblastic leukemia, and myelodysplastic syndrome. Regarding other post-HSCT complications, 5-allele - AA patients had a higher incidence of primary graft failure (GF) than 5-allele + AA patients, even when deaths before engraftment were censored and when compared to the propensity score-matched patients. Nevertheless, the 5 HLA alleles continued to stratify the survival among patients who achieved engraftment ( P < .0001). There were no significant differences in the incidence of acute and chronic graft-versus-host disease, late GF, or cause of death between 5-allele -and 5-allele + AA patients. To further stratify the survival rate in 5-allele - AA patients with a short TTT, we examined the impact of other HLA class I alleles and found that patients carrying HLA-B*07:02 or HLA-B*48:01, in which HLA loss had been previously detected in some AA patients, showed a particularly poor survival (Figure 1C; P = .0082). This association remained significant even after propensity score matching ( P = .031). Conclusions Significant associations between specific loss-prone HLA class I alleles and diverse survival outcomes after HSCT in AA patients with short TTT indicate the relevance of HLA information in patient selection for allogeneic HSCT. These findings also support the existence of a distinct immune pathogenesis affecting the clinical manifestations of AA differently, as observed in IST-treated patients. Further investigations in other ethnic groups are required to validate these observations.

HLA Class I Allele-Specific Pathology Defines Clinical Manifestations of Immune Aplastic Anemia

Leukocytes that have lost the cell surface expression of an HLA class I allele due to somatic HLA gene mutations or 6p loss of heterozygosity (LOH) are often present in patients with immune aplastic anemia (AA), consistent with T cell-mediated bone marrow cell destruction. A recent NIH study suggested that the clinical significance and underlying pathology of HLA loss may differ according to the type of HLA allele affected (Zaimoku et al. Blood 2021) because (1) the significant correlation of HLA loss with clonal evolution was primarily attributed to patients who lost HLA-B*14:02 and A*02:01; (2) all patients who lost B*40:02 and A*02:06 achieved a hematologic response to immunosuppressive therapy (IST), though HLA loss in general was not correlated with the IST response; (3) loss of B*07:02 and B*40:01 was associated with older age of onset; (4) patients who retained B*14:02, B*07:02, and B*40:01 also showed clinical features of patients who lost respective HLA alleles. We aimed to confirm the HLA class I allele-related clinical manifestations in Japanese AA patients. HLA loss was assessed by SNP array, HLA flow cytometry, digital PCR, or deep nucleotide sequencing. An HLA allele was classified as "affected by loss" in individual patients when the single allele was lost or inactivated by somatic mutations or when both HLA-A and HLA-B alleles in a haplotype were lost due to 6p LOH with no evidence of loss or inactivation of a single HLA allele. Clinical characteristics were compared according to the affected HLA alleles. In 496 Japanese patients with AA (severe, n = 368; non-severe, n = 128; median age, 59 [range, 1-89] years), there were 146 (29%) patients with HLA loss. HLA-B*40:02, A*02:06, A*31:01, and B*54:01 were more frequently affected in our cohort than in the NIH cohort, where B*14:02, A*02:01, B*08:01, and B*07:02 held the majority of lost alleles (Figure 1). The median age of patients with B*40:01 loss (75 years) was highest among the groups of patients with HLA allele loss, consistent with the NIH study. The prevalence of ≥0.003% paroxysmal nocturnal hemoglobinuria (PNH) clones was significantly reduced in patients with B*40:02 loss but not in others. Sex and severity of AA were not markedly different across the groups of patients with HLA allele loss. Outcomes after anti-thymocyte globulin (ATG)-based IST were evaluable in 263 of the 496 patients. Although HLA loss was correlated with the IST response at 6 months (as reported by our group), loss of HLA-A*02:01 did not contribute to the high response, consistent with the NIH study. Loss of frequently affected HLA alleles in Japanese patients (B*40:02, A*02:06, B*54:01, etc.) was generally correlated with the IST-response. Further, patients who carried 5 HLA-B alleles (B*40:01, B*40:02, B*44:03, B*54:01, and B*56:01) without loss still displayed higher response rates than other patients. In contrast, the favorable effect of A*02:06 loss was not seen in patients who carried A*02:06 without loss. A*02:06 loss, the presence of the 5 HLA-B alleles (irrespective of loss), and previously reported HLA class II alleles (DRB1*13 and DRB1*15) were independently correlated with the IST response. The incidence of clonal evolution to myeloid neoplasms after ATG-based IST was significantly higher in patients with HLA-A*02:01 loss than other patients (Figure 2), as observed in the NIH study; A*02:01 was affected in 4 of 7 patients showing both HLA loss and clonal evolution in our cohort; B*40:02, B*44:02, and B*52:01 were affected in other 3 patients. In summary, our study in Japanese AA patients broadly confirmed the associations of loss of specific HLA alleles with clinical manifestations observed in American patients, including older age of onset in patients with HLA-B*40:01 loss, high IST response rates in patients who lost B*40:02 and A*02:06 but not in patients with A*02:01 loss, and frequent clonal evolution in patients with A*02:01 loss. We also revealed the correlation of 5 HLA-B alleles with IST response, irrespective of loss, similar to the relationship of B*14:02 to clonal evolution in the United States, and the reduced prevalence of PNH clones in patients with B*40:02 loss. These findings strongly suggest that the heterogeneous pathology of AA can be classified by affected HLA alleles. This should be useful for studies to unravel the pathophysiology and improve the outcomes of patients with immune AA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Frequency of FMR1 Premutation Alleles in Patients with Undiagnosed Cerebellar Ataxia and Multiple System Atrophy in the Japanese Population.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder caused by FMR1 premutation expansion of CGG repeats. FXTAS can be misdiagnosed with many neurodegenerative disorders manifesting with cerebellar ataxias owing to their overlapping clinical and radiological features. The frequency of the FMR1 premutation allele in Japan has not been fully determined. Herein, we aimed to determine the frequency of FMR1 premutation alleles in Japanese patients with undiagnosed cerebellar ataxia and multiple system atrophy, using repeat-primed PCR in 186 patients with adult onset of undiagnosed cerebellar ataxia and 668 patients with multiple system atrophy, to identify expanded CGG repeats as well as to detect AGG interruptions within the expanded alleles. The size of expansions was estimated using fragment length analysis of PCR products obtained by conventional PCR employing a pair of unique primers flanking the repeat sequence. We identified FMR1 premutation alleles in three male patients. One patient revealed 84 repeat units with one AGG interruption and another patient showed 103 repeat units. Both had presented with sporadic cerebellar ataxia, giving an estimated frequency of 3.7% among Japanese male patients with sporadic cerebellar ataxia with age at onset above 50years. One patient with the clinical diagnosis of multiple system atrophy harbored 60 repeat units with four AGG interruptions. FMR1 intermediate alleles were observed in two males and one female among the multiple system atrophy patients. We found that genetic tests for FMR1 premutation should be considered in Japanese male patients with cerebellar ataxia with the age at onset above 50years.

HLA-DQB1 DPB1 alleles in Japanese patients with adult-onset Still’s disease

Objective: HLA class II alleles are major determinants of genetic predisposition to rheumatic diseases. Predisposing effects of HLA had been suggested in AOSD, however, ethnic differences may account for variations in AOSD association with HLA. We determined the contribution of HLA-DQB1, DPB1 alleles to susceptibility to Adult-onset Still’s disease (AOSD) in the Japanese population.Methods: HLA-DQB1 and DPB1 alleles were analyzed in 87 Japanese patients with AOSD and 413 Japanese healthy subjects.Results: We found significant association between HLA-DQB1*06:02 (Pc = 0.010, odds ratio: 2.54) and AOSD, whereas there was no association between the DQB1*06:02 allele and disease phenotypes of AOSD. Moreover, we did not find a predisposing effect of the HLA-DPB1 allele to AOSD. Haplotype analysis showed that presence of DRB1*15:01–DQB1*06:02 was associated with Japanese patients with AOSD. However, conditional logistic regression tests were unable to demonstrate independent association between DRB1*1501 or DQB1*0602 and AOSD.Conclusions: Our results show significant association between AOSD and the HLA DQB1*06:02 allele, and between the DRB1*1501–DQB1*06:02 haplotype and AOSD susceptibility. These findings suggest that genetic susceptibility to AOSD depends on the genotype combinations of HLA DRB1 and DQB1 alleles.

HLA-DQB1*03:01 as a Biomarker for Genetic Susceptibility to Bullous Pemphigoid Induced by DPP-4 Inhibitors

HLA-DQB1*03:01 as a Biomarker for Genetic Susceptibility to Bullous Pemphigoid Induced by DPP-4 Inhibitors

Clinical and molecular investigation of 14 Japanese patients with complete TFP deficiency: a comparison with Caucasian cases

Mitochondrial trifunctional protein (TFP) deficiency is an inherited metabolic disorder of mitochondrial fatty-acid oxidation. Isolated long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency is often reported in Caucasian countries due to a common mutation. However, the molecular and clinical basis of complete TFP deficiency has not been extensively reported. In this study, 14 Japanese cases (13 families) with complete TFP deficiency, including 9 previously reported cases, were analyzed to clarify the clinical and molecular characteristics of TFP deficiency. The clinical types of the 14 patients were as follows: 12 cases of neonatal (n=7) or myopathic (n=5) types and 2 cases of intermediate type. Peripheral neuropathy was found in four cases and hypocalcemia due to hypoparathyroidism, which is rarely reported in Caucasian patients, had developed in four cases. Maternal hemolysis, elevated liver enzymes and low platelet count syndrome and acute fatty liver of pregnancy were noted in two and one mothers, respectively. Fourteen mutations were identified in 26 alleles in Japanese patients, including two novel mutations (HADHA: c.361C>T, and HADHA-HADHB: g.26233880_ 26248855del), although no common mutations were found. This study suggests that the molecular and clinical aspects of Japanese patients with TFP deficiencies differ from those of Caucasian patients.

The Severity of Takayasu Arteritis Is Associated with the HLA-B52 Allele in Japanese Patients

Takayasu arteritis (TA) is a type of vasculitis that affects the large elastic arteries, specifically the aorta and its main branches. It has been reported that TA occurred most frequently in Nagasaki Prefecture, the western area in Japan. We retrospectively collected the information of 34 patients with TA, diagnosed using the American College of Rheumatology 1990 criteria for the classification of TA, from the medical records of Nagasaki University Hospital from 2003 to 2015, and we investigated the clinical characteristics of these TA patients. Among the 35 patients, 25 patients were examined for the existence of the HLA-B52 allele that has been reported to influence TA susceptibility. Seventeen patients (68.0%) of the 25 patients were HLA-B52-allele-positive, which was defined as the state of having at least one HLA-B52 allele. There was a significant difference in the rate of smokers: HLA-B52-allele-positive: six patients (35.3%) vs. HLA-B52-allele-negative: 0 (0.0%). The C-reactive protein level in the HLA-B52-positive patients (9.0 ± 6.4 mg/dL) was significantly higher than that in the HLA-B52-negative patients (3.2 ± 3.9 mg/dL). All HLA-B52-allele-positive patients were found to be active according to Kerr's criteria. The HLA-B52-positive patients' initial prednisolone dosage (37.7 ± 8.6 mg/day) was significantly higher than that of the HLA-B52-allele-negative patients (23.1 ± 13.1 mg/day). Thus, the HLA-B52 allele is associated with the disease activity and the steroid requirements of TA patients. Furthermore, our present findings have revealed for the first time that the HLA-B52 allele and smoking might be associated with the onset of TA.

Association between HLA-DRB1*0405, -DQB1*0401 and -DQA1*0303 alleles and lamotrigine-induced cutaneous adverse drug reactions. A pilot case-control study from Japan

BackgroundHuman leukocyte antigen (HLA) genotypes in lamotrigine -induced (LTG-induced) cutaneous adverse drug reactions (cADRs) have been described in several reports but controversy remains even for a given ethnic group. We attempted to clarify a possible association between LTG-induced cADRs and HLA alleles in Japanese patients. MethodSixteen subjects, including eight patients with LTG-induced cADRs and eight LTG-tolerant controls were included in this study. All eight patients with LTG-induced cADRs gave positive results in a drug-induced lymphocyte stimulation test (DLST) with LTG. We performed HLA-typing for HLA-A, -B, -C, -DRB1, -DQA1, -DQB1, -DPA1 and -DPB1, using PCR with sequence-specific oligonucleotide probes and multiple analyte profiling (xMAP) technology (Luminex System; Luminex Corporation, Austin, TX).We examined differences between allele frequencies in our two groups of subjects and the allele frequencies in the general Japanese population. ResultsThe frequencies of HLA-DRB1*0405, and HLA-DQB1*0401 alleles were higher in our LTG-cADRs patients than the reference frequencies in the general Japanese population. We also detected HLA-DQA1*0303 frequently in our LTG-cADRs patients, but data for this allele in the Japanese population was not available. Our observation was presumably due to the linkage disequilibrium among the three alleles. The haplotype frequency of HLA-DRB1*0405, DQB1*0401 and DQA1*0303 in our LTG-cADRs subjects was also different from the corresponding haplotype frequency in the database for the Japanese population and the difference was statistically significant. One patient with the HLA-DRB1*0405, -DQB1*0401 and DQA1*0303 haplotype was safely re-treated with LTG after results of a DLST with LTG ceased to be positive about 4 months after discontinuation of LTG. LimitationsOur analysis included only 16 patients. Associations between LTG-induced cADRs and specific HLA loci will have to be confirmed in larger studies. ConclusionsLTG-induced cADRs are associated with HLA-DRB1*0405, -DQB1*0401 and -DQA1*0303.

Associations of HLA class I alleles in Japanese patients with Crohn's disease.

Previous studies have suggested that the human leukocyte antigen (HLA) is involved in the etiology of Crohn's disease (CD); however, few reports are available on the association between HLA class I antigens and CD in Japan. In this study, we performed association analysis of HLA class I antigens in CD using 208 Japanese patients and 384 healthy controls. We identified novel positive associations between CD and HLA-A*02:01 (odds ratio (OR)=1.64, P=0.016) and HLA-A*02:07 (OR=2.31, P=0.0067) and confirmed previously reported positive associations between CD and HLA-Cw*14:02 (OR=2.18, P=0.0021) and HLA-B*51:01 (OR=1.70, P=0.033). We also identified novel negative associations between CD and HLA-A*24:02 (OR=0.60, P=0.0047) and HLA-B*07:02 (OR=0.38, P=0.0041). Although the associations were not significant after full Bonferroni correction, we suggested that HLA class I genes have dual functions, susceptibility and resistance in controlling the development of CD.

Association between severe toxicity of nilotinib and UGT1A1 polymorphisms in Japanese patients with chronic myelogenous leukemia

Nilotinib is a BCR-ABL kinase inhibitor approved for the treatment of Philadelphia chromosome-positive chronic myelogenous leukemia (CML). The UDP-glucuronosyltransferase 1A1 (UGT1A1) polymorphism UGT1A1*28 (*28)/*28 has been linked to an increased risk of hyperbilirubinemia in patients with CML who receive nilotinib. Beside *28, UGT1A1*6 (*6) is another important variant allele in Japanese patients because it is associated with adverse events of irinotecan, metabolized by UGT1A1. We retrospectively investigated the association between severe toxicity of nilotinib and UGT1A1 polymorphisms (*6 and*28) in Japanese patients with CML. Eight patients with cytogenetically confirmed CML who were receiving nilotinib were studied to explore the association of UGT1A1 polymorphisms with severe nilotinib-related toxicity. Genotyping analyses were determined for *6 and *28. All 3 patients with the *6/*6 or *6/*28 genotype had severe toxicity, including QT interval prolongation (grade 3), elevated lipase levels (grade 3) plus hyperbilirubinemia (grade 2), and anemia (grade 3) plus hepatic cyst hemorrhage (grade 2) in 1 patient each. Among the 5 patients with the *6/*1 or *1/*1 genotype, 1 had elevated lipase levels (grade 3) and another had severe pain in the lower extremities (grade 3). These findings suggest that UGT1A1 polymorphisms are important determinants of severe toxicity of nilotinib in Japanese patients.

Simple and rapid genetic testing for citrin deficiency by screening 11 prevalent mutations in SLC25A13

Citrin deficiency is an autosomal recessive disorder caused by mutations in the SLC25A13 gene and has two disease outcomes: adult-onset type II citrullinemia and neonatal intrahepatic cholestasis caused by citrin deficiency. The clinical appearance of these diseases is variable, ranging from almost no symptoms to coma, brain edema, and severe liver failure. Genetic testing for SLC25A13 mutations is essential for the diagnosis of citrin deficiency because chemical diagnoses are prohibitively difficult. Eleven SLC25A13 mutations account for 95% of the mutant alleles in Japanese patients with citrin deficiency. Therefore, a simple test for these mutations is desirable. We established a 1-hour, closed-tube assay for the 11 SLC25A13 mutations using real-time PCR. Each mutation site was amplified by PCR followed by a melting-curve analysis with adjacent hybridization probes (HybProbe, Roche). The 11 prevalent mutations were detected in seven PCR reactions. Six reactions were used to detect a single mutation each, and one reaction was used to detect five mutations that are clustered in a 21-bp region in exon 17. To test the reliability, we used this method to genotype blind DNA samples from 50 patients with citrin deficiency. Our results were in complete agreement those obtained using previously established methods. Furthermore, the mutations could be detected without difficulty using dried blood samples collected on filter paper. Therefore, this assay could be used for newborn screening and for facilitating the genetic diagnosis of citrin deficiency, especially in East Asian populations.

New Human Leukocyte Antigen Risk Allele in Japanese Patients With Takayasu Arteritis

The association of human leukocyte antigen (HLA) alleles and Takayasu arteritis (TA) is not fully understood. The aim of the present study was to investigate HLA alleles in Japanese patients with TA and the association of these alleles with clinical manifestations. A total of 96 patients diagnosed with TA according to the Guideline for Management of Vasculitis Syndrome (Japanese Circulation Society 2008) and 371 healthy controls were enrolled in the present study. HLA genotyping showed a significant association of HLA-B67 (P=0.00024, odds ratio [OR]=4.94), a novel locus, and B52 (P<0.0001; OR=3.35), a conventional locus, with TA using both sequence-based typing and PCR-SSP assay. The frequency of HLA-B39, an allele reportedly associated with TA in Asian populations, was not higher than controls in the present study (P=0.86, OR=1.07). B52 had higher prevalence than B67 but the OR was higher for B67. We next studied the association of HLA-B67 and -B52 with clinical characteristics: age at disease onset, distribution of arteritis, pulmonary involvement, aortic regurgitation, systemic hypertension, steroid resistance and recurrence rate in TA. There was no significant difference in these clinical parameters between HLA-B67-positive or HLA-B52-positive patients and other patients. The HLA-B67 allele could be a new and important marker of TA because of its high OR compared to HLA-B52, although its prevalence in TA is lower.

Peptides Presented by HLA-DR Molecules in Synovia of Patients with Rheumatoid Arthritis or Antibiotic-Refractory Lyme Arthritis

Disease-associated HLA-DR molecules, which may present autoantigens, constitute the greatest genetic risk factor for rheumatoid arthritis (RA) and antibiotic-refractory Lyme arthritis (LA). The peptides presented by HLA-DR molecules in synovia have not previously been defined. Using tandem mass spectrometry, rigorous database searches, and manual spectral interpretation, we identified 1,427 HLA-DR-presented peptides (220-464 per patient) from the synovia of four patients, two diagnosed with RA and two diagnosed with LA. The peptides were derived from 166 source proteins, including a wide range of intracellular and plasma proteins. A few epitopes were found only in RA or LA patients. However, two patients with different diseases who had the same HLA allele had the largest number of epitopes in common. In one RA patient, peptides were identified as originating from source proteins that have been reported to undergo citrullination under other circumstances, yet neither this post-translational modification nor anti-cyclic citrullinated peptide antibodies were detected. Instead, peptides with the post-translational modification of S-cysteinylation were identified. We conclude that a wide range of proteins enter the HLA-DR pathway of antigen-presenting cells in the patients' synovial tissue, and their HLA-DR genotype, not the disease type, appears to be the primary determinant of their HLA-DR-peptide repertoire. New insights into the naturally presented HLA-DR epitope repertoire in target tissues may allow the identification of pathogenic T cell epitopes, and this could lead to innovative therapeutic interventions.

Identification and functional characterization of novel telomerase variant alleles in Japanese patients with bone-marrow failure syndromes

As the incidence of bone-marrow failure syndromes (BMFS) is 2–3× higher in East Asia than in the West, we examined peripheral blood or marrow cells of 100 Japanese patients for possible pathogenic mutations in the two main components of the telomere-synthesizing enzyme telomerase (hTERC RNA and hTERT protein) that have recently been implicated in the disease pathogenesis. We analyzed samples collected from 34 patients with acquired aplastic anemia (AA), 66 patients with myelodysplastic syndromes (MDS) and 120 healthy controls. In addition to two polymorphic germ-line sequence changes (n-771A/G and n-714 C insertion) in the promoter region of hTERC and eleven hTERT polymorphisms that were identified in both patients and healthy individuals, we found a novel germ-line C323T mutation in the hTERC RNA in an MDS patient only. This heterozygous C323T mutation abolished telomerase enzymatic activity and functioned in a haploinsufficiency manner to modulate telomerase activity in cells. In summary, this study reports a novel telomerase natural variant that abolishes telomerase function, which may lead to telomere shortening and marrow hypocellularity in patients with BMFS. This study also highlights the rarity of genetic alterations in BMFS patients in Japan, which suggests that other factors may play a more prominent role in the disease pathogenesis in East Asia.

Identification and Functional Characterization of Telomerase RNA Variant Alleles in Japanese Patients with Aplastic Anemia or Myelodysplastic Syndrome.

Dyskeratosis Congenita (DKC) is an inherited bone marrow failure (BMF) characterized by abnormal skin pigmentation, nail dystrophy and mucosa leucoplakia. Mutations in the DKC1 and TERC genes occur in the X-linked and autosomal dominant DKC, respectively. Several groups have recently reported that mutations of the telomerase TERC RNA and of the telomerase catalytic TERT protein caused short telomere and developed DKC and other BMF syndromes, such as aplastic anemia (AA) and myelodysplasia (MDS). As the incidence for BMF seems to be higher in East Asia than in the West, we investigated whether mutations in TERC and TERT genes are indeed associated with BMF patients in Japan.We examined blood or marrow cells from 73 BMF patients; 27 samples collected from patients with acquired AA (n=27) who did not respond to immunosuppressive therapy, and 46 samples from patients with MDS (RA). We also analyzed blood samples from 120 healthy donors as controls. Polymerase-chain-reaction (PCR) amplification of TERC and TERT genes was performed with DNA samples extracted from peripheral blood or bone marrow cells. PCR products were purified and direct sequencing was performed. Altered sequences were transfected into telomerase-negative cell line in order to address the effect of the mutations on telomerase enzymatic function. We found two polymorphic sequence changes (n-717A/G and n-713 deletion) in the promoter region of the hTERC gene and nine TERT polymorphic sequences in the hTERT gene. These hTERT sequence polymorphisms, which were also identified in healthy individuals, did not introduce an amino acid change in the corresponding protein. To our knowledge, the two TERC polymorphisms and three of the nine TERT polymorphisms (IVS6 −93 G/A, codon837 CTC/CTG and codon 840 CTG/CTA) have not been reported elsewhere. Among the 73 BMF patients, 1(1.4%) heterozygous TERC mutation (C323T) was identified in an MDS patient. This mutation was not observed in normal controls. The C323T mutation is located on the P5 stem of the predicted hTERC RNA secondary structure, which suggests that the mutation may disrupt basepairing interactions of this stem. We introduced this C233T mutation, as well as additional sequence changes to test for the effect of the mutation in the formation of the P5 stem, into VA13+hTERT cells. We found that the mutations located on the individual strands of the P5 stem (either the natural C323T(rt) mutation or the corresponding C323T(lt) mutation located at position 246 alone) abolished telomerase activity to about the same degree. Interestingly, the compensatory mutation (C323T(comp)), in which the natural C323T mutation was accompanied by complementary mutation on the opposite strand of the P5 stem, did not restore telomerase activity, suggesting that both the primary and secondary structure of the P5 stem are important to confer optimal telomerase enzymatic activity. Moreover, no effects were observed between cells that carried only the wild type TERC vector and those that carried both the wild type and the individual mutated TERC copy. These results suggest that the natural hTERC variant functions in a haploinsufficient manner to modulate wild type telomerase function.

The Clinical Significance of Some Specific HLA Class I Alleles in Japanese Patients with Bone Marrow Failure (BMF).

Autoreactive T lymphocytes are implicated in the immune mechanisms involved in the bone marrow failure (BMF) syndrome, including aplastic anemia (AA), paroxysmal nocturnal hemoglobinuria (PNH), and myelodysplastic syndrome (MDS). However, the significance of the HLA class I alleles remains unknown in the BMF syndrome. Nevertheless, from many clinical and basic studies, it is certain that CD8+ T lymphocytes are implicated in some of the immune mechanisms involved in the occurrence of AA. To clarify some clinical significance of the HLA class I alleles in the BMF syndrome, we investigated the alleles using a high-resolution method of genotyping in 78 Japanese patients with BMF, including 32 AA, 24 PNH, and 22 MDS patients. Subsequently, we compared various clinical findings, including age, sex, white blood cell counts, absolute neutrophil counts, hemoglobin concentrations, reticulocyte counts, platelet counts, values of lactate dehydrogenase, durations of illness, chromosomal findings, and proportions of CD55− and CD59− erythrocytes, between the groups with and without some alleles. The diagnosis and grading of the severity of AA were based on the criteria of the International Agranulocytosis and Aplastic Anemia Study Group (Blood 1987; 70: 1718–21) and that of Frickhofen et al (N Engl J Med 1991; 324: 1297–304), respectively. A patient with a CD55− and CD59− population of more than 1% was judged to have PNH erythrocytes (Blood 1996; 87: 5332–40). The diagnosis of MDS was determined according to the FAB criteria (Br J Haematol 1982; 51: 189–99). The frequencies of the HLA-B* 4002 allele in AA patients (21.9%) and of the HLA-A* 0206 allele in PNH patients (22.9%) were significantly different from those in controls (n=371; 8.6%, p<0.002 and 7.7%, p<0.001, respectively), while we found no specific HLA class I alelles in MDS patients. The frequency of the HLA-DRB1*1501 allele in PNH patients (31.3%) was significantly higher than that in controls (6.1%, p<0.0001), while we could not find the high frequencies of the HLA-DRB1*1501 (10.9%) and *1502 (10.9%) alleles in AA patients. The proportion of severe or very severe AA patients with the HLA-B* 4002 allele (10/17, 58.8%) was significantly higher than that of non-severe AA patients with the allele (3/15, 20%; p<0.05). In contrast, the proportion of severe or very severe AA patients (4/17; 23.5%) with the HLA-DRB1* 1501 allele was not different from that of non-severe AA patients (3/15; 20%) with the allele. Subsequently, the reticulocyte counts (138 ± 73 x 10 9/L) and values of lactate dehydrogenase (2399 ± 235 IU/L) at the time of examination in PNH patients (n=10) with the HLA-A* 0206 allele were significantly higher than those in PNH patients (n=14) without the allele (78 ± 34 x 109/L, p<0.02 and 972 ± 770 IU/L, p<0.05, respectively). In addition, the frequency of PNH patients with over 30% of complement-sensitive erythrocytes, consisting of intermediate and negative populations of CD55 and CD59 expressions on erythrocytes by flow cytometry, was significantly higher in PNH patients with the HLA-A* 0206 than in those without the allele (80% versus 28.6%, p<0.05). In conclusions, our results suggest that the HLA-B* 4002 allele in AA or the HLA-A* 0206 allele in PNH is related to grading of the severity of AA or grading of hemolysis of PNH, respectively.

High Frequency of Hermansky–Pudlak Syndrome Type 1 (HPS1) Among Japanese Albinism Patients and Functional Analysis of HPS1 Mutant Protein

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by oculocutaneous albinism (OCA), bleeding tendency, and lysosomal accumulation of ceroid-like material. Seven genetically distinct subtypes of HPS are known in humans; most are rare outside of Puerto Rico. Here, we describe the analysis of the HPS1 gene in 24 Japanese OCA patients who lacked mutations in the four genes known to cause OCA (TYR/OCA1, P/OCA2, TYRP1/OCA3, and MATP/OCA4), and the identification of eight different HPS1 mutations in ten of these patients, four of which were novel (W583X, L668P, 532insC, 1691delA). An IVS5+5G --> A splice consensus mutation was particularly frequent, the result of a founder effect for this allele in Japanese patients. Functional analysis by transfection of the L668P variant into Hps1-mutant melan-ep mouse melanocytes showed that this missense substitution is pathologic, resulting in an Hps-1 protein that is unable to assemble into the biogenesis of lysosome-related organelles complex-3.

The High Frequency of HLA-A*0206 Allele in Japanese Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) and Its Significance.

PNH is an acquired hematologic disorder which is characterized by complement-mediated hemolysis, thrombosis, and bone marrow failure. Also, PNH is one disorder of bone marrow failure syndromes, including aplastic anemia (AA) and myelodysplastic syndrome (MDS). It is well known that immunologic mechanisms by cytotoxic T lymphocytes (CTLs) contribute to pathophysiology of these disorders. In fact, some reports (Maciejewski et al, Blood, 2001; Shichishima et al, Blood, 2002) showed that HLA-DR*1501 is related with clinical pathophysiology of PNH. In this study, to clarify significance of CD8+ CTLs in pathophysiology of PNH, we investigated HLA class I (A and B) alleles in Japanese patients with PNH (female: male=7:17), AA (female: male=14:15), and MDS (female: male=6:16) by high-resolution method using polymerase-chain reaction after obtaining informed consent and approval from the institutional Human Research Committee. Mean age ± standard deviation of PNH, AA, and MDS patients was 52 ± 16, 54 ± 20, and 59 ± 18, respectively. HLA genotyping showed that the frequency of HLA-A*0206 allele in PNH patients (22.9%) was significantly different from those in 309 unrelated Japanese individuals (Saito et al, Tissue Antigens, 2000) (7.7%; p<0.02) and AA patients (5.2%; p<0.01). In contrast, we found no significant differences in the frequencies of the other alleles between PNH, AA, or MDS patients and the controls or between these disorders, except for high frequency of HLA-B40 alleles in AA patients (Nakamura et al, Blood, 2003). Then, various clinical parameters, including peripheral blood, bone marrow blood, and laboratory findings, proportions of glycosylphosphatidylinositol protein-negative population in erythrocytes, granulocytes, and monocytes, findings of chromosomal analyses and HLA-DR alleles, transfusion requirements, past history of AA, and history of thrombosis, were statistically compared between HLA-A*0206-positive group (n=10) and -negative group (n=14) in PNH patients. Statistical analyses showed that the reticulocyte counts , the values of lactate dehydrogenase, and the frequency of PNH patients with over 30% of CD59− erythrocytes in HLA-A*0206-positive group were significantly higher than in HLA-A*0206-negative group (121 ± 49 x 109/L vs 76.9 ± 43.9 x 109/L, p<0.03; 2866 ± 2606 IU/L vs 938 ± 775, p<0.02; and 80.0 % vs 28.6 %, p<0.02, respectively). Moreover, we found no AA (n=3) and MDS (n=5) patients with both the HLA-A*0206 allele and more than 1% of CD59− granulocytes. In conclusion, our findings suggest that the HLA-A*0206 allele in PNH may be correlated with the grade of the disease by complement-mediated hemolysis during negative selection of PNH clones, probably due to immunologic mechanisms by CD8+ CTLs.

Antibodies to type II collagen and their association with HLA DR1 alleles in Japanese patients with rheumatoid arthritis

Antibodies to type II collagen and their association with HLA DR1 alleles in Japanese patients with rheumatoid arthritis

Antibodies to type II collagen and their association with HLA DR1 alleles in Japanese patients with rheumatoid arthritis

To investigate whether immunological responses to type II collagen (CII) play an important role in the pathogenesis of rheumatoid arthritis (RA), the presence of anti-CII antibodies was examined by enzyme immunoassay in 130 Japanese patients with RA, 10 systemic lupus erythematosus (SLE) patients, and 30 healthy subjects. In addition, the HLA-DRB1 genes of 40 RA patients were determined, and their association with positive findings of anti-CII antibodies was examined. A significantly high frequency of positive findings of anti-CII antibodies was detected in sera from RA patients (19%, P < 0.05) in comparison with that in sera from healthy subjects (3%). High frequencies of DRB1 *0405 and 0101 alleles were observed in the 40 RA patients examined (40.0% and 30.0%, respectively). Patients with DRB1 *0101 had a significantly higher rate of positive findings of anti-CII antibodies than those without DRB1 *0101 (66.7% and 28.6%, respectively, P < 0.05). No such association was observed for DRB1 *0405. From these findings, we suggest that immunological responses to CII may play an important role in the development of arthritis in some RA patients.