Genomics of Immune Diseases and New Therapies.

Genomic DNA sequencing technologies have been one of the great advances of the 21st century, having decreased in cost by seven orders of magnitude and opening up new fields of investigation throughout research and clinical medicine. Genomics coupled with biochemical investigation has allowed the molecular definition of a growing number of new genetic diseases that reveal new concepts of immune regulation. Also, defining the genetic pathogenesis of these diseases has led to improved diagnosis, prognosis, genetic counseling, and, most importantly, new therapies. We highlight the investigational journey from patient phenotype to treatment using the newly defined XMEN disease, caused by the genetic loss of the MAGT1 magnesium transporter, as an example. This disease illustrates how genomics yields new fundamental immunoregulatory insights as well as how research genomics is integrated into clinical immunology. At the end, we discuss two other recently described diseases, CHAI/LATAIE (CTLA-4 deficiency) and PASLI (PI3K dysregulation), as additional examples of the journey from unknown immunological diseases to new precision medicine treatments using genomics.

Congenital iRHOM2 deficiency causes ADAM17 dysfunction and environmentally directed immunodysregulatory disease.

We report a pleiotropic disease due to loss-of-function mutations in RHBDF2, the gene encoding iRHOM2, in two kindreds with recurrent infections in different organs. One patient had recurrent pneumonia but no colon involvement, another had recurrent infectious hemorrhagic colitis but no lung involvement and the other two experienced recurrent respiratory infections. Loss of iRHOM2, a rhomboid superfamily member that regulates the ADAM17 metalloproteinase, caused defective ADAM17-dependent cleavage and release of cytokines, including tumor-necrosis factor and amphiregulin. To understand the diverse clinical phenotypes, we challenged Rhbdf2-/- mice with Pseudomonas aeruginosa by nasal gavage and observed more severe pneumonia, whereas infection with Citrobacter rodentium caused worse inflammatory colitis than in wild-type mice. The fecal microbiota in the colitis patient had characteristic oral species that can predispose to colitis. Thus, a human immunodeficiency arising from iRHOM2 deficiency causes divergent disease phenotypes that can involve the local microbial environment.

Loss of the TRPM4 channel in humans causes immune dysregulation with defective monocyte migration.

BACKGROUND: TRPM4 is a broadly expressed, calcium-activated, monovalent cation channel that regulates immune cell function in mice and cell lines. Clinically, however, partial loss- or gain-of-function mutations in TRPM4 lead to arrhythmia and heart disease, with no documentation of immunologic disorders. OBJECTIVE: To characterize functional cellular mechanisms underlying the immune dysregulation phenotype in a proband with a mutated TRPM4 gene. METHODS: We employed a combination of biochemical, cell biological, imaging, omics analyses, flow cytometry, and gene editing approaches. RESULTS: We report the first human cases to our knowledge with complete loss of the TRPM4 channel, leading to immune dysregulation with frequent bacterial and fungal infections. Single-cell and bulk RNA sequencing point to altered expression of genes affecting cell migration, specifically in monocytes. Inhibition of TRPM4 in T cells and the THP-1 monocyte cell line reduces migration. More importantly, primary T cells and monocytes from TRPM4 patients migrate poorly. Finally, CRISPR knockout of TRPM4 in THP-1 cells greatly reduces their migration potential. CONCLUSION: Our results demonstrate that TRPM4 plays a critical role in regulating immune cell migration, leading to increased susceptibility to infections.

Novel Synonymous Variant in IL7R Causes Preferential Expression of the Soluble Isoform.

PURPOSE: The interleukin-7 receptor (IL-7R) is primarily expressed on lymphoid cells and plays a crucial role in the development, proliferation, and survival of T cells. Autosomal recessive mutations that disrupt IL-7Rα chain expression give rise to a severe combined immunodeficiency (SCID), which is characterized by lymphopenia and a T-B+NK+ phenotype. The objective here was to diagnose two siblings displaying the T-B+NK+ SCID phenotype as initial clinical genetic testing did not detect any variants in known SCID genes. METHODS: Whole genome sequencing (WGS) was utilized to identify potential variants causing the SCID phenotype. Splicing prediction tools were employed to assess the deleterious impact of the mutation. Polymerase Chain Reaction (PCR), Sanger sequencing, flow cytometry, and ELISA were then used to validate the pathogenicity of the detected mutation. RESULTS: We discovered a novel homozygous synonymous mutation in the IL7R gene. Our functional studies indicate that this variant is pathogenic, causing exon 6, which encodes the transmembrane domain, to be preferentially spliced out. CONCLUSION: In this study, we identified a novel rare synonymous mutation causing a loss of IL-7Rα expression at the cellular membrane. This case demonstrates the value of reanalyzing genetic data based on the clinical phenotype and highlights the significance of functional studies in determining the pathogenicity of genetic variants.

Uniparental Disomy of Chromosome 4: A Case of Whole Chromosome UPD Presenting with LRBA Deficiency.

PURPOSE: Lipopolysaccharide-responsive beige-like anchor protein (LRBA) encodes a widely expressed cytosolic protein that participates in polarized vesicle trafficking. Homozygous loss-of-function LRBA mutations can lead to immune deficiency due to the lack of immune regulation, classified as a part of Tregopathies. We present a case of a 49-year-old female, with polyarthralgia in metacarpophalangeal and proximal interphalangeal joints bilaterally, and morning stiffness, leading to the diagnosis of rheumatoid arthritis treated with pulse steroid therapy. She had experienced sepsis and in-depth scrutiny revealed panhypogammaglobulinemia. After being referred to the immunology clinic, she was followed under the diagnosis of common variable immunodeficiency (CVID)-like inborn errors of immunity (IEI). METHODS AND RESULTS: Physical examination and diagnostic follow-up revealed massive splenomegaly accompanied by portal hypertension, and ulcerations in the colon. She also presented with periodic hematuria and dysuria. Cystoscopic biopsy revealed mast cell-derived interstitial cystitis which has not been previously reported in LRBA deficiency in the literature to our knowledge. A multi-gene next-generation sequencing panel performed for immune deficiencies (264 genes and 524 amplicons), resulted in the identification of an apparently homozygous LRBA mutation (p.Arg722His) in the Beige and Chediak-Higashi (BEACH) domain. The SNP array showed copy neutral absence of heterozygosity of the entire chromosome 4, which is consistent with uniparental isodisomy of chromosome 4. CONCLUSION: In conclusion, this case study underscores the critical role of LRBA in immune regulation and highlights the clinical heterogeneity associated with LRBA deficiency. The patient's presentation with severe immune dysregulation, including massive splenomegaly, portal hypertension, and the novel finding of mast cell-derived interstitial cystitis, expands the clinical spectrum of LRBA mutations. The identification of an apparently homozygous LRBA mutation via next-generation sequencing further emphasizes the importance of genetic analysis in diagnosing monogenic defects manifested as CVID-like phenotype. This is the first reported case of LRBA deficiency due to whole chromosome UPD to our knowledge. Future research should focus on elucidating the full range of clinical manifestations and developing targeted therapies for patients with LRBA deficiency.

Biallelic PI4KA Mutations Disrupt B-Cell Metabolism and Cause B-Cell Lymphopenia and Hypogammaglobulinemia.

PURPOSE: PI4KA-related disorder is a highly clinically variable condition characterized by neurological (limb spasticity, developmental delay, intellectual disability, seizures, ataxia, nystagmus) and gastrointestinal (inflammatory bowel disease and multiple intestinal atresia) manifestations. Although features consistent with immunodeficiency (autoimmunity/autoinflammation and recurrent infections) have been reported in a subset of patients, the burden of B-cell deficiency and hypogammaglobulinemia has not been extensively investigated. We sought to describe the clinical presentation and manifestations of patients with PI4KA-related disorder and to investigate the metabolic consequences of biallelic PI4KA variants in B cells. METHODS: Clinical data from patients with PI4KA variants were obtained. Multi-omics analyses combining transcriptome, proteome, lipidome and metabolome analyses in conjunction with functional assays were performed in EBV-transformed B cells. RESULTS: Clinical and laboratory data of 13 patients were collected. Recurrent infections (7/13), autoimmune/autoinflammatory manifestations (5/13), B-cell deficiency (8/13) and hypogammaglobulinemia (8/13) were frequently observed. Patients' B cells frequently showed increased transitional and decreased switched memory B-cell subsets. Pathway analyses based on differentially expressed transcripts and proteins confirmed the central role of PI4KA in B cell differentiation with altered B-cell receptor (BCR) complex and signalling. By altering lipids production and tricarboxylic acid cycle regulation, and causing increased endoplasmic reticulum stress, biallelic PI4KA mutations disrupt B cell metabolism inducing mitochondrial dysfunction. As a result, B cells show hyperactive PI3K/mTOR pathway, increased autophagy and deranged cytoskeleton organization. CONCLUSION: By altering lipid metabolism and TCA cycle, impairing mitochondrial activity, hyperactivating mTOR pathway and increasing autophagy, PI4KA-related disorder causes a syndromic inborn error of immunity presenting with B-cell deficiency and hypogammaglobulinemia.

Biallelic TLR4 deficiency in humans.

BACKGROUND: Toll-like receptors (TLRs) mediate functions for host defense and inflammatory responses. TLR4 recognizes LPS, a component of gram-negative bacteria as well as host-derived endogenous ligands such as S100A8 and S100A9 proteins. OBJECTIVE: We sought to report phenotype and cellular function of individuals with complete TLR4 deficiency. METHODS: We performed genome sequencing and investigated exome and genome sequencing databases. Cellular responses were studied on primary monocytes, macrophages, and neutrophils, as well as cell lines using flow cytometry, reporter, and cytokine assays. RESULTS: We identified 2 individuals in a family of Qatari origin carrying a homozygous stop codon variant p.Q188X in TLR4 presenting with a variable phenotype (asymptomatic and inflammatory bowel disease consistent with severe perianal Crohn disease). A third individual with homozygous p.Y794X was identified in a population database. In contrast to hypomorphic polymorphisms p.D299G and p.T399I, the variants p.Q188X and p.Y794X completely abrogated LPS-induced cytokine responses whereas TLR2 response was normal. TLR4 deficiency causes a neutrophil CD62L shedding defect, whereas antimicrobial activity toward intracellular Salmonella was intact. CONCLUSIONS: Biallelic TLR4 deficiency in humans causes an inborn error of immunity in responding to LPS. This complements the spectrum of known primary immunodeficiencies, in particular myeloid differentiation primary response 88 (MYD88) or the IL-1 receptor-associated kinase 4 (IRAK4) deficiency that are downstream of TLR4 and TLR2 signaling.

Zero tolerance! A perspective on monogenic disorders with defective regulatory T cells and IBD-like disease.

Recently, several studies have investigated a number of rare monogenic autoimmune disorders, in which the causative genetic defects were identified and found to affect the development or function of regulatory T cells (Tregs). The studies of these disorders have facilitated a deeper understanding of the mechanisms involved in immune regulation and tolerance. Furthermore, these studies have highlighted the importance of Tregs in maintaining homeostasis at the mucosal interface between the host and microbiome. Here, we offer our perspective on these monogenic autoimmune disorders, highlighting their overlapping clinical features with inflammatory bowel disease.

Comparing the levels of CTLA-4-dependent biological defects in patients with LRBA deficiency and CTLA-4 insufficiency.

BACKGROUND: Lipopolysaccharide-responsive beige-like anchor protein (LRBA) deficiency and cytotoxic T-lymphocyte protein-4 (CTLA-4) insufficiency are recently described disorders that present with susceptibility to infections, autoimmunity, and lymphoproliferation. Clinical and immunological comparisons of the diseases with long-term follow-up have not been previously reported. We sought to compare the clinical and laboratory manifestations of both diseases and investigate the role of flow cytometry in predicting the genetic defect in patients with LRBA deficiency and CTLA-4 insufficiency. METHODS: Patients were evaluated clinically with laboratory assessments for lymphocyte subsets, T follicular helper cells (TFH ), LRBA expression, and expression of CD25, FOXP3, and CTLA4 in regulatory T cells (Tregs) at baseline and 16 h post-stimulation. RESULTS: LRBA-deficient patients (n = 29) showed significantly early age of symptom onset, higher rates of pneumonia, autoimmunity, chronic diarrhea, and failure to thrive compared to CTLA-4 insufficiency (n = 12). In total, 29 patients received abatacept with favorable responses and the overall survival probability was not different between transplanted versus non-transplanted patients in LRBA deficiency. Meanwhile, higher probability of survival was observed in CTLA-4-insufficient patients (p = 0.04). The T-cell subsets showed more deviation to memory cells in CTLA-4-insufficiency, accompanied by low percentages of Treg and dysregulated cTFH cells response in both diseases. Cumulative numbers of autoimmunities positively correlated with cTFH frequencies. Baseline CTLA-4 expression was significantly diminished in LRBA deficiency and CTLA-4 insufficiency, but significant induction in CTLA-4 was observed after short-term T-cell stimulation in LRBA deficiency and controls, while this elevation was less in CTLA-4 insufficiency, allowing to differentiate this disease from LRBA deficiency with high sensitivity (87.5%) and specificity (90%). CONCLUSION: This cohort provided detailed clinical and laboratory comparisons for LRBA deficiency and CTLA-4 insufficiency. The flow cytometric approach is useful in predicting the defective gene; thus, targeted sequencing can be conducted to provide rapid diagnosis and treatment for these diseases impacting the CTLA-4 pathway.

Evolution and long-term outcomes of combined immunodeficiency due to CARMIL2 deficiency.

BACKGROUND: Biallelic loss-of-function mutations in CARMIL2 cause combined immunodeficiency associated with dermatitis, inflammatory bowel disease (IBD), and EBV-related smooth muscle tumors. Clinical and immunological characterizations of the disease with long-term follow-up and treatment options have not been previously reported in large cohorts. We sought to determine the clinical and immunological features of CARMIL2 deficiency and long-term efficacy of treatment in controlling different disease manifestations. METHODS: The presenting phenotypes, long-term outcomes, and treatment responses were evaluated prospectively in 15 CARMIL2-deficient patients, including 13 novel cases. Lymphocyte subpopulations, protein expression, regulatory T (Treg), and circulating T follicular helper (cTFH ) cells were analyzed. Three-dimensional (3D) migration assay was performed to determine T-cell shape. RESULTS: Mean age at disease onset was 38 ± 23 months. Main clinical features were skin manifestations (n = 14, 93%), failure to thrive (n = 10, 67%), recurrent infections (n = 10, 67%), allergic symptoms (n = 8, 53%), chronic diarrhea (n = 4, 27%), and EBV-related leiomyoma (n = 2, 13%). Skin manifestations ranged from atopic and seborrheic dermatitis to psoriasiform rash. Patients had reduced proportions of memory CD4+ T cells, Treg, and cTFH cells. Memory B and NK cells were also decreased. CARMIL2-deficient T cells exhibited reduced T-cell proliferation and cytokine production following CD28 co-stimulation and normal morphology when migrating in a high-density 3D collagen gel matrix. IBD was the most severe clinical manifestation, leading to growth retardation, requiring multiple interventional treatments. All patients were alive with a median follow-up of 10.8 years (range: 3-17 years). CONCLUSION: This cohort provides clinical and immunological features and long-term follow-up of different manifestations of CARMIL2 deficiency.

Biallelic PI4KA variants cause neurological, intestinal and immunological disease.

Phosphatidylinositol 4-kinase IIIα (PI4KIIIα/PI4KA/OMIM:600286) is a lipid kinase generating phosphatidylinositol 4-phosphate (PI4P), a membrane phospholipid with critical roles in the physiology of multiple cell types. PI4KIIIα's role in PI4P generation requires its assembly into a heterotetrameric complex with EFR3, TTC7 and FAM126. Sequence alterations in two of these molecular partners, TTC7 (encoded by TTC7A or TCC7B) and FAM126, have been associated with a heterogeneous group of either neurological (FAM126A) or intestinal and immunological (TTC7A) conditions. Here we show that biallelic PI4KA sequence alterations in humans are associated with neurological disease, in particular hypomyelinating leukodystrophy. In addition, affected individuals may present with inflammatory bowel disease, multiple intestinal atresia and combined immunodeficiency. Our cellular, biochemical and structural modelling studies indicate that PI4KA-associated phenotypical outcomes probably stem from impairment of PI4KIIIα-TTC7-FAM126's organ-specific functions, due to defective catalytic activity or altered intra-complex functional interactions. Together, these data define PI4KA gene alteration as a cause of a variable phenotypical spectrum and provide fundamental new insight into the combinatorial biology of the PI4KIIIα-FAM126-TTC7-EFR3 molecular complex.

Novel ORAI1 Mutation Disrupts Channel Trafficking Resulting in Combined Immunodeficiency.

Store-operated Ca2+ entry (SOCE) represents a predominant Ca2+ influx pathway in non-excitable cells. SOCE is required for immune cell activation and is mediated by the plasma membrane (PM) channel ORAI1 and the endoplasmic reticulum (ER) Ca2+ sensor STIM1. Mutations in the Orai1 or STIM1 genes abolish SOCE leading to combined immunodeficiency (CID), muscular hypotonia, and anhidrotic ectodermal dysplasia. Here, we identify a novel autosomal recessive mutation in ORAI1 in a child with CID. The patient is homozygous for p.C126R mutation in the second transmembrane domain (TM2) of ORAI1, a region with no previous loss-of-function mutations. SOCE is suppressed in the patient's lymphocytes, which is associated with impaired T cell proliferation and cytokine production. Functional analyses demonstrate that the p.C126R mutation does not alter protein expression but disrupts ORAI1 trafficking. Orai1-C126R does not insert properly into the bilayer resulting in ER retention. Insertion of an Arg on the opposite face of TM2 (L135R) also results in defective folding and trafficking. We conclude that positive side chains within ORAI1 TM2 are not tolerated and result in misfolding, defective bilayer insertion, and channel trafficking thus abolishing SOCE and resulting in CID.

A Novel STK4 Mutation Impairs T Cell Immunity Through Dysregulation of Cytokine-Induced Adhesion and Chemotaxis Genes.

PURPOSE: Human serine/threonine kinase 4 (STK4) deficiency is a rare, autosomal recessive genetic disorder leading to combined immunodeficiency; however, the extent to which immune signaling and host defense are impaired is unclear. We assessed the functional consequences of a novel, homozygous nonsense STK4 mutation (NM_006282.2:c.871C > T, p.Arg291*) identified in a pediatric patient by comparing his innate and adaptive cell-mediated and humoral immune responses with those of three heterozygous relatives and unrelated controls. METHODS: The genetic etiology was verified by whole genome and Sanger sequencing. STK4 gene and protein expression was measured by quantitative RT-PCR and immunoblotting, respectively. Cellular abnormalities were assessed by high-throughput RT-RCR, RNA-Seq, ELISA, and flow cytometry. Antibody responses were assessed by ELISA and phage immunoprecipitation-sequencing. RESULTS: The patient exhibited partial loss of STK4 expression and complete loss of STK4 function combined with recurrent viral and bacterial infections, notably persistent Epstein-Barr virus viremia and pulmonary tuberculosis. Cellular and molecular analyses revealed abnormal fractions of T cell subsets, plasmacytoid dendritic cells, and NK cells. The transcriptional responses of the patient's whole blood and PBMC samples indicated dysregulated interferon signaling, impaired T cell immunity, and increased T cell apoptosis as well as impaired regulation of cytokine-induced adhesion and leukocyte chemotaxis genes. Nonetheless, the patient had detectable vaccine-specific antibodies and IgG responses to various pathogens, consistent with a normal CD19 + B cell fraction, albeit with a distinctive antibody repertoire, largely driven by herpes virus antigens. CONCLUSION: Patients with STK4 deficiency can exhibit broad impairment of immune function extending beyond lymphoid cells.

Identifying Novel Mutations in Iranian Patients with LPS-responsive Beige-like Anchor Protein (LRBA) Deficiency.

LPS-responsive beige-like anchor protein (LRBA) deficiency is a monogenic primary immunodeficiency characterized by a heterogeneous spectrum of clinical manifestations associated with immune dysregulation. In this study, we reported clinical, immunologic, and genetic evaluation of two Iranian patients from unrelated families, both suffering from recurrent respiratory tract infections, failure to thrive, interstitial lung disease, autoimmune cytopenia, and hypogammaglobulinemia. Pulmonary abscess in one patient and persistent enteropathy in another were also observed. Further investigations revealed causative mutations in the exon (c.2166_2766del) and intron (c.4730-3 T > G) of the LRBA gene. These results may provide further elucidation of the clinical phenotypes and responsible genetic factors of LRBA deficiency.

A Spectrum of Clinical Findings from ALPS to CVID: Several Novel LRBA Defects.

INTRODUCTION: Autosomal recessively inherited lipopolysaccharide-responsive beige-like anchor (LRBA) protein deficiency was shown to be responsible for different types of inborn errors of immunity, such as common variable immunodeficiency (CVID) and autoimmune lymphoproliferative syndrome (ALPS). The aim of this study was to compare patients with LRBA-related ALPS and LRBA-related CVID, to describe their clinical and laboratory phenotypes, and to prepare an algorithm for their diagnosis and management. METHODS: Fifteen LRBA-deficient patients were identified among 31 CVID and 14 possible ALPS patients with Western blotting (WB), primary immunodeficiency disease (PIDD) gene, next-generation panel screening (NGS), and whole exome sequencing (WES). RESULTS: The median age on admission and age of diagnosis were 7 years (0.3-16.5) and 11 years (5-44), respectively. Splenomegaly was seen in 93.3% (14/15) of the patients on admission. Splenectomy was performed to 1/5. Recurrent upper respiratory tract infections (93.3% (14/15)), autoimmune cytopenia (80% (12/15)), chronic diarrhea (53.3% (8/15)), lower respiratory tract infections (53.3% (8/15)), lymphoma (26.6% (4/15)), Evans syndrome (26.6% (4/15)), and autoimmune thyroiditis (20% (3/15)) were common clinical findings and diseases. Lymphopenia (5/15), intermittant neutropenia (4/15), eosinophilia (4/15), and progressive hypogammaglobulinemia are recorded in given number of patients. Double negative T cells (TCRαß+CD4-CD8-) were increased in 80% (8/10) of the patients. B cell percentage/numbers were low in 60% (9/15) of the patients on admission. Decreased switched memory B cells, decreased naive and recent thymic emigrant (RTE) Thelper (Th) cells, markedly increased effector memory/effector memory RA+ (TEMRA) Th were documented. Large PD1+ population, increased memory, and enlarged follicular helper T cell population in the CD4+ T cell compartment was seen in one of the patients. Most of the deleterious missense mutations were located in the DUF1088 and BEACH domains. Interestingly, one of the two siblings with the same homozygous LRBA defect did not have any clinical symptom. Hematopoietic stem cell transplantation (HSCT) was performed to 7/15 (46.6%) of the patients. Transplanted patients are alive and well after a median of 2 years (1-3). In total, one patient died from sepsis during adulthood before HSCT. CONCLUSION: Patients with LRBA deficiency may initially be diagnosed as CVID or ALPS in the clinical practice. Progressive decrease in B cells as well as IgG in ALPS-like patients and addition of IBD symptoms in the follow-up should raise the suspicion for LRBA deficiency. Decreased switched memory B cells, decreased naive and recent thymic emigrant (RTE) Th cells, and markedly increased effector memory/effector memory RA+ Th cells (TEMRA Th) cells are important for the diagnosis of the patients in addition to clinical features. Analysis of protein by either WB or flow cytometry is required when the clinicians come across especially with missense LRBA variants of uncertain significance. High rate of malignancy shows the regulatory T cell's important role of immune surveillance. HSCT is curative and succesful in patients with HLA-matched family donor.

CHAI and LATAIE: new genetic diseases of CTLA-4 checkpoint insufficiency.

CTLA-4 is a critical inhibitory "checkpoint" molecule of immune activation. Several recent reports have described patients with immune dysregulation and lymphoproliferative disease resulting from 2 different genetic diseases that directly or indirectly cause CTLA-4 deficiency. Numerous articles have also been published describing CTLA-4 blockade in cancer immunotherapy and its side effects, which are ultimately the consequence of treatment-induced CTLA-4 deficiency. Here, we review these 2 diseases and CTLA-4 blockade therapy, emphasizing the crucial role of CTLA-4 in immune checkpoint regulation.

Two hits in one: whole genome sequencing unveils LIG4 syndrome and urofacial syndrome in a case report of a child with complex phenotype.

BACKGROUND: Ligase IV syndrome, a hereditary disease associated with compromised DNA damage response mechanisms, and Urofacial syndrome, caused by an impairment of neural cell signaling, are both rare genetic disorders, whose reports in literature are limited. We describe the first case combining both disorders in a specific phenotype. CASE PRESENTATION: We report a case of a 7-year old girl presenting with a complex phenotype characterized by multiple congenital abnormalities and dysmorphic features, microcephaly, short stature, combined immunodeficiency and severe vesicoureteral reflux. Whole Genome Sequencing was performed and a novel ligase IV homozygous missense c.T1312C/p.Y438H mutation was detected, and is believed to be responsible for most of the clinical features of the child, except vesicoureteral reflux which has not been previously described for ligase IV deficiency. However, we observed a second rare damaging (nonsense) homozygous mutation (c.C2125T/p.R709X) in the leucine-rich repeats and immunoglobulin-like domains 2 gene that encodes a protein implicated in neural cell signaling and oncogenesis. Interestingly, this mutation has recently been reported as pathogenic and causing urofacial syndrome, typically displaying vesicoureteral reflux. Thus, this second mutation completes the missing genetic explanation for this intriguing clinical puzzle. We verified that both mutations fit an autosomal recessive inheritance model due to extensive consanguinity. CONCLUSIONS: We successfully identified a novel ligase IV mutation, causing ligase IV syndrome, and an additional rare leucine-rich repeats and immunoglobulin-like domains 2 gene nonsense mutation, in the context of multiple autosomal recessive conditions due to extensive consanguinity. This work demonstrates the utility of Whole Genome Sequencing data in clinical diagnosis in such cases where the combination of multiple rare phenotypes results in very intricate clinical pictures. It also reports a novel causative mutation and a clinical phenotype, which will help in better defining the essential features of both ligase IV and leucine-rich repeats and immunoglobulin-like domains 2 deficiency syndromes.

Natural history of autoimmune lymphoproliferative syndrome associated with FAS gene mutations.

Autoimmune lymphoproliferative syndrome (ALPS) presents in childhood with nonmalignant lymphadenopathy and splenomegaly associated with a characteristic expansion of mature CD4 and CD8 negative or double negative T-cell receptor αß(+) T lymphocytes. Patients often present with chronic multilineage cytopenias due to autoimmune peripheral destruction and/or splenic sequestration of blood cells and have an increased risk of B-cell lymphoma. Deleterious heterozygous mutations in the FAS gene are the most common cause of this condition, which is termed ALPS-FAS. We report the natural history and pathophysiology of 150 ALPS-FAS patients and 63 healthy mutation-positive relatives evaluated in our institution over the last 2 decades. Our principal findings are that FAS mutations have a clinical penetrance of <60%, elevated serum vitamin B12 is a reliable and accurate biomarker of ALPS-FAS, and the major causes of morbidity and mortality in these patients are the overwhelming postsplenectomy sepsis and development of lymphoma. With longer follow-up, we observed a significantly greater relative risk of lymphoma than previously reported. Avoiding splenectomy while controlling hypersplenism by using corticosteroid-sparing treatments improves the outcome in ALPS-FAS patients. This trial was registered at www.clinicaltrials.gov as #NCT00001350.