ALBERT

Description: INTRODUCTION Severe combined immunodeficiency disease (SCID) is the most severe form of primary immunodeficiency disorders (PIDs). Impaired cellular and humoral immunity renders the affected infants susceptible to various infections and results in death within the first 2 years of life. Affected infants are asymptomatic at birth, untreated disease leads to death, and prompt treatment (i.e., hematopoietic stem cell transplantation, gene therapy, or enzyme replacement therapy) is linked to significant improvement in outcome. Thus, SCID meets the disease criteria for newborn screening (NBS). The T-cell receptor excision circle (TREC) is an excellent marker of recently formed T cells, and quantitative PCR-based measurement of TREC is an excellent tool in population-based NBS for SCID. Recent progress in next-generation sequencing (NGS) has enabled the simultaneous sequencing of numerous nucleic acids, detecting single nucleotide changes as well as copy number variants. We launched a pilot newborn optional screening program for SCID, combining the measurement of TREC and NGS in Japan. PATIENTS AND METHODS We measured TREC copy number using the Enlite™ Neonatal TREC assay (Perkin Elmer, Turku, Finland), which utilizes the duplex amplification of TREC and beta-actin in the same reaction for each specimen. We used TREC negative cutoffs as follows: TREC copy number of

Description: Minor histocompatibility antigens (mHags) are molecular targets of allo-immunity associated with hematopoietic stem cell transplantation (HSCT) and involved in graft-versus-host disease, but they also have beneficial antitumor activity. mHags are typically defined by host SNPs that are not shared by the donor and are immunologically recognized by cytotoxic T cells isolated from post-HSCT patients. However, the number of molecularly identified mHags is still too small to allow prospective studies of their clinical importance in transplantation medicine, mostly due to the lack of an efficient method for isolation. Here we show that when combined with conventional immunologic assays, the large data set from the International HapMap Project can be directly used for genetic mapping of novel mHags. Based on the immunologically determined mHag status in HapMap panels, a target mHag locus can be uniquely mapped through whole genome association scanning taking advantage of the unprecedented resolution and power obtained with more than 3 000 000 markers. The feasibility of our approach could be supported by extensive simulations and further confirmed by actually isolating 2 novel mHags as well as 1 previously identified example. The HapMap data set represents an invaluable resource for investigating human variation, with obvious applications in genetic mapping of clinically relevant human traits.

Description: Background Severe combined immune deficiency (SCID) is a potentially fatal primary immunodeficiency due to the absence of T and B lymphocyte function. Early intervention for patients with SCID results in a higher survival rate. From 2017, we launched the first optional newborn screening (NBS) for SCID in Japan based on the detection of T-cell receptor excision circles (TREC). However, NBS for severe B-cell lymphopenia, such as X-linked agammaglobulinemia (XLA), has not been a standard screening test because of a high false-positive rate of Kappa-deleting recombination excision circles (KREC), which reflects the replication of B cells. XLA is characterized by severe B-cell lymphopenia and marked reduction of all classes of serum immunoglobulins. Patients with XLA require early diagnosis and immunoglobulin replacement therapy to prevent the development of bronchiectasis caused by recurrent infections. This study aimed to analyze the results of NBS for SCID and elucidate the utility of NBS for SCID and XLA using the TREC/KREC assay. Patients and Methods We enrolled infants who received NBS for SCID (n = 29,447) between April 2017 and June 2018. Using the EnLiteTM TREC kit, we measured TRECA and β-actin, which are used as controls for monitoring sample amplification. Samples with less than 30 copies/µL and adequate β-actin were defined as positive TRECA. All infants with positive TRECA were followed up for at least 12 months. We measured TRECB and KREC using the EnLiteTM TREC/KREC kit in these infants. As positive controls, we used TRECB and KREC in patients with SCID and XLA, respectively. Furthermore, all infants with positive TRECA were evaluated using flow cytometric analysis and target capture-based next-generation sequencing (NGS) analysis covering 349 primary immunodeficiency- and bone marrow failure-related genes to evaluate CD4+CD45RA+ T-cell counts and identify diagnostic variants. This study was approved by the institutional review board of Nagoya University Graduate School of Medicine. Results Of the infants who underwent NBS for SCID, 43 (0.15%) infants showed positive TRECA. All 43 infants were followed up in Nagoya University for at least 12 months. Of these, we identified one case with DiGeorge syndrome showing severe lymphopenia but did not identify typical SCID. TRECB and KREC were measured in 43 infants with positive TRECA. To determine which kit is more useful to detect T-cell lymphopenia, we compared TRECA with TRECB in 1454 infants with normal TRECA and 43 with positive TRECA. All healthy infants with normal TRECA showed TRECB with more than 30 copies/µL but nine patients with SCID showed extremely low TRECB (median [range], 0 [0-3] copies/µL). Only 6 of 43 (14%) infants showed TRECB with less than 30 copies/µL. Moreover, we analyzed the correlation between CD4+CD45RA+ T-cell counts and TRECB. Compared with 37 infants with normal TRECB, 6 infants with positive TRECB demonstrated significantly lower CD4+CD45RA+ T-cell counts (P = 0.026). However, target capture-based NGS did not identify any diagnostic variants among them. This finding suggested that using this kit, false-positive rates might be decreased from 0.15% (43/29,447) to 0.02% (6/29,447). Using this kit, we assessed KREC in 1454 infants with normal TRECA and 43 with positive TRECA. Of these, we identified one case with less than 30 copies/µL KREC who was diagnosed with congenital asplenia. As positive controls, all six patients with XLA showed quite low KREC (0 [0-9] copies/µL). Compared with previously reported KREC assay, this kit may result in lower false-positive rates. Furthermore, to demonstrate whether KREC reflects the replication of B cells, we analyzed the correlation with CD19+ B-cell counts and KREC. Among 43 infants with positive TRECA, infants with less than 500/µL CD19+ B cells showed significantly lower KREC than those with more than 500/µL CD19+ B cells (P = 0.014). Conclusion We conducted the first large-scale study to evaluate the utility of the newly released EnLiteTM TREC/KREC kit. This kit may be more useful than the current TREC kit to identify infants with T-cell lymphopenia and to avoid unnecessary follow up. Compared with previous NBS for XLA, the false-positive rate of this assay was within an acceptable range. Furthermore, TRECB and KREC were assessed with almost the same screening cost and labor. Therefore, we are considering switching from the current TREC kit to this TREC/KREC kit. Disclosures No relevant conflicts of interest to declare.

Description: Minor histocompatibility antigens (mHags) are the molecular targets of allo-immunity associated with major anti-tumor activities in hematopoietic stem cell transplantation (HSCT), but are also involved in the pathogenesis of graft-versus-host disease (GVHD). They are typically defined by the host’s SNPs that are not shared by the donor and immunologically recognized by cytotoxic T-cells isolated from the post-HSCT patients. However, despite their critical importance in transplantation medicine, fewer than 20 mHags have been identified during the past 20 years due to the lack of an efficient method for their isolation. Here we developed a novel method in which the large data set from the International HapMap Project can be directly used for genetic mapping of novel mHags. Concretely, immortalized B lymphoblastoid cell lines (LCLs) from a HapMap panel are grouped into mHag positive (mHag+) and negative (mHag−) subpanels according to their susceptibility to a cytotoxic T-cells (CTL) clone as determined by conventional chromium release cytotoxicity assays (CRAs), and the target mHag locus could be directly identified by association scan (indicated by χ2 statistic) using the highly qualified HapMap data set having over 3,000,000 SNP markers. The major concern about this approach arises from the risk of overfitting observed phenotypes to one or more incidental SNPs from this large number of the HapMap SNPs. To address this problem, we first estimated the maximum sizes of the test statistics under the null hypothesis (i.e., no associated SNPs within the HapMap set) empirically by simulating 10,000 case-control HapMap panels in different experimental conditions, and compared them with the expected size of test statistic values from the marker SNPs associated with the target SNP, assuming different linkage disequilibrium (LD), or values in between. Except for those mHags having very low minor allele frequencies (MAF) below ~0.05, the possibility of overfitting is progressively reduced as the number of LCLs increases, allowing for unique identification of the target locus in a broad range of values. To demonstrate the feasibility of this method, we tried to map the locus for HA-1H mHag, by actually immunophenotyping 58 LCLs from the JPT+CHB HapMap panel with CRAs using HLA-A*0206-restricted LCL (CTL-4B1). As expected, the genome-wide scan clearly indicated a unique association within the HMHA1 gene, showing a peak χ2 statistic of 52.8 (not reached in 100,000 permutations) at rs10421359. Next, we applied this method to mapping novel mHags recognized by HLA-B*4002-restricted CTL-3B6 and HLA-A*0206-restricted CTL-1B2, both of whose target mHags had not been identified. The peak in chromosome 19q13.3 for the CTL-3B6 set showed the theoretically maximum χ2 value of 50 (not reached in 100,000 permutations) at rs3027952, which was mapped within a small LD block of ~182kb containing a single gene, SLC1A5, as a candidate mHag gene. In fact, when expressed in HEK293T with HLA-B*4002 transgene, recipient-derived, but not donor-derived, SLC1A5 cDNA was able to stimulate interferon-γ secretion from CTL-3B6, indicating that SLC1A5 encodes the target mHag recognized by CTL-3B6. Conventional epitope mapping finally identified an undecameric peptide, AEATANGGLAL, which was further confirmed by epitope reconstitution assays. The target mHag locus for CTL-1B2 was identified at the peak (max χ2 = 44, not reached in 100,000 permutations) within a 598 kb block on chromosome 4q13.1, and coincides with the locus for a previously reported mHag, UGT2B17. Our epitope mapping by using UGT2B17 cDNA deletion mutants, prediction of candidate epitopes by HLA-binding algorithms and epitope reconstitution assays successfully identified a novel nonameric peptide, CVATMIFMI. Our results demonstrate how effectively the HapMap resources could be used for genetic mapping of clinically relevant human traits. This method may be also applied to disclosing other relevant human variations, if an accurate bioassay is applied to discriminate them. We anticipate our method based on the HapMap scan greatly accelerates isolation of novel mHags, which could be used for the development of selective allo-immune therapies to intractable blood cancers, circumventing potentially life-threatening GVHD, while harnessing its anti-tumor effects. Such knowledge on mHags should also promote our understanding of allo-immunity.