Homozygous Loss Of Function Mutations Research Articles

Linking Oxidative Stress to Cell Fate-Ipsc-Based Disease Modeling Identifies New Therapeutic Target in Reticular Dysgenesis

Reticular Dysgenesis (RD) is one of the most serious forms of severe combined immune deficiency (SCID). It is characterized by complete absence of circulating lymphocytes and neutrophils. In addition, patients suffer from sensorineural hearing loss. Before newborn screening for SCID was implemented, the majority of patients succumbed to infection long before hematopoietic cell transplantation (HCT) could be attempted. To this date, the prognosis for RD remains grim. RD is caused by mutations in the mitochondrial ADP-generator Adenylate Kinase 2 (AK2). AK1 is a cytosolic protein that may compensate in various tissues for the lack of AK2. However, AK1 is not expressed in leukocytes and the stria vascularis of the inner ear [1]. While this observation may explain where AK2 defects manifest, the molecular mechanisms how AK2 defects take effect, remain largely obscure. Significant obstacles to elucidating disease pathology have been the lack of a suitable animal models and the unavailability of patient specimens. Using skin fibroblasts from an RD-patient we have recently identified at Boston Children’s Hospital [2], we have generated induced pluripotent stem cells (iPSC) with homozygous loss of function mutation in AK2. In-vitro myeloid differentiation of AK2-mutated iPSCs recapitulates the characteristic maturation arrest at the promyelocyte stage observed in-vivo in patients with this condition. AK2 is expressed in the intermitochondrial space and serves as primary mitochondrial ADP generator by promoting the reversible reaction AMP + ATP = 2 ADP. Maintenance of adequate levels of ADP is critical to support ATP synthase activity. Using Mass Spectrometry, we have shown that decreased AK2 activity leads to an increase in the AMP/ADP ratio in iPS-derived myeloid cells, indicating that AK2 is indispensable in maintaining ADP supply in the myeloid lineage. We have also performed transcriptome analysis of AK2- mutated myeloid cells compared to control and found a significant down regulation of ATP-dependant transporters. Based on this data, we hypothesized that in patients with RD, ADP-depletion in myeloid progenitors leads to stage 4 respiration, a well defined state in mitochondrial biology, in which the ATP-synthase lacks substrate and decreases its activity. This causes a reduction in proton flux from the intermitochondrial space back into the matrix, transient rise in membrane potential, and an escalation in the formation of reactive oxygen species (ROS). The cell responds by activating “inducible uncoupling”, the opening of alternative proton pores, which allows proton flux back into the matrix, bypassing the ATP-synthase and foregoing the use of energy stored in the proton gradient. While this represents a cellular rescue mechanism in response to acute oxidative stress, extended oxidative-stress-induced uncoupling eventually leads to a decline in proton gradient and membrane potential and ultimately in demise of the cell. To test this hypothesis, we have added Glutathione, the primary endogenous cellular antioxidant, to the culture conditions. We also tested G-CSF and all-trans-retinoic acid (ATRA), agents known to promote promyelocyte differentiation to mature neutrophils in other conditions. While G-CSF had no, and ATRA clearly deleterious effects on myeloid maturation in AK2-mutated cells, Glutathione led to a significant improvement in differentiation, allowing development of mature neutrophils in-vitro. Our results suggest that cell fate in RD is linked to oxidative stress and identify antioxidants as a possible therapeutic approach that may help reduce early mortality due to severe infections in patients with RD. Disclosures No relevant conflicts of interest to declare.

Exome sequencing identifies a recessive PIGN splice site mutation as a cause of syndromic Congenital Diaphragmatic Hernia

Using exome sequencing we identify a homozygous splice site mutation in the PIGN gene in a foetus with multiple congenital anomalies including bilateral diaphragmatic hernia, cardiovascular anomalies, segmental renal dysplasia, facial dysmorphism, cleft palate, and oligodactyly. This finding expands the phenotypic spectrum associated with homozygous loss of function mutations in PIGN, and adds further support for defective GPI anchor biosynthesis as a cause of developmental abnormalities. We demonstrate that exome sequencing is a valuable approach for the identification of a genetic cause in sporadic cases of multiple congenital anomalies (MCA) due to inherited mutations.

Deletion of MgcRacGAP in the male germ cells impairs spermatogenesis and causes male sterility in the mouse

MgcRacGAP (RACGAP1) is a GTPase Activating Protein (GAP), highly produced in the mouse embryonic brain and in the human and mouse post-natal testis. MgcRacGAP negatively controls the activity of Rac and Cdc42, which are key molecular switches acting on the microtubule and actin cytoskeleton and controlling various cell processes such as proliferation, adhesion and motility. Previous studies demonstrated that MgcRacGAP plays a critical role in the cytokinesis of somatic cells; hence homozygous inactivation of the gene in the mouse and mutation in Caenorhabditis elegans led to embryonic lethality due to the inability of MgcRacGAP-null embryos to assemble the central spindle and to complete cytokinesis.In the testis, the germ cells do not complete cytokinesis and remain connected as a syncytium throughout the entire process of spermatogenesis. Interestingly, MgcRacGAP was shown to locate to the intercellular bridges, connecting these germ cells. In order to determine the function(s) of MgcRacGAP in the male germline, we generated a conditional knock-out mouse using Stra8 promoter driven Cre recombinase to induce the specific deletion of MgcRacGAP in the pre-meiotic germ cells. We found that the absence of MgcRacGAP induced a germline depletion and male sterility. Consistent with the role of MgcRacGAP in the establishment of the cytoplasm constriction during cytokinesis of the somatic cells, we observed that MgcRacGAP deletion in the germ cells prevented the formation of the intercellular bridges and induced a proliferation arrest. While we assume that inherited homozygous loss of function mutations in MgcRacGAP would be lethal in human, de novo mutations in the testis might account for some cases of non-obstructive oligo- and/or azoo-spermia syndromes, whose genetic causes are altogether still poorly defined.

Human L-ferritin deficiency is characterized by idiopathic generalized seizures and atypical restless leg syndrome

The ubiquitously expressed iron storage protein ferritin plays a central role in maintaining cellular iron homeostasis. Cytosolic ferritins are composed of heavy (H) and light (L) subunits that co-assemble into a hollow spherical shell with an internal cavity where iron is stored. The ferroxidase activity of the ferritin H chain is critical to store iron in its Fe3+ oxidation state, while the L chain shows iron nucleation properties. We describe a unique case of a 23-yr-old female patient affected by a homozygous loss of function mutation in the L-ferritin gene, idiopathic generalized seizures, and atypical restless leg syndrome (RLS). We show that L chain ferritin is undetectable in primary fibroblasts from the patient, and thus ferritin consists only of H chains. Increased iron incorporation into the FtH homopolymer leads to reduced cellular iron availability, diminished levels of cytosolic catalase, SOD1 protein levels, enhanced ROS production and higher levels of oxidized proteins. Importantly, key phenotypic features observed in fibroblasts are also mirrored in reprogrammed neurons from the patient's fibroblasts. Our results demonstrate for the first time the pathophysiological consequences of L-ferritin deficiency in a human and help to define the concept for a new disease entity hallmarked by idiopathic generalized seizure and atypical RLS.

Abstract PR02: Targeting altered SWI/SNF function through synthetic lethal RNAi screening

Abstract The SWI/SNF family of chromatin remodeling complexes is critical for the mobilization of nucleosomes during transcription, DNA replication and repair, and cell division. SWI/SNF consists of a central ATPase subunit (BRG1 or BRM) and 10-12 additional subunits whose identity lends specificity to the complex. Abundant genomic data indicates that SWI/SNF components are frequent targets of homozygous loss of function mutations in a variety of tumor types. These loss of function mutations alter the composition and activity of the SWI/SNF complex, resulting in a tumor-specific chromatin state. In this work we describe a strategy for targeting this tumor-specific chromatin state using synthetic lethal RNAi screening. We have assembled and validated a focused shRNA library targeting 120 factors that act on chromatin, including histone methyltransferases, histone demethylases, and bromodomain-containing proteins. The Constellation shRNA library was transduced into several cell lines containing mutations in SWI/SNF components in addition to a panel of reference cell lines in order to identify factors that are specifically essential in those cell lines with altered SWI/SNF activity. Primary screening generated a list of genes that are potentially synthetically lethal with loss of function mutations in SWI/SNF components. Experiments are underway to confirm these genetic relationships and to obtain mechanistic insight into the role of these relationships in the context of chromatin biology. Most importantly, we will evaluate these genes as novel therapeutic targets in cancers with altered SWI/SNF function. This abstract is also presented as Poster A19. Citation Format: Andrew R. Conery, Adrianne Neiss, Charlie Hatton, Barbara M. Bryant, Robert J. Sims, III. Targeting altered SWI/SNF function through synthetic lethal RNAi screening. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr PR02.

Alzheimer's disease risk alleles in TREM2 illuminate innate immunity in Alzheimer's disease

Genetic studies have provided the best evidence for cause and effect relationships in Alzheimer's disease (AD). Indeed, the identification of deterministic mutations in the APP, PSEN1 and PSEN2 genes and subsequent preclinical studies linking these mutations to alterations in Aβ production and aggregation have provided pivotal support for the amyloid cascade hypothesis. In addition, genetic, pathologic and biological studies of APOE have also indicated that the genetic risk for AD associated with APOE4 can be attributed, at least in part, to its pro-amyloidogenic effect on Aβ. In recent years a number of SNPs that show unequivocal genome-wide association with AD risk have implicated novel genetic loci as modifiers of AD risk. However, the functional implications of these genetic associations are largely unknown. For almost all of these associations, the functional variants have not been identified. Very recently, two large consortiums demonstrated that rare variants in the triggering receptor expressed on myeloid cells 2 (TREM2) gene confer significant risk for AD. TREM2 is a type 1 membrane receptor protein primarily expressed on microglia in the central nervous system that has been shown to regulate phagocytosis and activation of monocytes. Previously it had been shown that homozygous loss of function mutations in TREM2 cause polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL, Nasu Hakola disease) and also a pure form of early-onset dementia. The association of TREM2 variants with AD brings innate immune signaling into the light, affirming innate immunity's role as a significant factor in AD pathogenesis.

PAPSS2 mutations cause autosomal recessive brachyolmia

BackgroundBrachyolmia is a heterogeneous group of skeletal dysplasias that primarily affects the spine. Clinical and genetic heterogeneity have been reported; at least three types of brachyolmia are known. TRPV4 mutations...

A tale of two glucose transporters: how GLUT2 re-emerged as a contender for glucose transport into the human beta cell

Finding novel causes for monogenic forms of diabetes is important as, alongside the clinical implications of such a discovery, it can identify critical proteins and pathways required for normal beta cell function in humans. It is increasingly apparent that there are significant differences between rodent and human islets. One example that has generated interest is the relative importance of the glucose transporter GLUT2 in rodent and human beta cells. The central role of GLUT2 in rodent beta cells is well established, but a number of studies have suggested that other glucose transporters, namely GLUT1 and GLUT3, may play an important role in facilitating glucose transport into human beta cells. In this issue of Diabetologia Sansbury et al (DOI: 10.1007/s00125-012-2595-0 ) report homozygous loss of function mutations in SLC2A2, which encodes GLUT2, as a rare cause of neonatal diabetes. Evidence for a beta cell defect in these subjects comes from very low birthweights, lack of endogenous insulin secretion and a requirement for insulin therapy. Neonatal diabetes is not a consistent feature of SLC2A2 mutations. It is only found in a small percentage of cases (~4%) and the diabetes largely resolves before 18months of age. This discovery is significant as it suggests that GLUT2 plays an important role in human beta cells, but the interplay and relative roles of other transporters differ from those in rodents. This finding should encourage efforts to delineate the precise role of GLUT2 in the human beta cell at different developmental time points and is a further reminder of critical differences between human and rodent islets.

Loss of Interleukin-10 Signaling and Infantile Inflammatory Bowel Disease: Implications for Diagnosis and Therapy

Homozygous loss of function mutations in interleukin-10 (IL10) and interleukin-10 receptors (IL10R) cause severe infantile (very early onset) inflammatory bowel disease (IBD). Allogeneic hematopoietic stem cell transplantation (HSCT) was reported to induce sustained remission in 1 patient with IL-10R deficiency. We investigated heterogeneity among patients with very early onset IBD, its mechanisms, and the use of allogeneic HSCT to treat this disorder. We analyzed 66 patients with early onset IBD (younger than 5 years of age) for mutations in the genes encoding IL-10, IL-10R1, and IL-10R2. IL-10R deficiency was confirmed by functional assays on patients' peripheral blood mononuclear cells (immunoblot and enzyme-linked immunosorbent assay analyses). We assessed the therapeutic effects of standardized allogeneic HSCT. Using a candidate gene sequencing approach, we identified 16 patients with IL-10 or IL-10R deficiency: 3 patients had mutations in IL-10, 5 had mutations in IL-10R1, and 8 had mutations in IL-10R2. Refractory colitis became manifest in all patients within the first 3 months of life and was associated with perianal disease (16 of 16 patients). Extraintestinal symptoms included folliculitis (11 of 16) and arthritis (4 of 16). Allogeneic HSCT was performed in 5 patients and induced sustained clinical remission with a median follow-up time of 2 years. In vitro experiments confirmed reconstitution of IL-10R-mediated signaling in all patients who received the transplant. We identified loss of function mutations in IL-10 and IL-10R in patients with very early onset IBD. These findings indicate that infantile IBD patients with perianal disease should be screened for IL-10 and IL-10R deficiency and that allogeneic HSCT can induce remission in those with IL-10R deficiency.

Loss of NPP1 induces cartilage remodelling into bone and OA-like changes in mice

BackgroundCartilage calcification is common in osteoarthritis (OA) and correlates with the severity of cartilage breakdown. Nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) contributes to maintain an optimal balance of pyrophosphate (PPi) and...

Impact on Bone of an Estrogen Receptor-α Gene Loss of Function Mutation

The kindred described is the only known instance of a germ line loss of function mutation of estrogen receptor (ER)-alpha. Our objective was to assess the impact of a loss of function mutation in the ER-alpha gene on histomorphometry, bone volumetric density, bone geometry and skeletal growth, and ER-alpha heterozygosity on spine density and adult height in an extended pedigree. A longitudinal follow-up of the propositus with homozygous loss of function mutation of ER-alpha and single contact evaluation of the kindred were performed. Iliac crest bone biopsy and peripheral quantitative computed tomography of propositus with serial measures of areal spine bone mineral density (aBMD) by dual-energy x-ray absorptiometry and bone age were performed. Members of pedigree were evaluated for ER-alpha mutation carrier status and spine aBMD. Bone biopsy revealed marked osteopenia (cortex: 641 microm), low trabecular volume (10.6%), decreased thickness (76.2 microm), normal trabecular number, and low activation frequency (0.099/yr). Radial periosteal circumference was similar, endosteal circumference larger, and trabecular and cortical volumetric bone mineral density markedly lower (158 and 1092 mg/cm(3), respectively) than controls. Spine aBMD at age 28.5 yr (0.745 g/cm(2)) decreased to 0.684 g/cm(2) (Z score -3.85) in 3.5 yr. Bone age advanced from 15-17.5 yr. Kindred analysis revealed that gene carriers had spine aBMD Z scores less than zero (P = 0.003), but carriers and nonmutant members were similar (-0.84 +/- 0.26 vs. -0.64 +/- 0.16). Homozygous ER-alpha disruption markedly affects bone growth, mineral content, and structure but not periosteal circumference. ER-alpha heterozygosity appears to not impair spine aBMD.

A new SBF2 mutation in a family with recessive demyelinating Charcot-Marie-Tooth (CMT4B2).

Charcot-Marie-Tooth (CMT) 1type 4 is a group of autosomal recessive progressive motor and sensory neuropathies characterized by distal muscle weakness and atrophy associated with sensory loss and, frequently, pes cavus foot deformity. CMT4 includes clinically and genetically heterogeneous disorders. Seven genes responsible for the different CMT4 subtypes (CMT4A, B1, B2, C, D, E, and F) have been identified so far.1 CMT4B, characterized by focally folded myelin sheaths on sural nerve biopsy, was first described in an Italian family2 and then in several other families of different ancestry. CMT4B is genetically heterogeneous. One form of CMT4B is caused by homozygous loss of function mutations in the myotubularin-related 2 gene (MTMR2) on chromosome 11q23 (CMT4B1).3 A second locus responsible for CMT4B has been mapped …

Generation of normal lymphocyte populations by Rb-deficient embryonic stem cells.

Mice homozygous for a loss-of-function mutation of the recombination-activating gene-2 (RAG 2), which is required for the rearrangement of antigen receptor genes, do not produce mature B and T lymphocytes. But chimeric mice that result from injection of normal embryonic stem (ES) cells into blastocysts from RAG2-deficient mice develop normal mature lymphocyte populations, all of which are derived from the injected ES cells; we have called this process RAG2-deficient blastocyst complementation. Using ES cells with homozygous mutations, RAG-2-deficient blastocyst complementation could provide a physiological assay with which to determine the potential role of almost any gene in the development and/or function of lymphocytes. To test the general utility of this system, we have used it to test the differentiation-potential of ES cells that harbor homozygous loss-of function mutations of their retinoblastoma susceptibility (Rb) gene loci. We chose Rb for this analysis because of its widespread function in the control of the cell cycle and cell differentiation, the adverse effect of homozygous germline mutations of Rb on hematopoiesis in fetal liver, and the embryonic lethality that results when the homozygous Rb mutation is introduced into the germline. Homozygous Rb mutant ES cells can develop into phenotypically normal, mature B and T lymphocytes in the RAG-2-deficient background. Strikingly, Rb-deficient B and T cells do not have major defects in either activation or function. We have demonstrated the efficacy of the RAG-2-deficient blastocyst complementation system for evaluating the role of critical genes in lymphocyte development. Our results indicate that Rb expression is not intrinsically required for B-cell or T-cell function, despite the normally high levels of Rb expressed in lymphoid cells.