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The past quarter of a century has witnessed an inordinate increase in our understanding of primary immunodeficiencies / inborn errors of immunity. These include a significant increase in the number of identified conditions, broadening the phenotypes of existing entities, delineation of classical inborn errors of immunity from those with a narrow phenotype, and a gradual shift from supportive to definitive care in patients afflicted with these diseases. It has also seen the discovery of conditions broadly defined as phenocopies of primary immunodeficiencies, where somatic mutations or autoantibodies mimic a recognised primary immunodeficiency's presentation in the absence of the underlying genetic basis for that disease. This article will provide a review of the anti-cytokine autoantibody-mediated phenocopies of inborn errors of immunity and discuss the therapeutic and laboratory aspects of this group of diseases.

CXXC5 is a member of the CXXC-type zinc finger epigenetic regulators. Various hematopoietic and nonhematopoietic roles have been assigned to CXXC5. In the present study, the role of Cxxc5 in myelopoiesis was studied using overexpression and short hairpin RNA-mediated knockdown in mouse early stem and progenitor cells defined as Lineage− Sca-1+c-Kit+ (LSK) cells. Knockdown of Cxxc5 in mouse progenitor cells reduced monocyte and increased granulocyte development in ex vivo culture systems. In addition, ex vivo differentiation and proliferation experiments demonstrated that the expression of Cxxc5 affects the cell cycle in stem/progenitor cells and myeloid cells. Flow cytometry-based analyses revealed that down-regulation of Cxxc5 leads to an increase in the percentage of cells in the S phase, whereas overexpression results in a decrease in the percentage of cells in the S phase. Progenitor cells proliferate more after Cxxc5 knockdown, and RNA sequencing of LSK cells, and single-cell RNA sequencing of differentiating myeloid cells showed up-regulation of genes involved in the regulation of cell cycle after Cxxc5 knockdown. These results provide novel insights into the physiologic function of Cxxc5 during hematopoiesis, and demonstrate for the first time that it plays a role in monocyte development.

Genetic testing has become an integral component of the diagnostic evaluation of patients with suspected primary immunodeficiency diseases. Results of genetic testing can have a profound effect on clinical management decisions. Therefore clinical providers must demonstrate proficiency in interpreting genetic data. Because of the need for increased knowledge regarding this practice, the American Academy of Allergy, Asthma & Immunology Primary Immunodeficiency Diseases Committee established a work group that reviewed and summarized information concerning appropriate methods, tools, and resources for evaluating variants identified by genetic testing. Strengths and limitations of tests frequently ordered by clinicians were examined. Summary statements and tables were then developed to guide the interpretation process. Finally, the need for research and collaboration was emphasized. Greater understanding of these important concepts will improve the diagnosis and management of patients with suspected primary immunodeficiency diseases.

B cells are one of the fundamental components of the adaptive immune system and are best known for their ability to produce antibodies. Among the various types of inborn errors of immunity, antibody deficiencies represent the largest group in terms of the number of affected individuals. Not all antibody deficiencies are due to B cell intrinsic defects but investigating B cell number and function are a critical part of the diagnostic process. B cells studies in clinical practice almost always rely on flow cytometry as the main tool of investigation. The advantage of flow cytometry is that it allows absolute and relative counting of B cells, and their phenotypic and functional evaluation at a single-cell level, while allowing the analysis of a large number of cells. Although versatile and broad in its utility, clinical flow cytometry has both theoretical and practical limitations. These include lack of consensus about definitions and classifications, and the use of non-standardized methods. Patients in all age groups, from newborns to the elderly, may require testing, yet B cells show significant changes in both numbers and subset distribution over the lifespan, requiring distinct reference ranges for narrowly defined age brackets for accurate interpretation. Sampling for testing is usually restricted to peripheral blood samples, and the number of markers routinely used are limited. This paper will provide a brief overview of flow cytometry and B cell biology, describe the human peripheral B cell subsets most commonly identified in clinical flow cytometry and discuss their clinical relevance in different settings.

Knowledge related to the biology of inborn errors of immunity and associated laboratory testing methods continues to expand at a tremendous rate. Despite this, many patients with inborn errors of immunity suffer for prolonged periods of time before identification of their underlying condition, thereby delaying appropriate care. Understanding that test selection and optimal evaluation for patients with recurrent infections or unusual patterns of inflammation can be unclear, we present a document that distills relevant clinical features of immunologic disease due to inborn errors of immunity and related appropriate and available test options. This document is intended to serve the practicing clinical immunologist and, in turn, patients by describing best available test options for initial and expanded immunologic evaluations across the disease spectrum. Our goal is to demystify the process of evaluating patients with suspected immune dysfunction and to enable more rapid and accurate diagnosis of such individuals.

We report a novel variant in IKZF1 associated with IKAROS haploinsufficiency in a patient with familial immune thrombocytopenia (ITP). IKAROS, encoded by the IKZF1 gene, is a hematopoietic zinc-finger transcription factor that can directly bind to DNA. We show that the identified IKZF1 variant (p.His195Arg) alters a completely conserved histidine residue required for the folding of the third zinc-finger of IKAROS protein, leading to a loss of characteristic immunofluorescence nuclear staining pattern. In our case, genetic testing was essential for the diagnosis of IKAROS haploinsufficiency, of which known presentations include infections, aberrant hematopoiesis, leukemia, and age-related decrease in humoral immunity. Our family study underscores that, after infections, ITP is the second most common clinical manifestation of IKAROS haploinsufficiency.

In the era of newborn screening (NBS) for severe combined immunodeficiency (SCID) and the possibility of gene therapy (GT), it is important to link SCID phenotype to the underlying genetic disease. In western countries, X-linked interleukin 2 receptor gamma chain (IL2RG) and adenosine deaminase (ADA) deficiency SCID are two of the most common types of SCID and can be treated by GT. As a challenge, both IL2RG and ADA genes are highly polymorphic and a gene–based diagnosis may be difficult if the variant is of unknown significance or if it is located in non-coding areas of the genes that are not routinely evaluated with exon-based genetic testing (e.g., introns, promoters, and the 5′and 3′ untranslated regions). Therefore, it is important to extend evaluation to non-coding areas of a SCID gene if the exon-based sequencing is inconclusive and there is strong suspicion that a variant in that gene is the cause for disease. Functional studies are often required in these cases to confirm a pathogenic variant. We present here two unique examples of X-linked SCID with variable immune phenotypes, where IL2R gamma chain expression was detected and no pathogenic variant was identified on initial genetic testing. Pathogenic IL2RG variants were subsequently confirmed by functional assay of gamma chain signaling and maternal X-inactivation studies. We propose that such tests can facilitate confirmation of suspected cases of X-linked SCID in newborns when initial genetic testing is inconclusive. Early identification of pathogenic IL2RG variants is especially important to ensure eligibility for gene therapy.

Implementation of human leukocyte antigen (HLA) genotyping by next-generation sequencing (NGS) in the clinical lab brings new challenges to the laboratories performing this testing. With the advent of commercially available HLA-NGS typing kits, labs must make numerous decisions concerning capital equipment and address labor considerations. Therefore, careful and unbiased evaluation of available methods is imperative. In this report, we compared our in-house developed HLA NGS typing with two commercially available kits from Illumina and Omixon using 10 International Histocompatibility Working Group (IHWG) and 36 clinical samples. Although all three methods employ long range polymerase chain reaction (PCR) and have been developed on the Illumina MiSeq platform, the methodologies for library preparation show significant variations. There was 100% typing concordance between all three methods at the first field when a HLA type could be assigned. Overall, HLA typing by NGS using in-house or commercially available methods is now feasible in clinical laboratories. However, technical variables such as hands-on time and indexing strategies are sufficiently different among these approaches to impact the workflow of the clinical laboratory.

Human leukocyte antigen (HLA) allele ambiguities are the result of limitations of current HLA typing methodologies. Ambiguities maybe due to polymorphisms in unsequenced regions of HLA genes or cis/trans variants that cannot be distinguished by Sanger sequencing. Next generation sequencing (NGS) can resolve these two sources of ambiguity because the entire gene can be sequenced. Commercially available HLA NGS genotyping kits enable laboratories to deliver high-quality and unambiguous HLA typing results at an affordable cost. Third generation sequencing technologies are poised to further improve sequencing quality, shorten turn-around and library preparation times, as well as provide full-gene phasing.

Background Mass cytometry can differentiate more channels than conventional flow cytometry. However, for clinical use, standardization and agreement with well-established methods is paramount. We compared mass cytometry to standard clinical flow cytometry. Methods Mass and flow cytometry were performed in parallel on peripheral blood samples from 25 healthy individuals. Antibody staining was performed on the same samples at the same time, and analyzed for granulocyte, monocyte, lymphocyte, T, B, NK, CD4 and CD8 percentages. Validation parameters included comparison to flow cytometry, inter- and intra-assay precision and establishment of reference intervals. Results There was a positive correlation between mass and flow cytometry for the eight populations studied (R2 between 0.26 and 0.97). Slopes of the best-fit lines varied from 0.50 to 1.21 (fluorescence/mass). No significant differences in variance were found (F-test, P > 0.05). However, paired t-tests were significantly different for four of the eight markers (granulocytes, NK cells, T cells and CD4 cells), resulting in different reference intervals. Signal intensities were correlated for monocytes, lymphocytes, T, CD4 and CD8 cells (R2 = 0.41-0.57). The mass cytometry intra-assay precisions were 0.7-8.5% and inter-assay precisions 1.5-13.8%. Conclusion Mass and flow cytometry evaluations of whole blood for major cell populations correlate with similar precision and signal intensity. However, for clinical use, separate reference interval studies are required. Cell population identification should rely on gating strategies that take advantage of the characteristics offered by each method. © 2019 International Clinical Cytometry Society.

Combined immunodeficiency (CID) presents a unique challenge to clinicians. Two patients presented with the prior clinical diagnosis of common variable immunodeficiency (CVID) disorder marked by an early age of presentation, opportunistic infections, and persistent lymphopenia. Due to the presence of atypical clinical features, next generation sequencing was applied documenting RAG deficiency in both patients.Two different genetic analysis techniques were applied in these patients including whole exome sequencing in one patient and the use of a gene panel designed to target genes known to cause primary immunodeficiency disorders (PIDD) in a second patient. Sanger dideoxy sequencing was used to confirm RAG1 mutations in both patients.Two young adults with a history of recurrent bacterial sinopulmonary infections, viral infections, and autoimmune disease as well as progressive hypogammaglobulinemia, abnormal antibody responses, lymphopenia and a prior diagnosis of CVID disorder were evaluated. Compound heterozygous mutations in RAG1 (1) c256_257delAA, p86VfsX32 and (2) c1835AG, pH612R were documented in one patient. Compound heterozygous mutations in RAG1 (1) c.1566GT, p.W522C and (2) c.2689CT, p. R897X) were documented in a second patient post-mortem following a fatal opportunistic infection.Astute clinical judgment in the evaluation of patients with PIDD is necessary. Atypical clinical findings such as early onset, granulomatous disease, or opportunistic infections should support the consideration of atypical forms of late onset CID secondary to RAG deficiency. Next generation sequencing approaches provide powerful tools in the investigation of these patients and may expedite definitive treatments.

Three related males presented with a newly recognized x-linked syndrome associated with neurodegeneration, cutaneous abnormalities, and systemic iron overload. Linkage studies demonstrated that they shared a haplotype on Xp21.3-Xp22.2 and exome sequencing was used to identify candidate variants. Of the segregating variants, only a PIGA mutation segregated with disease in the family. The c.328_330delCCT PIGA variant predicts, p.Leu110del (or c.1030_1032delCTT, p.Leu344del depending on the reference sequence). The unaffected great-grandfather shared his X allele with the proband but he did not have the PIGA mutation, indicating that the mutation arose de novo in his daughter. A single family with a germline PIGA mutation has been reported; affected males had a phenotype characterized by multiple congenital anomalies and severe neurologic impairment resulting in infantile lethality. In contrast, affected boys in the family described here were born without anomalies and were neurologically normal prior to onset of seizures after 6 months of age, with two surviving to the second decade. PIGA encodes an enzyme in the GPI anchor biosynthesis pathway. An affected individual in the family studied here was deficient in GPI anchor proteins on granulocytes but not erythrocytes. In conclusion, the PIGA mutation in this family likely causes a reduction in GPI anchor protein cell surface expression in various cell types, resulting in the observed pleiotropic phenotype involving central nervous system, skin, and iron metabolism.

Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by antibody deficiency, poor humoral response to antigens, and recurrent infections. To investigate the molecular cause of CVID, we carried out exome sequence analysis of a family diagnosed with CVID and identified a heterozygous frameshift mutation, c.2564delA (p.Lys855Serfs(∗)7), in NFKB2 affecting the C terminus of NF-κB2 (also known as p100/p52 or p100/p49). Subsequent screening of NFKB2 in 33 unrelated CVID-affected individuals uncovered a second heterozygous nonsense mutation, c.2557CT (p.Arg853(∗)), in one simplex case. Affected individuals in both families presented with an unusual combination of childhood-onset hypogammaglobulinemia with recurrent infections, autoimmune features, and adrenal insufficiency. NF-κB2 is the principal protein involved in the noncanonical NF-κB pathway, is evolutionarily conserved, and functions in peripheral lymphoid organ development, B cell development, and antibody production. In addition, Nfkb2 mouse models demonstrate a CVID-like phenotype with hypogammaglobulinemia and poor humoral response to antigens. Immunoblot analysis and immunofluorescence microscopy of transformed B cells from affected individuals show that the NFKB2 mutations affect phosphorylation and proteasomal processing of p100 and, ultimately, p52 nuclear translocation. These findings describe germline mutations in NFKB2 and establish the noncanonical NF-κB signaling pathway as a genetic etiology for this primary immunodeficiency syndrome.

To the editor: The multiple zinc finger-containing transcription factor, IKAROS/IKZF1, has multifaceted roles in hematopoiesis.[1][1] Numerous studies using both human primary hematopoietic cells and mouse models have suggested that IKZF1 plays a key role in erythropoiesis and the fetal-to-adult

Illumina first introduced their TruSight human leucocyte antigen (HLA) next-generation sequencing (NGS) typing kit in 2015 and subsequently followed up with a new version in 2016. Here we report on our experience comparing the two versions of the Illumina HLA NGS kits.

Common variable immunodeficiency (CVID) is characterized by late-onset hypogammaglobulinemia in the absence of predisposing factors. The genetic cause is unknown in the majority of cases, and less than 10% of patients have a family history of the disease. Most patients have normal numbers of B cells but lack plasma cells.We used whole-exome sequencing and array-based comparative genomic hybridization to evaluate a subset of patients with CVID and low B-cell numbers. Mutant proteins were analyzed for DNA binding with the use of an electrophoretic mobility-shift assay (EMSA) and confocal microscopy. Flow cytometry was used to analyze peripheral-blood lymphocytes and bone marrow aspirates.Six different heterozygous mutations in IKZF1, the gene encoding the transcription factor IKAROS, were identified in 29 persons from six families. In two families, the mutation was a de novo event in the proband. All the mutations, four amino acid substitutions, an intragenic deletion, and a 4.7-Mb multigene deletion involved the DNA-binding domain of IKAROS. The proteins bearing missense mutations failed to bind target DNA sequences on EMSA and confocal microscopy; however, they did not inhibit the binding of wild-type IKAROS. Studies in family members showed progressive loss of B cells and serum immunoglobulins. Bone marrow aspirates in two patients had markedly decreased early B-cell precursors, but plasma cells were present. Acute lymphoblastic leukemia developed in 2 of the 29 patients.Heterozygous mutations in the transcription factor IKAROS caused an autosomal dominant form of CVID that is associated with a striking decrease in B-cell numbers. (Funded by the National Institutes of Health and others.).

Patients with mutations of the recombination-activating genes (RAG) present with diverse clinical phenotypes, including severe combined immune deficiency (SCID), autoimmunity, and inflammation. However, the incidence and extent of immune dysregulation in RAG-dependent immunodeficiency have not been studied in detail. Here, we have demonstrated that patients with hypomorphic RAG mutations, especially those with delayed-onset combined immune deficiency and granulomatous/autoimmune manifestations (CID-G/AI), produce a broad spectrum of autoantibodies. Neutralizing anti–IFN-α or anti–IFN-ω antibodies were present at detectable levels in patients with CID-G/AI who had a history of severe viral infections. As this autoantibody profile is not observed in a wide range of other primary immunodeficiencies, we hypothesized that recurrent or chronic viral infections may precipitate or aggravate immune dysregulation in RAG-deficient hosts. We repeatedly challenged Rag1S723C/S723C mice, which serve as a model of leaky SCID, with agonists of the virus-recognizing receptors TLR3/MDA5, TLR7/-8, and TLR9 and found that this treatment elicits autoantibody production. Altogether, our data demonstrate that immune dysregulation is an integral aspect of RAG-associated immunodeficiency and indicate that environmental triggers may modulate the phenotypic expression of autoimmune manifestations.

Aim Detection of circulating donor-derived cell-free DNA (cfDNA) as a non-invasive biomarker of graft injury shows tremendous potential for transplant monitoring. Increases in the amount of cfDNA in blood or urine indicate injury to the transplanted organ, including rejection. Also known as a liquid biopsy, this method is less invasive and more sensitive than an actual biopsy to detect early rejection. We proposed to develop a clinical test based on the detection and quantification of donor single-nucleotide polymorphisms (SNPs) using cfDNA extracted from plasma and urine of solid organ transplant patients. Methods Using genomic DNA, informative SNP markers from donor and recipient (ThermoFisher) will be identified using real-time PCR (Roche). cfDNA from transplant recipients will be extracted from plasma and urine (Qiagen), and informative SNPs will be quantified using digital-droplet PCR (ddPCR), (Bio-Rad). For development of the assay blood and urine samples were collected from 25 healthy individuals, genomic and cfDNA were extracted, and analytical parameters determined including limits of detection (LOD) and limits of quantification (LOQ) using ddPCR. Results cfDNA extraction was successfully performed from blood and urine. The average cfDNA concentration was 1.36 ± 0.54 ng/ul from plasma, and 1.15 ± 2.1 ng/ul from urine. Determined from genomic DNA, an average of 4.9 informative markers was present between any two of the 25 individuals, although one pairing had no informative markers present, indicating the need for a larger panel using more than 36 SNPs. LOD studies determined the false positivity rate to be less than 0.0025%. Preliminary LOQ testing indicates that 0.5–0.75% donor-derived DNA can be detected. Conclusions We have established a laboratory developed test to detect donor-derived cfDNA from multiple sample types such as blood and urine at a sensitivity that appears acceptable for clinical testing. In collaboration with the local solid organ transplant program, the clinical utility of this assay will be evaluated in kidney transplant recipients.

Aim HLA associations have been linked to more than 100 diseases and are also important targets for pharmacogenomic risk assessment for drug hypersensitivity reactions. Traditionally, sequence-specific oligonucleotide probe (SSOP) genotyping have been used to identify HLA alleles associated with diseases. Other methods include real-time PCR with melting curve analysis used to determine the presence of HLA-DQB1*02, DQB1*03:02 and DQA1*05 for celiac disease, DQB1*06:02 for narcolepsy, B*27 for ankylosing spondylitis, and B*57:01 for abacavir hypersensitivity. The diagnostic value of HLA disease association testing is only as good as the accuracy of the methodology used. The increasing number of discovered HLA alleles has generated a growing list of ambiguities that cannot be ruled out with the current assays. Methods We proposed to evaluate the NGS-based HLA-B and DQ Monotype kits (Omixon) for HLA-disease association testing using a cohort of 72 samples. Method comparison was performed with both SSOP genotyping (One Lambda), and real-time PCR with melting curve analysis (laboratory-developed). Results Multiple discrepancies were observed between NGS and the existing methods. One sample was called DQB1*03:02 positive by the real-time PCR assay but was actually homozygous for DQB1*03:03, an allele not associated with celiac disease. DQB1*03:02 and 03:03 only differ by one base pair and that difference lies in the reverse primer. Likewise, a DQB1*03:04 allele was called DQB1*03:02 positive by the melting curve assay. A similar situation occurred with the B*57:01 real-time PCR assay; a sample was called weak positive but was in fact homozygous for B*57:03, an allele not associated with abacavir hypersensitivity. Limitations of the SSOP genotyping method included an ambiguous false positive DQB1*03:02, but NGS genotyped it as DQB1*03:40, an allele not known to be associated with celiac disease. The NGS kit correctly genotyped all samples without ambiguity. Conclusions Our study demonstrates that HLA typing by NGS is the most accurate method for identifying alleles associated with disease or drug hypersensitivity. The kit allows for testing of 96–192 individual patients per run, and HLA B and DQ samples can be combined, making it a viable approach for high volume clinical diagnostic laboratories.

The Job or hyper-immunoglobulinemia E syndrome is a primary immunodeficiency that is usually inherited in an autosomal dominant fashion. With the discovery of mutations in the STAT3 gene in the majority of autosomal dominant cases, it is now possible to make a molecular diagnosis of hyper-IgE syndrome. Both primary and secondary immunodeficiencies, including hyper-IgE syndrome, may predispose for malignancies, especially lymphomas, mainly mature B cell lymphomas, and classical Hodgkin lymphoma. Here, we report of a 48-year-old male with hyper-IgE syndrome who developed a primary parotid gland diffuse large B cell lymphoma. Analysis for STAT3 mutations demonstrated that the causal mutation of hyper-IgE syndrome, R382Q, arose de novo in the patient and it was transmitted to three of his five children, all three of whom are clinically affected. We review the literature regarding lymphoma in hyper-IgE syndrome and the possible etiologic relationship with STAT3 mutations.

With the recent discovery of mutations in the STAT3 gene in the majority of patients with classic Hyper-IgE syndrome, it is now possible to make a molecular diagnosis in most of these cases. We have developed a PCR-based high-resolution DNA-melting assay to scan selected exons of the STAT3 gene for mutations responsible for Hyper-IgE syndrome, which is then followed by targeted sequencing. We scanned for mutations in 10 unrelated pedigrees, which include 16 patients with classic Hyper-IgE syndrome. These pedigrees include both sporadic and familial cases and their relatives, and we have found STAT3 mutations in all affected individuals. High-resolution melting analysis allows a single day turn-around time for mutation scanning and targeted sequencing of the STAT3 gene, which will greatly facilitate the rapid diagnosis of the Hyper-IgE syndrome, allowing prompt and appropriate therapy, prophylaxis, improved clinical outcome, and accurate genetic counseling.

Iron is an essential nutrient but in excess may damage cells by generating reactive oxygen species due to Fenton reaction or by substituting for other transition metals in essential proteins. The budding yeast Saccharomyces cerevisiae detoxifies cytosolic iron by storage in the vacuole. Deletion of CCC1, which encodes the vacuolar iron importer, results in high iron sensitivity due to increased cytosolic iron. We selected mutants that permitted Δccc1 cells to grow under high iron conditions by UV mutagenesis. We identified a mutation (N44I) in the vacuolar zinc transporter ZRC1 that changed the substrate specificity of the transporter from zinc to iron. COT1, a vacuolar zinc and cobalt transporter, is a homologue of ZRC1 and both are members of the cation diffusion facilitator family. Mutation of the homologous amino acid (N45I) in COT1 results in an increased ability to transport iron and decreased ability to transport cobalt. These mutations are within the second hydrophobic domain of the transporters and show the essential nature of this domain in the specificity of metal transport.

The nature of the connection between mitochondrial Fe-S cluster synthesis and the iron-sensitive transcription factor Aft1 in regulating the expression of the iron transport system in Saccharomyces cerevisiae is not known. Using a genetic screen, we identified two novel cytosolic proteins, Fra1 and Fra2, that are part of a complex that interprets the signal derived from mitochondrial Fe-S synthesis. We found that mutations in FRA1 (YLL029W) and FRA2 (YGL220W) led to an increase in transcription of the iron regulon. In cells incubated in high iron medium, deletion of either FRA gene results in the translocation of the low iron-sensing transcription factor Aft1 into the nucleus, where it occupies the FET3 promoter. Deletion of either FRA gene has the same effect on transcription as deletion of both genes and is not additive with activation of the iron regulon due to loss of mitochondrial Fe-S cluster synthesis. These observations suggest that the FRA proteins are in the same signal transduction pathway as Fe-S cluster synthesis. We show that Fra1 and Fra2 interact in the cytosol in an iron-independent fashion. The Fra1-Fra2 complex binds to Grx3 and Grx4, two cytosolic monothiol glutaredoxins, in an iron-independent fashion. These results show that the Fra-Grx complex is an intermediate between the production of mitochondrial Fe-S clusters and transcription of the iron regulon.

YIL023C encodes a member of the SLC39A, or ZIP, family, which we refer to as yeast KE4 (YKE4) after its mouse ortholog. Yke4p was localized to the endoplasmic reticulum (ER) membrane using Yke4p-specific antiserum. YKE4 is not an essential gene; however, deletion of YKE4 resulted in a sensitivity to calcofluor white and poor growth at 36 degrees C on respiratory substrates containing high zinc. Overexpression of transition metal transporters Zrc1p and Cot1p or the mouse orthologue mKe4 in Deltayke4 suppressed the poor growth at 36 degrees C on respiratory substrates. We found that the role of Yke4p depends on the zinc status of the cells. In a zinc-adequate environment, Yke4p transports zinc into the secretory pathway, and the deletion of YKE4 leads to a zinc-suppressible cell wall defect. In high zinc medium, transport of zinc into the secretory pathway through Yke4p is a way to eliminate zinc from the cytosol, and deletion of YKE4 leads to toxic zinc accumulation in the cytosol. Under low cytosolic zinc conditions, however, Yke4p removes zinc from the secretory pathway, and deletion of YKE4 partially compensates for the loss of Msc2p, an ER zinc importer, and therefore helps to alleviate ER stress. In our model, Yke4p balances zinc levels between the cytosol and the secretory pathway, whereas the previously described Msc2p-Zrg17p ER zinc importer complex functions mainly in zinc-depleted conditions to ensure a ready supply of zinc essential for ER functions, such as phospholipid biosynthesis and unfolded protein response.

As part of an ongoing elucidation of rainbow trout major histocompatibility complex (MHC) class I, the polymorphism of two MHC class Ib loci was analyzed. These loci, Onmy-UCA and Onmy-UDA, are situated head-to-tail and share more than 89% nucleotide identity in their open reading frames. They share 80% identity with some trout Ia alleles. The deduced amino acid sequences suggest that the UCA and UDA molecules are transported to endosomal compartments and may bind peptides in their binding groove. Our survey revealed seven UCA and eight UDA alleles. Similarity indices overlap when comparing within and between UCA and UDA alleles and some cross-locus motif variation is observed. In most trout both UCA and UDA transcripts were found. However, there probably is functional redundancy, because some trout lacked transcription of one of the two loci. Furthermore, for some UCA and UDA alleles, splicing deficiencies, early stop codons, and upstream start codons were found, which may interfere with efficient protein expression. The present study is the first extensive report on MHC class Ib polymorphism assigned to locus in ectotherm species.

Single-molecule sequencing should allow for unambiguous, accurate, and high-throughput HLA typing. In this proof of principle study, we investigated the effects of fragment size for library preparation, indexing strategy, and read length on HLA typing. Whole gene amplicons of HLA-A, B, C, DRB1, and DQB1 were obtained by long-range PCR. For library preparation, two fragment sizes were evaluated: 100-300bp and 300-600bp. For sample multiplexing, two indexing strategies were compared: indexing-by-amplicon, where each individual amplicon is barcoded, and indexing-by-patient, where each patient's five loci are equimolarly pooled after PCR and indexed with the same barcode. Sequencing was performed on an Illumina MiSeq instrument using paired-end 150bp and 250bp read lengths. Our results revealed that the 300-600bp fragments in the 2×250 MiSeq group gave the most accurate sequencing results. There was no difference in HLA typing results between the two indexing strategies, suggesting that indexing-by-patient, which is much simpler, is a viable option. In conclusion, enzymatic fragmentation of pooled whole gene amplicons is a suitable strategy for HLA typing by next-generation sequencing on the Illumina MiSeq.

Recent large-scale sequencing and comparative analyses of the major histocompatibility complex (Mhc) provide a novel view of this long-studied region. The main insight is that even though Mhcs are defined by the presence of the Mhc class I and II genes, the regions encoding class I/II histocompatibility antigens are the least conserved among the species; hence the difficulty of modeling the human class I/II-linked diseases. Fortunately, the majority of the genes in the Mhc, the non-class I/II genes, are conserved among the investigated mammals. The full set of Mhc genes in their evolutionary context presents new possibilities to study Mhc-linked diseases by allowing systematic evaluation of the various experimental animals and approaches.

A 5-Mb YAC contig, partly supplemented with BAC contigs, was created from the distal Mhc class I region on mouse Chr 17. The gene order of Znf173-Tctex5-Mog-D17Tu42-D17Leh 89 is conserved between mouse and human but not the physical distance, supporting the independent expansion of Mhc class I genes in the so-called accordion model of Mhc evolution. The distal H2-M region includes the breakpoint of conserved synteny between mouse and human as well as the In(17)4 t-inversion. The H2-M region is rich in L1 repeats, implying that the insertion of L1 repeats may be associated with the evolutionary flexibility to break a chromosome.

We report the 897 kb sequence of a cluster of olfactory receptor (OR) genes located at the distal end of the major histocompatibility complex (MHC) class I region on mouse chromosome 17 of strain 129/SvJ (H2bc). With additional information from the mouse genome draft sequence, we identified 59 OR loci (approximately 20% pseudogenes) in contrast to only 25 OR loci (approximately 50% pseudogenes) in the corresponding centromeric OR cluster that is part of the 'extended MHC class I region' on human chromosome 6. Comparative analysis leads to three major observations: (i) most of the OR subfamilies have evolved independently in the two species, expanding more in the mouse, and resulting in co-orthologs--subfamilies of highly similar paralogs that keep orthologous relationships with their human counterparts; (ii) three of the mouse OR subfamilies have no orthologs in humans; and (iii) MHC class I loci are interspersed in the OR cluster in mouse but not in human, and were subjected to co-duplication with OR genes. Screening of our sequence against the available sequences of other strains/haplotypes revealed that most of the OR loci are polymorphic and that the number of OR loci may vary among strains/haplotypes. Our findings that MHC-linked OR loci share duplication with MHC class I loci, have duplicated extensively and are polymorphic revives questions about potential reciprocal influences acting on the dynamics and evolution of the H2 region and the H2-linked OR loci.

The Human Genome Project transformed the quest of more than 50 years to understand the major histocompatibility complex (Mhc). The sequence of the Mhc from human and mouse, together with a large amount of sequence and mapping information from several other species, allows us to draw general conclusions about the organization and origin of this crucial part of the immune system. The Mhc is a mosaic of stretches formed by conserved and nonconserved genes. Surprisingly, of the approximately 3.6-Mb Mhc, the stretches that encode the class I and class II genes, which epitomize the Mhc, are the least conserved part, whereas the approximately 1.7-Mb stretches that encode at least 115 other genes are highly conserved. We summarize the available data to answer the questions (a) What is the Mhc? and (b) How can we define it in a general, not species-specific, way? Knowing what is essential and what is incidental helps us understand the fundamentals of the Mhc, and defining the species differences makes the model organisms more useful.

We have determined the complete sequence of 951,695 bp from the class I region ofH2,the mouse major histocompatibility complex (Mhc) from strain 129/Sv (haplotypebc). The sequence contains 26 genes. The sequence spans from the last 50 kb of theH2-Tregion, including 2 class I genes and 3 class I pesudogenes, and includes theH2-Mregion up toGabbr1.A 500-kb stretch of theH2-Mregion contains 9 class I genes and 4 pseudogenes, which fall into two subfamilies,M1andM10, distinct from other mouse class I genes. ThisM1/M10class I gene-cluster is separated from the centromericH2-Tand the telomericH2-M4, -5 and-6class I genes by “nonclass I genes”. Comparison with the corresponding 853-kb region of the humanMhc,which includes theHLA-Aregion, shows a mosaic of conserved regions of orthologous nonclass I genes separated by regions of species-specific expansion of paralogousMhcclass I genes. The analysis of this mosaic structure illuminates the dynamic evolution of theMhcclass I region among mammals and provides evidence for the framework hypothesis.[Supplemental material is available online atwww.genome.org. The sequence data from this study have been submitted to GenBank under accession nos.AC005413,AC005665,AF532111–AF532117. A preliminary draft sequence was earlier submitted asAC002615and replaced this year by NT002615.]

The H2-D and -Q regions of the mouse major histocompatibility complex (Mhc or H2) have been sequenced from strain 129/SvJ (haplotype bc), revealing a D/Q region different from all other investigated haplotypes, including the closely related b haplotype. The 300-kb class I-rich region consists of the classical class I, H2-D, and 11 non-classical class I genes. The Q region was formed by two series of tandem duplications. Comparison of the segment between the D and Q1 genes with the H2-K region provides evidence that class I genes were translocated from the K region to the D region, and gives a new explanation for the weak locus specificity of the H-K and H2-D alleles.

The six-transmembrane channels are thought to be composed of two modules: pore and sensor. Whereas the modular design of the pore has been established, the modularity of the sensor remains hypothetical. As a first step toward establishing the modularity of this region, we searched for genes where the sensor is found independent of the pore and have identified new members of the sensor superfamily. Analysis of these sensors reveals a motif shared among not only these newly discovered members and voltage-gated, transient receptor potential, and polycystin channel sensors, but also MscL, a bacterial mechanosensitive channel. Mutational analyses presented here and in previous studies demonstrate that highly conserved residues within this motif are required for normal channel activity; mutations of residues within this motif in different subfamilies lead to consistent channel phenotypes. Previous studies have demonstrated that peptides containing this motif and the adjacent conserved transmembrane domain elicit channel activities when reconstituted into lipid membranes. These data provide evidence for the modularity of the sensor, imply a model for its evolution, suggest a common origin for mechano- and voltage-sensing, and may offer a glimpse of the properties of the first sensor/channel.

RAG deficiency is emerging as one of the leading causes of SCID and leaky SCID with an estimated incidence of 1:336,000. Hypomorphic mutations in the RAG genes can also lead to highly variable delayed-onset combined immunodeficiency diseases. We estimate the population genetic frequency of these hypomorphic diseases as up to 1:181,000, suggesting that RAG1/2 mutations are likely to contribute to undiagnosed cases of combined immunodeficiencies.

The mouse MHC class Ib gene H2-T11 is 95% identical at the DNA level to H2-T23, which encodes Qa-1, one of the most studied MHC class Ib molecules. H2-T11 mRNA was observed to be expressed widely in tissues of C57BL/6 mice, with the highest levels in thymus. To circumvent the availability of a specific mAb, cells were transduced with cDNA encoding T11 with a substituted α3 domain. Hybrid T11D3 protein was expressed at high levels similar to control T23D3 molecules on the surface of both TAP+ and TAP− cells. Soluble T11D3 was generated by folding in vitro with Qa-1 determinant modifier, the dominant peptide presented by Qa-1. The circular dichroism spectrum of this protein was similar to that of other MHC class I molecules, and it was observed to bind labeled Qa-1 determinant modifier peptide with rapid kinetics. By contrast to the Qa-1 control, T11 tetramers did not react with cells expressing CD94/NKG2A, supporting the conclusion that T11 cannot replace Qa-1 as a ligand for NK cell inhibitory receptors. T11 also failed to substitute for Qa-1 in the presentation of insulin to a Qa-1–restricted T cell hybridoma. Despite divergent function, T11 was observed to share peptide-loading specificity with Qa-1. Direct analysis by tandem mass spectrometry of peptides eluted from T11D3 and T23D3 isolated from Hela cells demonstrated a diversity of peptides with a clear motif that was shared between the two molecules. Thus, T11 is a paralog of T23 encoding an MHC class Ib molecule that shares peptide-binding specificity with Qa-1 but differs in function.

The development of B cells is accompanied by their ability to specifically enter the peripheral lymphoid tissues. Recently, we described a novel rat monoclonal antibody (IBL-2; IgG2b/kreacting with a 26/29-kD heterodimeric structure of the cell surface. This mAb has been found to recognize differentially the peripheral B cells of mice depending on their tissue origin. The majority of splenic B cells as well as the mature B cells in the bone marrow were stained with this mAb, whereas the B lymphocytes isolated from LN or Peyer's patches displayed only negligible reactivity. We extended these observations by analyzing the relationship between the expression of IBL-2 antigen and L-selection on the surface of B-cell precursors in the bone marrow by multiparameter flow cytometry. Within the B220 positive compartment, a significant difference of L-selectin expression could be observed between the various IBL-2-reactive subsets. Furthermore, we investigated whether evidences for the establishment of tissue-associated phenotypic heterogeneity similar to that found in normal mice could be found upon the adoptive transfer of normal unselected splenic lymphocytes into SCID recipients (Spl-SCID). It has been found that a large part of the splenic B cells preserved their IBL-2 reactivity, whereas the LN B cells had lost the IBL-2 antigen in Spl-SCID. These data indicate that the phenotypic difference within the SCID mice may be the result of the migration of B lymphocytes from the spleen toward the lymph nodes, and the altered expression of the IBL-2 antigen correlates with this process.

Disorders of leukocyte function conventionally include the primary (or genetic) immunodeficiencies of phagocytes (granulocytes and monocytes). Phagocyte deficiencies can arise from insufficient number of cells or cellular dysfunctions. Phagocyte functional defects include leukocyte adhesion deficiencies, disorders of chemotaxis, microbicidal activity as well as deficiencies of specific antigen-recognition receptors. Deficiencies of many proteins involved in general cellular functions (e.g. intracellular trafficking) also lead to phagocyte dysfunction as part of a syndrome. These disorders lead to increased risk of mainly bacterial and fungal infections.