RelB-deficient autoinflammatory pathology presents as interferonopathy, but in mice is interferon-independent.

BACKGROUND: Autoimmune diseases are leading causes of ill health and morbidity and have diverse etiology. Two signaling pathways are key drivers of autoimmune pathology, interferon and nuclear factor-κB (NF-κB)/RelA, defining the 2 broad labels of interferonopathies and relopathies. Prior work has established that genetic loss of function of the NF-κB subunit RelB leads to autoimmune and inflammatory pathology in mice and humans. OBJECTIVE: We sought to characterize RelB-deficient autoimmunity by unbiased profiling of the responses of immune sentinel cells to stimulus and to determine the functional role of dysregulated gene programs in the RelB-deficient pathology. METHODS: Transcriptomic profiling was performed on fibroblasts and dendritic cells derived from patients with RelB deficiency and knockout mice, and transcriptomic responses and pathology were assessed in mice deficient in both RelB and the type I interferon receptor. RESULTS: We found that loss of RelB in patient-derived fibroblasts and mouse myeloid cells results in elevated induction of hundreds of interferon-stimulated genes. Removing hyperexpression of the interferon-stimulated gene program did not ameliorate the autoimmune pathology of RelB knockout mice. Instead, we found that RelB suppresses a different set of inflammatory response genes in a manner that is independent of interferon signaling but associated with NF-κB binding motifs. CONCLUSION: Although transcriptomic profiling would describe RelB-deficient autoimmune disease as an interferonopathy, the genetic evidence indicates that the pathology in mice is interferon-independent.

Deep phenotypic characterization of the retinal dystrophy in patients with RNU4ATAC-associated Roifman syndrome.

PURPOSE: To characterize the retinal phenotype in RNU4ATAC-associated Roifman syndrome. METHODS: Ten patients (including 8 males) with molecularly confirmed Roifman syndrome underwent detailed ophthalmologic evaluation including fundus imaging, fundus autofluorescence (FAF) imaging, spectral-domain optical coherence tomography (SD-OCT), and electroretinography (ERG). Six patients had follow-up eye exams. All patients also underwent comprehensive examination for features of extra-retinal Roifman syndrome. RESULTS: All patients had biallelic RNU4ATAC variants. Nyctalopia was common (7/10). Visual acuity at presentation ranged from 20/20 to 20/200 (Age Range: 5-41 years). Retinal exam revealed features of generalized retinopathy with mid-peripheral pigment epithelial changes. A para or peri-foveal ring of hyper-autofluorescence was the commonest FAF abnormality noted (6/8). The SD-OCT demonstrated relative preservation of the foveal ellipsoid zone in six cases; associated features included cystoid changes (5/10) and posterior staphyloma (3/10). The ERG was abnormal in all patients; nine showed generalized rod-cone dystrophy, whilst one patient with sectoral retinal involvement only had isolated rod dystrophy (20 years old). On follow-up examination (Mean duration: 8.16 years), progressive loss of visual acuity (2/6), mid-peripheral retinal atrophy (3/6) or shortening of ellipsoid zone width (1/6) were observed. CONCLUSION: This study has characterized the retinal phenotype in RNU4ATAC-associated Roifman syndrome. Retinal involvement is universal, early-onset, and overall, the retinal and FAF features are consistent with rod-cone degeneration that is slowly progressive over time. The sub-foveal retinal ultrastructure is relatively preserved in majority of patients. Phenotypic variability independent of age exists, and more study of allelic- and sex-based determinants of disease severity are necessary.

NFκB pathway dysregulation due to reduced RelB expression leads to severe autoimmune disorders and declining immunity.

BACKGROUND: Genetic aberrations in the NFκB pathway lead to primary immunodeficiencies with various degrees of severity. We previously demonstrated that complete ablation of the RelB transcription factor, a key component of the alternative pathway, results in an early manifested combined immunodeficiency requiring stem cell transplantation. OBJECTIVE: To study the molecular basis of a progressive severe autoimmunity and immunodeficiency in three patients. METHODS: Whole exome sequencing was performed to identify the genetic defect. Molecular and cellular techniques were utilized to assess the variant impact on NFκB signaling, canonical and alternative pathway crosstalk, as well as the resultant effects on immune function. RESULTS: Patients presented with multiple autoimmune progressive severe manifestations encompassing the liver, gut, lung, and skin, becoming debilitating in the second decade of life. This was accompanied by a deterioration of the immune system, demonstrating an age-related decline in naïve T cells and responses to mitogens, accompanied by a gradual loss of all circulating CD19+ cells. Whole exome sequencing identified a novel homozygous c. C1091T (P364L) transition in RELB. The P364L RelB protein was unstable, with extremely low expression, but retained some function and could be transiently and partially upregulated following Toll-like receptor stimulation. Stimulation of P364L patient fibroblasts resulted in a marked rise in a cluster of pro-inflammatory hyper-expressed transcripts consistent with the removal of RelB inhibitory effect on RelA function. This is likely the main driver of autoimmune manifestations in these patients. CONCLUSION: Incomplete loss of RelB provided a unique opportunity to gain insights into NFκB's pathway interactions as well as the pathogenesis of autoimmunity. The P364L RelB mutation leads to gradual decline in immune function with progression of severe debilitating autoimmunity.

Case Report: Eosinophilic Esophagitis in a Patient With a Novel STAT1 Gain-of-Function Pathogenic Variant.

Background: STAT1 gain-of-function (GOF) is a primary immune dysregulatory disorder marked by wide infectious predisposition (most notably chronic mucocutaneous Candidiasis), autoimmunity, vascular disease and malignant predisposition. While atopic features have been described in some STAT1 GOF patients, they are not considered a predominant feature of the disease. Additionally, while eosinophilic gastrointestinal infiltration has been reported in some cases, this has always been described in the context of pre-existing oropharyngeal and/or esophageal Candidiasis. Clinical cases: Herein, we report 3 members of a multi-generational family diagnosed with STAT1 GOF caused by a novel mutation in the N-terminal domain, c.194A>C (p.D65A). The proband presented initially with a long-standing history of treatment-refractory eosinophilic esophagitis (EoE) without preceding gastrointestinal tract fungal infections, and her mother was diagnosed with esophagitis as well. Conclusion: EoE has been previously associated with alterations to STAT6 and STAT3 signaling pathways. The current report expands the possible association between JAK/STAT-related disorders and EoE, suggesting that EoE could be a primary disease manifestation of STAT1 GOF, even in the absence of oropharyngeal and/or esophageal Candidiasis.

DNA-Binding domain mutations confer severe outcome at an early age among STAT1 gain-of-function patients.

BACKGROUND: STAT1 gain-of-function (GOF) is an immune dysregulatory disorder with poorly studied genotype-phenotype correlation, impeding prognostication and early intervention. Given previous mechanistic studies, as well as anecdotal clinical reports, we sought to systematically determine whether DNA-binding domain (DBD) mutations in STAT1 result in a different phenotype than mutations in other gene domains. METHODS: Negative prognostic features previously identified by the International STAT1 GOF Study Group (invasive infections, intracranial aneurysms, and malignancy), as well as other clinical features and mortality, were compared within a cohort of 30 patients with STAT1 GOF diagnosed at our center, consisting of 9 patients with DBD mutations and 21 patients with non-DBD mutations. We subsequently re-analyzed mortality data from a large, previously-published 274-patient cohort by the International STAT1 GOF Study Group. RESULTS: While no differences were noted with respect to malignancy or symptomatic aneurysms, invasive /opportunistic infections were substantially more common among DBD patients, as were sinopulmonary infections, bronchiectasis, enteropathy, endocrinopathies, lymphoproliferative manifestations, and recurrent fevers/HLH. DBD patients also had a lower probability of survival and younger age of mortality compared with non-DBD patients. Our re-evaluation of the published data from the International STAT1 GOF Study Group revealed a similar finding of earlier mortality among patients harboring DBD mutations. CONCLUSION: We report that STAT1 GOF patients with DBD mutations may be regarded as a unique subgroup, impacted more by early-onset profound combined immunodeficiency and with earlier mortality. These findings may impact clinical decision making with respect to early intervention, and in particular hematopoietic stem cell transplant considerations, in such patients.

Homozygous duplication identified by whole genome sequencing causes LRBA deficiency.

In more than one-third of primary immunodeficiency (PID) patients, extensive genetic analysis including whole-exome sequencing (WES) fails to identify the genetic defect. Whole-genome sequencing (WGS) is able to detect variants missed by other genomics platforms, enabling the molecular diagnosis of otherwise unresolved cases. Here, we report two siblings, offspring of consanguineous parents, who experienced similar severe events encompassing early onset of colitis, lymphoproliferation, and hypogammaglobulinemia, typical of lipopolysaccharide-responsive and beige-like anchor (LRBA) or cytotoxic T lymphocyte antigen 4 (CTLA4) deficiencies. Gene-panel sequencing, comparative genomic hybridization (CGH) array, and WES failed to reveal a genetic aberration in relevant genes. WGS of these patients detected a 12.3 kb homozygous tandem duplication that was absent in control cohorts and is predicted to disrupt the reading frame of the LRBA gene. The variant was validated by PCR and Sanger sequencing, demonstrating the presence of the junction between the reference and the tandem-duplicated sequence. Droplet digital PCR (ddPCR) further confirmed the copy number in the unaffected parents (CN = 3, heterozygous) and affected siblings (CN = 4, homozygous), confirming the expected segregation pattern. In cases of suspected inherited immunodeficiency, WGS may reveal a mutation when other methods such as microarray and WES analysis failed to detect an aberration.

Thymic Epithelial Cell Alterations and Defective Thymopoiesis Lead to Central and Peripheral Tolerance Perturbation in MHCII Deficiency.

Major Histocompatibility Complex (MHC) class II (MHCII) deficiency (MHCII-D), also known as Bare Lymphocyte Syndrome (BLS), is a rare combined immunodeficiency due to mutations in genes regulating expression of MHCII molecules. MHCII deficiency results in impaired cellular and humoral immune responses, leading to severe infections and autoimmunity. Abnormal cross-talk with developing T cells due to the absence of MHCII expression likely leads to defects in thymic epithelial cells (TEC). However, the contribution of TEC alterations to the pathogenesis of this primary immunodeficiency has not been well characterized to date, in particular in regard to immune dysregulation. To this aim, we have performed an in-depth cellular and molecular characterization of TEC in this disease. We observed an overall perturbation of thymic structure and function in both MHCII-/- mice and patients. Transcriptomic and proteomic profiling of murine TEC revealed several alterations. In particular, we demonstrated that impairment of lymphostromal cross-talk in the thymus of MHCII-/- mice affects mTEC maturation and promiscuous gene expression and causes defects of central tolerance. Furthermore, we observed peripheral tolerance impairment, likely due to defective Treg cell generation and/or function and B cell tolerance breakdown. Overall, our findings reveal disease-specific TEC defects resulting in perturbation of central tolerance and limiting the potential benefits of hematopoietic stem cell transplantation in MHCII deficiency.

STAT1 gain-of-function heterozygous cell models reveal diverse interferon-signature gene transcriptional responses.

Signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) is an autosomal dominant immune disorder marked by wide infectious predisposition, autoimmunity, vascular disease, and malignancy. Its molecular hallmark, elevated phospho-STAT1 (pSTAT1) following interferon (IFN) stimulation, is seen consistently in all patients and may not fully account for the broad phenotypic spectrum associated with this disorder. While over 100 mutations have been implicated in STAT1 GOF, genotype-phenotype correlation remains limited, and current overexpression models may be of limited use in gene expression studies. We generated heterozygous mutants in diploid HAP1 cells using CRISPR/Cas9 base-editing, targeting the endogenous STAT1 gene. Our models recapitulated the molecular phenotype of elevated pSTAT1, and were used to characterize the expression of five IFN-stimulated genes under a number of conditions. At baseline, transcriptional polarization was evident among mutants compared with wild type, and this was maintained following prolonged serum starvation. This suggests a possible role for unphosphorylated STAT1 in the pathogenesis of STAT1 GOF. Following stimulation with IFNα or IFNγ, differential patterns of gene expression emerged among mutants, including both gain and loss of transcriptional function. This work highlights the importance of modeling heterozygous conditions, and in particular transcription factor-related disorders, in a manner which accurately reflects patient genotype and molecular signature. Furthermore, we propose a complex and multifactorial transcriptional profile associated with various STAT1 mutations, adding to global efforts in establishing STAT1 GOF genotype-phenotype correlation and enhancing our understanding of disease pathogenesis.

Disruption of exon-bridging interactions between the minor and major spliceosomes results in alternative splicing around minor introns.

Vertebrate genomes contain major (>99.5%) and minor (<0.5%) introns that are spliced by the major and minor spliceosomes, respectively. Major intron splicing follows the exon-definition model, whereby major spliceosome components first assemble across exons. However, since most genes with minor introns predominately consist of major introns, formation of exon-definition complexes in these genes would require interaction between the major and minor spliceosomes. Here, we report that minor spliceosome protein U11-59K binds to the major spliceosome U2AF complex, thereby supporting a model in which the minor spliceosome interacts with the major spliceosome across an exon to regulate the splicing of minor introns. Inhibition of minor spliceosome snRNAs and U11-59K disrupted exon-bridging interactions, leading to exon skipping by the major spliceosome. The resulting aberrant isoforms contained a premature stop codon, yet were not subjected to nonsense-mediated decay, but rather bound to polysomes. Importantly, we detected elevated levels of these alternatively spliced transcripts in individuals with minor spliceosome-related diseases such as Roifman syndrome, Lowry-Wood syndrome and early-onset cerebellar ataxia. In all, we report that the minor spliceosome informs splicing by the major spliceosome through exon-definition interactions and show that minor spliceosome inhibition results in aberrant alternative splicing in disease.

Characterization of the clinical and immunologic phenotype and management of 157 individuals with 56 distinct heterozygous NFKB1 mutations.

BACKGROUND: An increasing number of NFKB1 variants are being identified in patients with heterogeneous immunologic phenotypes. OBJECTIVE: To characterize the clinical and cellular phenotype as well as the management of patients with heterozygous NFKB1 mutations. METHODS: In a worldwide collaborative effort, we evaluated 231 individuals harboring 105 distinct heterozygous NFKB1 variants. To provide evidence for pathogenicity, each variant was assessed in silico; in addition, 32 variants were assessed by functional in vitro testing of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) signaling. RESULTS: We classified 56 of the 105 distinct NFKB1 variants in 157 individuals from 68 unrelated families as pathogenic. Incomplete clinical penetrance (70%) and age-dependent severity of NFKB1-related phenotypes were observed. The phenotype included hypogammaglobulinemia (88.9%), reduced switched memory B cells (60.3%), and respiratory (83%) and gastrointestinal (28.6%) infections, thus characterizing the disorder as primary immunodeficiency. However, the high frequency of autoimmunity (57.4%), lymphoproliferation (52.4%), noninfectious enteropathy (23.1%), opportunistic infections (15.7%), autoinflammation (29.6%), and malignancy (16.8%) identified NF-κB1-related disease as an inborn error of immunity with immune dysregulation, rather than a mere primary immunodeficiency. Current treatment includes immunoglobulin replacement and immunosuppressive agents. CONCLUSIONS: We present a comprehensive clinical overview of the NF-κB1-related phenotype, which includes immunodeficiency, autoimmunity, autoinflammation, and cancer. Because of its multisystem involvement, clinicians from each and every medical discipline need to be made aware of this autosomal-dominant disease. Hematopoietic stem cell transplantation and NF-κB1 pathway-targeted therapeutic strategies should be considered in the future.

Prevalence and Clinical Features of Inflammatory Bowel Diseases Associated With Monogenic Variants, Identified by Whole-Exome Sequencing in 1000 Children at a Single Center.

BACKGROUND & AIMS: A proportion of infants and young children with inflammatory bowel diseases (IBDs) have subtypes associated with a single gene variant (monogenic IBD). We aimed to determine the prevalence of monogenic disease in a cohort of pediatric patients with IBD. METHODS: We performed whole-exome sequencing analyses of blood samples from an unselected cohort of 1005 children with IBD, aged 0-18 years (median age at diagnosis, 11.96 years) at a single center in Canada and their family members (2305 samples total). Variants believed to cause IBD were validated using Sanger sequencing. Biopsies from patients were analyzed by immunofluorescence and histochemical analyses. RESULTS: We identified 40 rare variants associated with 21 monogenic genes among 31 of the 1005 children with IBD (including 5 variants in XIAP, 3 in DOCK8, and 2 each in FOXP3, GUCY2C, and LRBA). These variants occurred in 7.8% of children younger than 6 years and 2.3% of children aged 6-18 years. Of the 17 patients with monogenic Crohn's disease, 35% had abdominal pain, 24% had nonbloody loose stool, 18% had vomiting, 18% had weight loss, and 5% had intermittent bloody loose stool. The 14 patients with monogenic ulcerative colitis or IBD-unclassified received their diagnosis at a younger age, and their most predominant feature was bloody loose stool (78%). Features associated with monogenic IBD, compared to cases of IBD not associated with a single variant, were age of onset younger than 2 years (odds ratio [OR], 6.30; P = .020), family history of autoimmune disease (OR, 5.12; P = .002), extra-intestinal manifestations (OR, 15.36; P < .0001), and surgery (OR, 3.42; P = .042). Seventeen patients had variants in genes that could be corrected with allogeneic hematopoietic stem cell transplantation. CONCLUSIONS: In whole-exome sequencing analyses of more than 1000 children with IBD at a single center, we found that 3% had rare variants in genes previously associated with pediatric IBD. These were associated with different IBD phenotypes, and 1% of the patients had variants that could be potentially corrected with allogeneic hematopoietic stem cell transplantation. Monogenic IBD is rare, but should be considered in analysis of all patients with pediatric onset of IBD.

Morbidity in an adenosine deaminase-deficient patient during 27 years of enzyme replacement therapy.

INTRODUCTION: Adenosine deaminase (ADA) deficiency causes severe immunodeficiency that is lethal in infancy. Enzyme replacement therapy (ERT) can improve the metabolic, immune and non-immune abnormalities in patients prior to transplantation, however, its benefits over extended periods are not well characterized. We describe a 28-year-old female who received ERT for 27 years. She suffered from EBV negative B cell lymphoma of the hip at 14 years of age and Guillian-Barre Syndrome 2 years later. At 22 years of age, she experienced a gastrointestinal infection with Mycobacterium genavense. At 26 years of age, lymphoma reoccurred with multiple liver lesions followed by Mycobacterium genavense infection with dissemination to the brain. Throughout this period, ADA activity in the plasma was within the therapeutic range. Repeated evaluations demonstrated very low lymphocyte counts and impaired T cell function. CONCLUSIONS: ERT might be insufficient to maintain normal immunity over extended periods in some ADA-deficient patients.

New insights into minor splicing-a transcriptomic analysis of cells derived from TALS patients.

Minor intron splicing plays a central role in human embryonic development and survival. Indeed, biallelic mutations in RNU4ATAC, transcribed into the minor spliceosomal U4atac snRNA, are responsible for three rare autosomal recessive multimalformation disorders named Taybi-Linder (TALS/MOPD1), Roifman (RFMN), and Lowry-Wood (LWS) syndromes, which associate numerous overlapping signs of varying severity. Although RNA-seq experiments have been conducted on a few RFMN patient cells, none have been performed in TALS, and more generally no in-depth transcriptomic analysis of the ∼700 human genes containing a minor (U12-type) intron had been published as yet. We thus sequenced RNA from cells derived from five skin, three amniotic fluid, and one blood biosamples obtained from seven unrelated TALS cases and from age- and sex-matched controls. This allowed us to describe for the first time the mRNA expression and splicing profile of genes containing U12-type introns, in the context of a functional minor spliceosome. Concerning RNU4ATAC-mutated patients, we show that as expected, they display distinct U12-type intron splicing profiles compared to controls, but that rather unexpectedly mRNA expression levels are mostly unchanged. Furthermore, although U12-type intron missplicing concerns most of the expressed U12 genes, the level of U12-type intron retention is surprisingly low in fibroblasts and amniocytes, and much more pronounced in blood cells. Interestingly, we found several occurrences of introns that can be spliced using either U2, U12, or a combination of both types of splice site consensus sequences, with a shift towards splicing using preferentially U2 sites in TALS patients' cells compared to controls.

NF-κB pathway and the Goldilocks principle: Lessons from human disorders of immunity and inflammation.

Nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling pathways play a key role in various cell processes related to host immunity. The last few years have seen an explosion of disorders associated with NF-κB components from core members of the canonical and noncanonical cascades to adaptor protein and ubiquitination-related enzymes. Disease phenotypes have extended beyond susceptibility to infections and include autoimmunity, lymphoproliferation, atopy, and inflammation. Concurrently, studies are unveiling a tightly regulated system marked by extensive cross-talk between the canonical and noncanonical pathways, as well as among the NF-κB and other signaling pathways. As the rate of discovery in the realm of NF-κB defects accelerates, this review presents a timely summary of major known defects causing human disease, as well as diagnostic, therapeutic, and research challenges and opportunities.

Ataxia Telangiectasia Diagnosed on Newborn Screening-Case Cohort of 5 Years' Experience.

Ataxia telangiectasia (AT) is a genetic condition caused by mutations involving ATM (Ataxia Telangiectasia Mutated). This gene is responsible for the expression of a DNA double stranded break repair kinase, the ATM protein kinase. The syndrome encompasses combined immunodeficiency and various degrees of neurological abnormalities and increased risk of malignancy. Typically, patients present early in life with delay in neurological milestones, but very infrequently, with life threatening infections typical of a profound T cell deficiency. It would therefore be unexpected to identify this condition immediately after birth using T cell receptor excision circle (TREC)-based newborn screening (NBS) for SCID. We sought to evaluate the frequency of AT detected by NBS, and to assess immunity as well as the genetic aberrations associated with this early presentation. Here, we describe the clinical, laboratory, and genetic features of patients diagnosed with AT through the Ontario NBS program for SCID, and followed in our center since its inception in 2013. Four patients were diagnosed with AT as a result of low TRECs on NBS. In each case, whole exome sequencing was diagnostic. All of our patients had compound heterozygous mutations involving the FRAP-ATM-TRRAP (FAT) domain of the ATM gene, which appears critical for kinase activity and is highly sensitive to mutagenesis. Our patients presented with profound lymphopenia involving both B and T cells. The ratio of naïve/memory CD45+RA/RO T cells population was variable. T cell repertoire showed decreased T cell diversity. Two out of four patients had decreased specific antibody response to vaccination and hypogammaglobulinemia requiring IVIG replacement. In two patients, profound decreased responses to phytohemagglutinin stimulation was observed. In the other two patients, the initial robust response declined with time. In summary, the rate of detection of AT through NBS had been surprisingly high at our center. One case was identified per year, while the total rate for SCID has been five new cases per year. This early detection may allow for better prospective evaluation of AT shortly after birth, and may assist in formulating early and more effective interventions both for the neurological as well as the immune abnormalities in this syndrome.

Hematopoietic stem cell transplantation in patients with gain-of-function signal transducer and activator of transcription 1 mutations.

BACKGROUND: Gain-of-function (GOF) mutations in signal transducer and activator of transcription 1 (STAT1) cause susceptibility to a range of infections, autoimmunity, immune dysregulation, and combined immunodeficiency. Disease manifestations can be mild or severe and life-threatening. Hematopoietic stem cell transplantation (HSCT) has been used in some patients with more severe symptoms to treat and cure the disorder. However, the outcome of HSCT for this disorder is not well established. OBJECTIVE: We sought to aggregate the worldwide experience of HSCT in patients with GOF-STAT1 mutations and to assess outcomes, including donor engraftment, overall survival, graft-versus-host disease, and transplant-related complications. METHODS: Data were collected from an international cohort of 15 patients with GOF-STAT1 mutations who had undergone HSCT using a variety of conditioning regimens and donor sources. Retrospective data collection allowed the outcome of transplantation to be assessed. In vitro functional testing was performed to confirm that each of the identified STAT1 variants was in fact a GOF mutation. RESULTS: Primary donor engraftment in this cohort of 15 patients with GOF-STAT1 mutations was 74%, and overall survival was only 40%. Secondary graft failure was common (50%), and posttransplantation event-free survival was poor (10% by 100 days). A subset of patients had hemophagocytic lymphohistiocytosis before transplant, contributing to their poor outcomes. CONCLUSION: Our data indicate that HSCT for patients with GOF-STAT1 mutations is curative but has significant risk of secondary graft failure and death.

Dual loss of p110δ PI3-kinase and SKAP (KNSTRN) expression leads to combined immunodeficiency and multisystem syndromic features.

BACKGROUND: We previously reported a novel syndrome characterized by combined immunodeficiency associated with severe developmental defects-subsequently known as Roifman-Chitayat syndrome (RCS; OMIM 613328). Linkage analysis identified 2 disease-associated loci. OBJECTIVES: We sought to identify the genetic defect in these patients and characterize their immunologic cellular abnormalities. METHODS: Genetic, immunologic, protein, and cellular functional analyses were used to identify and characterize patient genetic deficiencies and aberrant patient cell behavior. RESULTS: Deleterious variants were found at both loci identified by linkage analysis: a homozygous stop codon in PI3-kinase p110δ (PIK3CD) and a homozygous frame shift mutation in SKAP (KNSTRN), both ablating protein expression. Patients with RCS display aberrant B-cell development, similar to p110δ-deficient mice, but also aberrant T-cell spreading, cell-cell interaction, and migration. Patients also display significant developmental abnormalities not seen in p110δ knockouts (eg, optic nerve atrophy and skeletal anomalies) that we ascribe to loss of SKAP. Aberrant SKAP expression can prolong anaphase and this may contribute to developmental defects. However, we also identified microtubule-associated protein 4 microtubule-binding protein as a novel SKAP-binding partner and show that it undergoes relocalization in patient T cells, with associated areas of aberrant microtubule hyperstabilization, likely contributing not only to the altered properties of RCS lymphoid cells but also to developmental defects. CONCLUSIONS: The complex RCS presentation, with combined developmental and immunologic defects, is associated with a combined deficiency of 2 genes products, PI3-kinase p110δ and SKAP, both of which appear to play a significant role in the disease.

A homozygous mutation in the stem II domain of RNU4ATAC causes typical Roifman syndrome.

Roifman syndrome (OMIM# 616651) is a complex syndrome encompassing skeletal dysplasia, immunodeficiency, retinal dystrophy and developmental delay, and is caused by compound heterozygous mutations involving the Stem II region and one of the other domains of the RNU4ATAC gene. This small nuclear RNA gene is essential for minor intron splicing. The Canadian Centre for Primary Immunodeficiency Registry and Repository were used to derive patient information as well as tissues. Utilising RNA sequencing methodologies, we analysed samples from patients with Roifman syndrome and assessed intron retention. We demonstrate that a homozygous mutation in Stem II is sufficient to cause the full spectrum of features associated with typical Roifman syndrome. Further, we demonstrate the same pattern of aberration in minor intron retention as found in cases with compound heterozygous mutations.

Novel Combined Immune Deficiency and Radiation Sensitivity Blended Phenotype in an Adult with Biallelic Variations in ZAP70 and RNF168.

With the advent of high-throughput genomic sequencing techniques, novel genetic etiologies are being uncovered for previously unexplained Mendelian phenotypes, and the underlying genetic architecture of disease is being unraveled. Although most of these "mendelizing" disease traits represent phenotypes caused by single-gene defects, a percentage of patients have blended phenotypes caused by pathogenic variants in multiple genes. We describe an adult patient with susceptibility to bacterial, herpesviral, and fungal infections. Immunologic defects included CD8+ T cell lymphopenia, decreased T cell proliferative responses to mitogens, hypogammaglobulinemia, and radiation sensitivity. Whole-exome sequencing revealed compound heterozygous variants in ZAP70. Biallelic mutations in ZAP70 are known to produce a spectrum of immune deficiency that includes the T cell abnormalities observed in this patient. Analyses for variants in genes associated with radiation sensitivity identified the presence of a homozygous RNF168 variant of unknown significance. RNF168 deficiency causes radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties syndrome and may account for the radiation sensitivity. Thus, the patient was found to have a novel blended phenotype associated with multilocus genomic variation: i.e., separate and distinct genetic defects. These findings further illustrate the clinical utility of applying genomic testing in patients with primary immunodeficiency diseases.