Homozygous Loss Of Function Mutations Research Articles

A single center experience on PI3K/AKT/MTOR signaling defects: Towards pathogenicity assessment for four novel defects.

Phosphoinositide 3 kinases (PI3K) are lipid kinases expressed in lymphocytes/myeloid cells. PI3K/AKT/mTOR signaling defects present with recurrent infections, autoimmunity, lymphoproliferation, and agammaglobulinemia. To characterize the PI3K/AKT/mTOR pathway defects and perform pathway analyses to assess novel variant pathogenicity. We included 12 patients (heterozygous PIK3CD (n = 9) and PIK3R1 (n = 1) (activated PI3K delta syndrome (APDS) with gain-of-function mutations) and homozygous PIK3R1 variant (n = 2)), performed clinical/laboratory/genetic evaluation, and flow cytometric PI3K/AKT/mTOR pathway analyses. Median age at onset of complaints was 17.5 months (3 months to 12 years) and at diagnosis was 15.7 years (2.5-37) in APDS. Median diagnostic delay was 12.9 years (1.6-27). Recurrent respiratory tract infections (90%), lymphoproliferation (70%), autoimmune/inflammatory findings (60%), and allergy (40%) were common in APDS. Recurrent viral infections were present in 4/10 and malignancy (non-Hodgkin lymphoma and testicular yolk sac tumor) was present in 2/10 in APDS. Low CD4+ T cells(5/8) with increased CD4+ effector memory (8/8) and CD4+ TEMRA cells (6/8) were present in the given number of APDS patients. We diagnosed tubulointerstitial nephritis, Langerhans cell histiocytosis, and late-onset congenital adrenal hyperplasia in APDS. Allergic findings, lymphoproliferation/malignancy, and high IgM were present in the APDS but not in PIK3R1 deficiency. Low IgM/IgG/CD19+ B cell counts were characteristic in patients with PIK3R1 homozygous loss-of function mutations. Differential diagnosis with combined immunodeficiency and diseases of immune dysregulation make molecular genetic analysis crucial for diagnosing mTOR pathway defects. It is easy to differentiate APDS and homozygous PIK3R1 defects with specific laboratory features. Additionally, mTOR pathway functional analysis is a definitive diagnostic and pathogenicity assessment tool for novel APDS mutations.

Possible involvement of zinc transporter ZIP13 in myogenic differentiation

Ehlers–Danlos syndrome spondylodysplastic type 3 (EDSSPD3, OMIM 612350) is an inherited recessive connective tissue disorder that is caused by loss of function of SLC39A13/ZIP13, a zinc transporter belonging to the Slc39a/ZIP family. We previously reported that patients with EDSSPD3 harboring a homozygous loss of function mutation (c.221G > A, p.G64D) in ZIP13 exon 2 (ZIP13G64D) suffer from impaired development of bone and connective tissues, and muscular hypotonia. However, whether ZIP13 participates in the early differentiation of these cell types remains unclear. In the present study, we investigated the role of ZIP13 in myogenic differentiation using a murine myoblast cell line (C2C12) as well as patient-derived induced pluripotent stem cells (iPSCs). We found that ZIP13 gene expression was upregulated by myogenic stimulation in C2C12 cells, and its knockdown disrupted myotubular differentiation. Myocytes differentiated from iPSCs derived from patients with EDSSPD3 (EDSSPD3-iPSCs) also exhibited incomplete myogenic differentiation. Such phenotypic abnormalities of EDSSPD3-iPSC-derived myocytes were corrected by genomic editing of the pathogenic ZIP13G64D mutation. Collectively, our findings suggest the possible involvement of ZIP13 in myogenic differentiation, and that EDSSPD3-iPSCs established herein may be a promising tool to study the molecular basis underlying the clinical features caused by loss of ZIP13 function.

Clinical and cellular phenotypes resulting from a founder mutation in IL10RB.

Inborn errors of immunity are a group of rare genetically determined diseases that impair immune system development or function. Many of these diseases include immune dysregulation, autoimmunity, or autoinflammation as prominent clinical features. In some children diagnosed with very early onset inflammatory bowel disease (VEOIBD), monogenic inborn errors of immune dysregulation underlie disease. We report a case of VEOIBD caused by a novel homozygous loss of function mutation in IL10RB. We use cytometry by time-of-flight with a broad panel of antibodies to interrogate the immunophenotype of this patient and detect reduced frequencies of CD4 and CD8 T cells with additional defects in some populations of T helper cells, innate-like T cells, and memory B cells. Finally, we identify the patient's mutation as a founder allele in an isolated indigenous population and estimate the age of this variant by studying the shared ancestral haplotype.

Myofibrillar myopathy hallmarks associated with ZAK deficiency.

The ZAK gene encodes two functionally distinct kinases, ZAKα and ZAKβ. Homozygous loss of function mutations affecting both isoforms causes a congenital muscle disease. ZAKβ is the only isoform expressed in skeletal muscle and is activated by muscle contraction and cellular compression. The ZAKβ substrates in skeletal muscle or the mechanism whereby ZAKβ senses mechanical stress remains to be determined. To gain insights into the pathogenic mechanism, we exploited ZAK-deficient cell lines, zebrafish, mice and a human biopsy. ZAK-deficient mice and zebrafish show a mild phenotype. In mice, comparative histopathology data from regeneration, overloading, ageing and sex conditions indicate that while age and activity are drivers of the pathology, ZAKβ appears to have a marginal role in myoblast fusion in vitro or muscle regeneration in vivo. The presence of SYNPO2, BAG3 and Filamin C (FLNC) in a phosphoproteomics assay and extended analyses suggested a role for ZAKβ in the turnover of FLNC. Immunofluorescence analysis of muscle sections from mice and a human biopsy showed evidence of FLNC and BAG3 accumulations as well as other myofibrillar myopathy markers. Moreover, endogenous overloading of skeletal muscle exacerbated the presence of fibres with FLNC accumulations in mice, indicating that ZAKβ signalling is necessary for an adaptive turnover of FLNC that allows for the normal physiological response to sustained mechanical stress. We suggest that accumulation of mislocalized FLNC and BAG3 in highly immunoreactive fibres contributes to the pathogenic mechanism of ZAK deficiency.

Whole Exome Sequencing Identifies a Heterozygous Variant in the Cav1.3 Gene CACNA1D Associated with Familial Sinus Node Dysfunction and Focal Idiopathic Epilepsy.

Cav1.3 voltage-gated L-type calcium channels (LTCCs) are involved in cardiac pacemaking, hearing and hormone secretion, but are also expressed postsynaptically in neurons. So far, homozygous loss of function mutations in CACNA1D encoding the Cav1.3 α1-subunit are described in congenital sinus node dysfunction and deafness. In addition, germline mutations in CACNA1D have been linked to neurodevelopmental syndromes including epileptic seizures, autism, intellectual disability and primary hyperaldosteronism. Here, a three-generation family with a syndromal phenotype of sinus node dysfunction, idiopathic epilepsy and attention deficit hyperactivity disorder (ADHD) is investigated. Whole genome sequencing and functional heterologous expression studies were used to identify the disease-causing mechanisms in this novel syndromal disorder. We identified a heterozygous non-synonymous variant (p.Arg930His) in the CACNA1D gene that cosegregated with the combined clinical phenotype in an autosomal dominant manner. Functional heterologous expression studies showed that the CACNA1D variant induces isoform-specific alterations of Cav1.3 channel gating: a gain of ion channel function was observed in the brain-specific short CACNA1D isoform (Cav1.3S), whereas a loss of ion channel function was seen in the long (Cav1.3L) isoform. The combined gain-of-function (GOF) and loss-of-function (LOF) induced by the R930H variant are likely to be associated with the rare combined clinical and syndromal phenotypes in the family. The GOF in the Cav1.3S variant with high neuronal expression is likely to result in epilepsy, whereas the LOF in the long Cav1.3L variant results in sinus node dysfunction.

Transcription factor gene Pea3 regulates erectile function during copulation in mice.

Male mice with homozygous loss of function mutations of the transcription factor gene Pea3 (Pea3 null) are infertile due to their inability to inseminate females, however the specific deficits in male sexual behaviors that drive this phenotype are unknown. Here, the copulatory behavior of male mice (Pea3 null and control) with hormonally primed ovariectomized females was monitored via high-speed and high-resolution digital videography to assess for differences in female-directed social behaviors, gross sexual behaviors (mounting, thrusting), and erectile and ejaculatory function. Pea3 null male mice exhibit greatly reduced erectile function, with 44% of males displaying no visible erections during copulation, and 0% achieving sustained erections. As such, Pea3 null males are incapable of intromission and copulatory plug deposition, despite displaying largely normal female-directed social behaviors, mounting behaviors, and ejaculatory grasping behavior. Additionally, the organization and timing of thrusting behaviors is impaired in Pea3 null males. Our results show that the transcription factor gene Pea3 regulates the ability to achieve and maintain erections during copulation in mice.

Craniofacial Defects in Embryos with Homozygous Deletion of Eftud2 in Their Neural Crest Cells Are Not Rescued by Trp53 Deletion.

Embryos with homozygous mutation of Eftud2 in their neural crest cells (Eftud2ncc−/−) have brain and craniofacial malformations, hyperactivation of the P53-pathway and die before birth. Treatment of Eftud2ncc−/− embryos with pifithrin-α, a P53-inhibitor, partly improved brain and craniofacial development. To uncover if craniofacial malformations and death were indeed due to P53 hyperactivation we generated embryos with homozygous loss of function mutations in both Eftud2 and Trp53 in the neural crest cells. We evaluated the molecular mechanism underlying craniofacial development in pifithrin-α-treated embryos and in Eftud2; Trp53 double homozygous (Eftud2ncc−/−; Trp53ncc−/−) mutant embryos. Eftud2ncc−/− embryos that were treated with pifithrin-α or homozygous mutant for Trp53 in their neural crest cells showed reduced apoptosis in their neural tube and reduced P53-target activity. Furthermore, although the number of SOX10 positive cranial neural crest cells was increased in embryonic day (E) 9.0 Eftud2ncc−/−; Trp53ncc−/− embryos compared to Eftud2ncc−/− mutants, brain and craniofacial development, and survival were not improved in double mutant embryos. Furthermore, mis-splicing of both P53-regulated transcripts, Mdm2 and Foxm1, and a P53-independent transcript, Synj2bp, was increased in the head of Eftud2ncc−/−; Trp53ncc−/− embryos. While levels of Zmat3, a P53- regulated splicing factor, was similar to those of wild-type. Altogether, our data indicate that both P53-regulated and P53-independent pathways contribute to craniofacial malformations and death of Eftud2ncc−/− embryos.

ANGPTL3 and PCSK9 interact and show coordinated metabolic regulation in vitro

Background and Aims : ANGPTL3 and PCSK9 are known regulators of lipoprotein metabolism. Patients harboring homozygous loss of function mutations in the ANGPTL3 gene show reduced levels of circulating PCSK9, indicating a possible coordinate regulation of these two proteins. This study aimed to establish whether the two proteins can cross-regulate in different conditions of nutritional availability.

ANGPLT3 in cardio-metabolic disorders.

Dyslipidemia is associated with numerous health problems that include the combination of insulin resistance, hypertension and obesity, which is always grouped together asmetabolic syndrome. Given that metabolic syndrome leads to a high mortality and poses serious risks to human health worldwide, it is vital to explore the mechanisms whereby dyslipidemia modulates the risk and the severity of cardio-metabolic disorders. Recently, a specific secretory protein family, named angiopoietin-like protein (ANGPTL), is considered as one of the significant biomarkers which facilitate the development of angiogenesis. Among the eight proteins of ANGPTL family, ANGPTL3 has been demonstrated as an essential modulator of lipid catabolism within circulation by inhibiting the activity of lipoprotein lipase (LPL) and endothelial lipase (EL). Consistent with these notions, mice with ANGPTL3 gene-deficiency presented reduced circulating levels of low density lipoprotein cholesterol (LDL-C) and lower risk of atherosclerosis. On the other hand, participants carrying homozygous loss-of function (LOF) mutation in ANGPTL3 gene also displayed lower circulating LDL-C levels and atherosclerotic risk. In the current review, we summarized the recent understanding of ANGPTL3 in controlling the risk and the development of dyslipidemia and its related cardio-metabolic disorders. Moreover, we also provided the perspectives which potentially suggested that ANGPTL3 could be considered as a promising target in treating metabolic syndrome.

A brain penetrant progranulin biotherapeutic rescues lysosomal and inflammatory phenotypes in the brain of GRN knockout mice

AbstractBackgroundHeterozygous loss of function (LOF) mutations in GRN cause frontotemporal dementia (FTD), a neurodegenerative disorder associated with lysosomal dysfunction, TDP‐43 pathology and inflammation in the CNS. Additionally, homozygous LOF mutations cause neuronal ceroid lipofuscinosis, a lysosomal storage disorder. GRN encodes progranulin (PGRN), a soluble protein that is most abundantly expressed in microglia, where it localizes to lysosomes and can undergo secretion. Although the precise function of PGRN is unknown, growing evidence suggests that it regulates lysosomal function, inflammatory responses and promotes neuronal survival. Accordingly, PGRN LOF models are associated with lysosomal defects, hyperinflammatory responses, and decreased neuronal viability, both in vitro and in vivo. Because GRN‐FTD patients exhibit reduced levels of PGRN in biofluids and tissues, including the brain, a protein replacement therapy analogous to enzyme replacement therapy and capable of crossing the blood brain barrier (BBB) more efficiently may represent a powerful approach to slow or prevent disease progression.MethodHere we describe a novel therapeutic for increasing brain penetrance of PGRN, referred to as Protein Transport Vehicle (PTV):PGRN. PTV:PGRN consists of recombinant human PGRN fused to a modified Fc domain engineered to bind to the human transferrin receptor (huTfR), thus facilitating receptor‐mediated transcytosis across the BBB.ResultWe found that extracellular applications of PTV:PGRN rescue a range of Grn KO cell phenotypes in vitro, including lysosomal proteolysis and alteration in the levels of bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid involved in lysosomal lipid catabolism. Intravenously administered PTV:PGRN showed increased acute brain exposure in huTfR knock‐in (TfRmu/hu) mice relative to a regular Fc:PGRN fusion that does not bind to huTfR. Importantly, low doses of PTV:PGRN, but not Fc:PGRN, fully corrected lysosomal lipid alterations, including BMP deficiency, as well as inflammatory markers in the brain of Grn KO x TfRmu/hu mice.ConclusionOur data suggest that PTV:PGRN may represent a viable therapeutic strategy for the treatment of GRN‐FTD and potentially other disorders associated with PGRN deficiency.

Persistent Müllerian Duct Syndrome: A Rare But Important Etiology of Inguinal Hernia and Cryptorchidism

Homozygous loss of function mutations in genes encoding anti-Müllerian hormone (AMH) or its receptor (AMHRII) lead to persistent Müllerian duct syndrome (PMDS). PMDS is characterized by the presence of a uterus, fallopian tubes, cervix, and upper vagina in fully virilised 46,XY males. Both surgical management and long-term follow-up of these patients are challenging. Four cases with PMDS presented with cryptorchidism and inguinal hernia, and laparoscopic inguinal exploration revealed Müllerian remnants. Three of the patients had homozygous mutations in the AMH gene, one with a novel c.1673G>A (p.Gly558Asp) mutation, and one patient had an AMHRII mutation. All patients underwent a single-stage laparotomy in which the fundus of the uterus was split along the midline to release testes and to avoid damaging the vas deferens or the deferential artery. Biopsy of Müllerian remnants did not reveal any malignancy. The cases presented here expand the clinical and molecular presentation of PMDS. Cryptorchidism and inguinal hernia in the presence of Müllerian structures in an appropriately virilised 46,XY individual should suggest PMDS. Long-term reproductive and endocrinological surveillance is necessary.

Endogenous control of inflammatory visceral pain by T cell-derived opioids in IL-10-deficient mice.

The opioid-mediated analgesic activity of mucosal CD4+ T lymphocytes in colitis has been reported in immunocompetent mice so far. Here, we investigated whether CD4+ T lymphocytes alleviate from inflammation-induced abdominal pain in mice with defective immune regulation. Endogenous control of visceral pain by opioids locally produced in inflamed mucosa was assessed in IL-10-deficient mice. CD4+ T lymphocytes but not F4/80+ macrophages isolated from the lamina propria of IL-10-deficient mice with colitis express enkephalin-containing opioid peptides as assessed by cytofluorometry. Colitis in IL-10-/- mice was not associated with abdominal pain. Intraperitoneal injection of naloxone-methiodide, a peripheral opioid receptor antagonist, induced abdominal hypersensitivity in IL-10-/- mice with colitis. Opioid-mediated analgesic activity of mucosal T lymphocytes remains operating in IL-10-/- mice with impaired immune regulation. The data suggest that endogenous T cell-derived opioids might reduce inflammation-induced abdominal pain in inflammatory bowel diseases associated with homozygous "loss of function mutations" in interleukin-10.

Mimicry and well known genetic friends: molecular diagnosis in an Iranian cohort of suspected Bartter syndrome and proposition of an algorithm for clinical differential diagnosis

BackgroundBartter Syndrome is a rare, genetically heterogeneous, mainly autosomal recessively inherited condition characterized by hypochloremic hypokalemic metabolic alkalosis. Mutations in several genes encoding for ion channels localizing to the renal tubules including SLC12A1, KCNJ1, BSND, CLCNKA, CLCNKB, MAGED2 and CASR have been identified as underlying molecular cause. No genetically defined cases have been described in the Iranian population to date. Like for other rare genetic disorders, implementation of Next Generation Sequencing (NGS) technologies has greatly facilitated genetic diagnostics and counseling over the last years. In this study, we describe the clinical, biochemical and genetic characteristics of patients from 15 Iranian families with a clinical diagnosis of Bartter Syndrome.ResultsAge range of patients included in this study was 3 months to 6 years and all patients showed hypokalemic metabolic alkalosis. 3 patients additionally displayed hypercalciuria, with evidence of nephrocalcinosis in one case. Screening by Whole Exome Sequencing (WES) and long range PCR revealed that 12/17 patients (70%) had a deletion of the entire CLCNKB gene that was previously identified as the most common cause of Bartter Syndrome in other populations. 4/17 individuals (approximately 25% of cases) were found to suffer in fact from pseudo-Bartter syndrome resulting from congenital chloride diarrhea due to a novel homozygous mutation in the SLC26A3 gene, Pendred syndrome due to a known homozygous mutation in SLC26A4, Cystic Fibrosis (CF) due to a novel mutation in CFTR and apparent mineralocorticoid excess syndrome due to a novel homozygous loss of function mutation in HSD11B2 gene. 1 case (5%) remained unsolved.ConclusionsOur findings demonstrate deletion of CLCNKB is the most common cause of Bartter syndrome in Iranian patients and we show that age of onset of clinical symptoms as well as clinical features amongst those patients are variable. Further, using WES we were able to prove that nearly 1/4 patients in fact suffered from Pseudo-Bartter Syndrome, reversing the initial clinical diagnosis with important impact on the subsequent treatment and clinical follow up pathway. Finally, we propose an algorithm for clinical differential diagnosis of Bartter Syndrome.

Functional rare variants influence the clinical response to anti-TNF therapy in Crohn's disease.

Background:The effect of low-frequency functional variation on anti-tumor necrosis factor alpha (TNF) response in Crohn’s disease (CD) patients remains unexplored. The objective of this study was to investigate the impact of functional rare variants in clinical response to anti-TNF therapy in CD.Methods:CD anti-TNF naïve patients starting anti-TNF treatment due to active disease [Crohn’s Disease Activity Index (CDAI > 150)] were included. The whole genome was sequenced using the Illumina Hiseq4000 platform. Clinical response was defined as a CDAI score <150 at week 14 of anti-TNF treatment. Low-frequency variants were annotated and classified according to their damaging potential. The whole genome of CD patients was screened to identify homozygous loss-of-function (LoF) variants. The TNF signaling pathway was tested for overabundance of damaging variants using the SKAT-O method. Functional implication of the associated rare variation was evaluated using cell-type epigenetic enrichment analyses.Results:A total of 41 consecutive CD patients were included; 3250 functional rare variants were identified (2682 damaging and 568 LoF variants). Two homozygous LoF mutations were found in HLA-B and HLA-DRB1 genes associated with lack of response and remission, respectively. Genome-wide LoF variants were enriched in epigenetic marks specific for the gastrointestinal tissue (colon, p = 4.11e–4; duodenum, p = 0.011). The burden of damaging variation in the TNF signaling pathway was associated with response to anti-TNF therapy (p = 0.016); damaging variants were enriched in epigenetic marks from CD8+ (p = 6.01e–4) and CD4+ (p = 0.032) T cells.Conclusions:Functional rare variants are involved in the response to anti-TNF therapy in CD. Cell-type enrichment analysis suggests that the gut mucosa and CD8+ T cells are the main mediators of this response.

A CASE OF RECURRENT ASPIRATION PNEUMONIA AND PERSISTENT EOSINOPHILIA

Introduction Homozygous or compound heterozygous loss-of-function (LOF) mutations in CARMIL2 are associated with combined immunodeficiency disorder with a variable phenotypic presentation in 10 families (21 patients). Clinical manifestations include recurrent skin and lung infections, dermatitis, chronic diarrhea, esophagitis, and failure-to-thrive. Here we describe a case of immune deficiency associated with an apparently homozygous, novel variant c.1942delC (p.His648ThrfsX20) in the CARMIL2 gene (RLTPR). Case Description The patient presented with recurrent pneumonias from 6-29 months of age, 11 requiring hospitalization. Pneumonias and bronchiectasis were attributed to aspiration of liquids, confirmed on barium swallow. Whole exome sequencing identified an apparently homozygous pathogenic variant (c.1954delC, resulting in frameshift p.H648TfsX20) in CARMIL2 gene. Immune evaluation was significant for poor antibody response to 12 of 14 pneumococcal serotypes, absent lymphocyte proliferation to antigens (candida and tetanus) with reduced lymphocyte proliferation to mitogens (PHA and Con A), an excess of CD19+B cells (2769 cells/mcL), low-level EBV viremia (5806 copies), and persistent eosinophilia (1.82-6.18x10³/µL). Total immunoglobulin levels (IgG, IgM, IgA, IgE) and CD8+T-suppressor cell-, CD4+T-helper cell-, and CD56+NK cell-counts were within normal range. Discussion Here we report a case of combined immune deficiency with associated findings of aspiration, recurrent pneumonias, eosinophilia, and B-cell lymphocytosis with EBV viremia in a child with an apparently homozygous novel variant in the CARMIL2 gene. This case report provides further evidence that homozygous LOF mutations in CARMIL2 are associated with a combined immune defect with varied phenotypic features. Identification of this mutation may have prognostic value and will guide genetic counseling.

Whole-exome sequencing identifies mutations in FSIP2 as a recurrent cause of multiple morphological abnormalities of the sperm flagella.

Can whole-exome sequencing (WES) of infertile patients identify new genes responsible for multiple morphological abnormalities of the sperm flagella (MMAF)? WES analysis of 78 infertile men with a MMAF phenotype permitted the identification of four homozygous mutations in the fibrous sheath (FS) interacting protein 2 (FSIP2) gene in four unrelated individuals. The use of high-throughput sequencing techniques revealed that mutations in the dynein axonemal heavy chain 1 (DNAH1) gene, and in the cilia and flagella associated protein 43 (CFAP43) and 44 (CFAP44) genes account for approximately one-third of MMAF cases thus indicating that other relevant genes await identification. This was a retrospective genetics study of 78 patients presenting a MMAF phenotype who were recruited in three fertility clinics between 2008 and 2015. Control sperm samples were obtained from normospermic donors. Allelic frequency for control subjects was derived from large public databases. WES was performed for all 78 subjects. All identified variants were confirmed by Sanger sequencing. Relative mRNA expression levels for the selected candidate gene (FSIP2) was assessed by quantitative RT-PCR in a panel of normal human and mouse tissues. To characterize the structural and ultrastructural anomalies present in patients' sperm, immunofluorescence (IF) was performed on sperm samples from two subjects with a mutation and one control and transmission electron microscopy (TEM) analyses was performed on sperm samples from one subject with a mutation and one control. We identified four unrelated patients (4/78, 5.1%) with homozygous loss of function mutations in the FSIP2 gene, which encodes a protein of the sperm FS and is specifically expressed in human and mouse testis. None of these mutations were reported in control sequence databases. TEM analyses showed a complete disorganization of the FS associated with axonemal defects. IF analyses confirmed that the central-pair microtubules and the inner and outer dynein arms of the axoneme were abnormal in all four patients carrying FSIP2 mutations. Importantly, and in contrast to what was observed in patients with MMAF and mutations in other MMAF-related genes (DNAH1, CFAP43 and CFAP44), mutations in FSIP2 led to the absence of A-kinase anchoring protein 4 (AKAP4). The low number of biological samples and the absence of a reliable anti-FSIP2 antibody prevented the formal demonstration that the FSIP2 protein was absent in sperm from subjects with a FSIP2 mutation. Our findings indicate that FSIP2 is one of the main genes involved in MMAF syndrome. In humans, genes previously associated with a MMAF phenotype encoded axonemal-associated proteins (DNAH1, CFAP43 and CFAP44). We show here that FSIP2, a protein of the sperm FS, is also logically associated with MMAF syndrome as we showed that it is necessary for FS assembly and for the overall axonemal and flagellar biogenesis. As was suggested before in mouse and man, our results also suggest that defects in AKAP4, one of the main proteins interacting with FSIP2, would induce a MMAF phenotype. Finally, this work reinforces the demonstration that WES sequencing is a good strategy to reach a genetic diagnosis for patients with severe male infertility phenotypes. This work was supported by the following grants: the 'MAS-Flagella' project financed by the French ANR and the DGOS for the program PRTS 2014 (14-CE15) and the 'Whole genome sequencing of patients with Flagellar Growth Defects (FGD)' project financed by the Fondation Maladies Rares for the program Séquençage à haut débit 2012. The authors have no conflict of interest.

Genetic compensation triggered by actin mutation prevents the muscle damage caused by loss of actin protein

The lack of a mutant phenotype in homozygous mutant individuals’ due to compensatory gene expression triggered upstream of protein function has been identified as genetic compensation. Whilst this intriguing process has been recognized in zebrafish, the presence of homozygous loss of function mutations in healthy human individuals suggests that compensation may not be restricted to this model. Loss of skeletal α-actin results in nemaline myopathy and we have previously shown that the pathological symptoms of the disease and reduction in muscle performance are recapitulated in a zebrafish antisense morpholino knockdown model. Here we reveal that a genetic actc1b mutant exhibits mild muscle defects and is unaffected by injection of the actc1b targeting morpholino. We further show that the milder phenotype results from a compensatory transcriptional upregulation of an actin paralogue providing a novel approach to be explored for the treatment of actin myopathy. Our findings provide further evidence that genetic compensation may influence the penetrance of disease-causing mutations.

Insights into Clonal Relationships of Putative Adaptive Natural Killer Cells (NK) in Humans, Via Mapping of Somatic Piga Mutations in Patients with Paroxysmal Nocturna Hemoglobinuria (PNH)

NK cells play a central role in innate immunity, specifically in tumor surveillance and microbial pathogen control. Recent murine models and human studies have identified subsets of NK-cells with apparent memory cell function, strongly linked to CMV infection in humans and termed "adaptive" NK. Our recent clonal tracking studies following autologous hematopoietic stem cells transplantation (HSCT) of genetically-barcoded CD34+ cells in macaques revealed distinct clonal ontogeny of a subset of NK cells within the primate equivalent of the human CD56dim population (Wu et al., Cell Stem Cell, 2014), with little clonal overlap with T-, B-lymphoid or myeloid cells, suggesting a separate precursor pool for this NK subtype. Peripheral blood CD 56bright -NK cells have been previously hypothesized to be precursors for the main population of circulating cytotoxic CD56dim cells. To further investigate NK-cell ontogeny and clonal relationships in humans we took advantage of naturally-occurring somatic mutations in the X-linked phosphatidylinositol glycan class A (PIGA) gene in patients with the hemolytic disorder paroxysmal nocturnal hemoglobinuria. This gene codes for an enzyme required for cell surface localization of glycosylphophatidylinositol (GPI)-anchored proteins, and thus loss of function mutations result in hematopoietic cells lacking GPI-anchored proteins, and red cell hemolysis. PNH patients have not been reported to have immune dysfunction and can have stable disease for many years. Membrane bound GPI anchors can be detected on any cell via flow cytometry using a labeled inactive aerolysin (FLAER), and serves as a marker for the fraction of cells comprising the PIGA mutant (GPI negative) clonal compartment. The PNH clone sizes contributing to peripheral blood cells are variable in but can reach almost 100% in some patients, and can be stable over decades. We selected 9 PNH patients with GPI negative granulocytes ranging from 5% to 98% and a median time from diagnosis of 43.7 months (15-100) for this study. NK cells were defined as CD56+/CD16+/CD3-/CD20- lymphocytes. We observed disproportionally fewer GPI negative NK cells compared to granulocytes (Fig 1), with the discrepancy most marked the major peripheral blood CD56dim population (mean 65% vs 25% GPI negative granulocytes, p = 0.0028, paired t-test), in contrast to 46% GPI negative cells in the CD56bright population (p=0.057). Due to the prolonged life span of memory T and B cells, fewer GPI negative B and particularly T-lymphocytes have been reported in PNH patients. In our cohort 3.4% of CD3+ T-cells and 13.2% of CD20+ B-cells were GPI negative (p=0.0005 and 0.0014, respectively versus granulocytes). Compared to the NK subsets, the CD56bright population showed the most significant differences (p = 0.0063 versus CD3 and p=0.0151 versus CD20). To further characterize the phenotype of the GPI positive versus negative CD56dim cells, we analyzed cells co-expressing either the terminal differential marker CD57, the inhibitory receptor NKG2A, or activating receptor NKG2C for FLAER positivity. Prior studies have suggested that the human CMV-linked adaptive NK subset is CD57+, NKG2A- and NKG2C+. The NKG2C+ CD 56dim population was highly enriched for GPI positive cells (p=0.0024 vs granulocytes, Fig 1). This profile was most prominent in CMV-IgG positive patients who had also significantly more GPI positive CD 56dim/CD57+ cells compared to granulocytes (p=0.008). Interestingly, one CMV positive patient (#5) had a complete lack of NKG2C expression, most likely due to homozygous loss of function mutation, and this patient had almost 100% GPI negative NK cells, matching his neutrophil pattern. Compared to granulocytes, NKGA2A+ or CD57+ positive CD56dim cells were also mostly GPI negative (p=0.0081 and 0.028).Circulating NK cell turnover has been estimated to be about 14 days. The PNH patients studied had documented clonal PIG-A mutations for many years. Our observation that the majority of CD56dim NK cells, specifically the NKG2C subset, are not progeny of the same progenitors producing CD56bright NK cells or myeloid cells based on clonal disparity regarding the PNH clone is suggestive of an independent, very long-lived or self-renewing NK cell progenitor for a CMV-linked CD56dim/CD57+/NKG2C+ memory NK cell compartment. These observations provide novel further insights into the human adaptive NK cell subset. [Display omitted] DisclosuresWinkler:Novartis: Research Funding; GSK: Research Funding. Townsley:Novartis: Research Funding; GSK: Research Funding.

Autoimmune lymphoproliferative syndrome-like disease in patients with LRBA mutation

Mutations in LPS-responsive and beige-like anchor (LRBA) gene were recently described in patients with combined immunodeficiency, enteropathy and autoimmune cytopenia. Here, we extend the clinical and immunological phenotypic spectrum of LRBA associated disorders by reporting on three patients from two unrelated families who presented with splenomegaly and lymphadenopathy, cytopenia, elevated double negative T cells and raised serum Fas ligand levels resembling autoimmune lymphoproliferative syndrome (ALPS) and one asymptomatic patient. Homozygous loss of function mutations in LRBA were identified by whole exome analysis. Similar to ALPS patients, Fas mediated apoptosis was impaired in LRBA deficient patients, while apoptosis in response to stimuli of the intrinsic mitochondria mediated apoptotic pathway was even enhanced. This manuscript illustrates the phenotypic overlap of other primary immunodeficiencies with ALPS-like disorders and strongly underlines the necessity of genetic diagnosis in order to provide early correct diagnosis and subsequent care.

RAC2 loss-of-function mutation in 2 siblings with characteristics of common variable immunodeficiency

We report the first patients with a homozygous loss of function mutation in the RAC2 gene, presenting with clinical features of common variable immunodeficiency. In addition, the patients suffered from glomerulonephritis, coagulopathy, multiple hormone deficiencies potentially on the autoimmune basis and abnormalities of neutrophil granules.