Biochemical Defect Research Articles

The BiP Molecular Chaperone Plays Multiple Roles during the Biogenesis of TorsinA, an AAA+ ATPase Associated with the Neurological Disease Early-onset Torsion Dystonia

Early-onset torsion dystonia (EOTD) is a neurological disorder characterized by involuntary and sustained muscle contractions that can lead to paralysis and abnormal posture. EOTD is associated with the deletion of a glutamate (ΔE) in torsinA, an endoplasmic reticulum (ER) resident AAA(+) ATPase. To date, the effect of ΔE on torsinA and the reason that this mutation results in EOTD are unclear. Moreover, there are no specific therapeutic options to treat EOTD. To define the underlying biochemical defects associated with torsinAΔE and to uncover factors that might be targeted to offset defects associated with torsinAΔE, we developed a yeast torsinA expression system and tested the roles of ER chaperones in mediating the folding and stability of torsinA and torsinAΔE. We discovered that the ER lumenal Hsp70, BiP, an associated Hsp40, Scj1, and a nucleotide exchange factor, Lhs1, stabilize torsinA and torsinAΔE. BiP also maintained torsinA and torsinAΔE solubility. Mutations predicted to compromise specific torsinA functional motifs showed a synthetic interaction with the ΔE mutation and destabilized torsinAΔE, suggesting that the ΔE mutation predisposes torsinA to defects in the presence of secondary insults. In this case, BiP was required for torsinAΔE degradation, consistent with data that specific chaperones exhibit either pro-degradative or pro-folding activities. Finally, using two independent approaches, we established that BiP stabilizes torsinA and torsinAΔE in mammalian cells. Together, these data define BiP as the first identified torsinA chaperone, and treatments that modulate BiP might improve symptoms associated with EOTD.

Frataxin Silencing Inactivates Mitochondrial Complex I in NSC34 Motoneuronal Cells and Alters Glutathione Homeostasis

Friedreich’s ataxia (FRDA) is a hereditary neurodegenerative disease characterized by a reduced synthesis of the mitochondrial iron chaperon protein frataxin as a result of a large GAA triplet-repeat expansion within the first intron of the frataxin gene. Despite neurodegeneration being the prominent feature of this pathology involving both the central and the peripheral nervous system, information on the impact of frataxin deficiency in neurons is scant. Here, we describe a neuronal model displaying some major biochemical and morphological features of FRDA. By silencing the mouse NSC34 motor neurons for the frataxin gene with shRNA lentiviral vectors, we generated two cell lines with 40% and 70% residual amounts of frataxin, respectively. Frataxin-deficient cells showed a specific inhibition of mitochondrial Complex I (CI) activity already at 70% residual frataxin levels, whereas the glutathione imbalance progressively increased after silencing. These biochemical defects were associated with the inhibition of cell proliferation and morphological changes at the axonal compartment, both depending on the frataxin amount. Interestingly, at 70% residual frataxin levels, the in vivo treatment with the reduced glutathione revealed a partial rescue of cell proliferation. Thus, NSC34 frataxin silenced cells could be a suitable model to study the effect of frataxin deficiency in neurons and highlight glutathione as a potential beneficial therapeutic target for FRDA.

Insight into the lysosomal storage disorder cystinosis: genetic interactions with ERS1 in Saccharomyces cerevisiae (963.1)

Cystinosis is an inherited pediatric lysosomal storage disorder caused by loss‐of‐function mutations in the gene CTNS, which encodes the Cystinosin protein. While the causative gene and primary biochemical defect have been identified, it is still unclear exactly which pathways contribute to the cell death that is observed in cystinotic patients. To investigate the implications of the loss of Cystinosin, we have employed the power of the model organism Saccharomyces cerevisiae, a model organism for higher eukaryotes. Yeast is an organism that is amenable to genetic manipulation and has a fully sequenced and highly annotated genome. With the understanding that the yeast protein Ers1p is the ortholog of human Cystinosin, the overall aim of this research was to better understand the role of Ers1p in yeast by identifying genes that interact with the cognate gene ERS1. Using data derived during a functional genomics screen, we propose to investigate genes that exhibit increased or decreased expression in the absence of ERS1. Our findings will not only contribute to our understanding of cystinosis, but also to primordial pathways in yeast and other eukaryotes.Grant Funding Source: Supported by NIH Grant Number 2 P20 RR016479

A magic bullet to specifically eliminate mutated mitochondrial genomes from patients' cells

When mitochondrial diseases result from mutations found in the mitochondrial DNA, engineered mitochondrial-targeted nucleases such as mitochondrial-targeted zinc finger nucleases are shown to specifically eliminate the mutated molecules, leaving the wild-type mitochondrial DNA intact to replicate and restore normal copy number. In this issue, Gammage and colleagues successfully apply this improved technology on patients' cells with two types of genetic alterations responsible for neuropathy ataxia and retinitis pigmentosa (NARP) syndrome and Kearns Sayre syndrome and progressive external ophthalmoplegia (PEO).

Identification of TCIRG1 and CLCN7 gene mutations in a patient with autosomal recessive osteopetrosis

Osteopetrosis is a heritable bone disorder that exhibits highly clinical and genetical heterogeneity, and is caused by defective osteoclastic resorption. The three main forms are the autosomal recessive severe (ARO), the intermediate autosomal and the autosomal dominant benign osteopetrosis forms. In the present study, the clinical, biochemical and radiological manifestations were described in a patient with osteopetrosis. Sequence analysis identified the compound heterozygous mutations, c.909C>A (p.Tyr303X) and c.2008C>T (p.Arg670X), in TCIRG1, and a heterozygous splicing mutation, c.1798‑1G>T, in the chloride channel 7 gene (CLCN7). Two aberrant forms of the CLCN7 transcripts, c.1798_1883 (exon 20) deletion predicted to cause p.Leu601GlyfsX13, and the c.1798_1821 deletion, the first 24 bp of the exon 20, predicted to cause p.Gly600_Gln607del, were detected by further analysis of the splicing patterns in the leukocytes. The patient's asymptomatic mother carried the TCIRG1 c.909C>A (p.Tyr303X) and CLCN7 c.1798‑1G>T mutations, while the asymptomatic father carried the TCIRG1 c.2008C>T (p.Arg670X) mutation only. The patient was finally diagnosed with ARO on the basis of clinical and biochemical parameters, radiological changes and genetic defects. To the best of our knowledge, this is the first reported case of a patient with osteopetrosis who carries TCIRG1 and CLCN7 mutations. In addition, among the three mutations, TCIRG1 c.909C>A and CLCN7 c.1798‑1G>T were novel mutations.

Hereditary Rickets. How Genetic Alterations Explain the Biochemical and Clinical Phenotypes

The reemergence of vitamin D deficiency in the industrialized countries resurrects the "threat" of nutritional rickets, especially among pediatric populations, a fact that may lead to underdiagnosis of hereditary rickets. Today, hereditary rickets may be subdivided into two main groups according to their biochemical profile: the one associated with defects in vitamin D synthesis and action and the second associated with abnormal phosphorus metabolism. The classification of the patients in a particular group of hereditary rickets is determinative of the treatment to follow. This review, through the recent advances on vitamin D and P metabolism, discusses the molecular and biochemical defects associated to each group of inherited rickets, as well as the clinical phenotypes and the recommended therapeutic approaches.

Human mitochondrial leucyl tRNA synthetase can suppress non cognate pathogenic mt‐tRNA mutations

Disorders of the mitochondrial genome cause a wide spectrum of disease, these present mainly as neurological and/or muscle related pathologies. Due to the intractability of the human mitochondrial genome there are currently no effective treatments for these disorders. The majority of the pathogenic mutations lie in the genes encoding mitochondrial tRNAs. Consequently, the biochemical deficiency is due to mitochondrial protein synthesis defects, which manifest as aberrant cellular respiration and ATP synthesis. It has previously been reported that overexpression of mitochondrial aminoacyl tRNA synthetases has been effective, in cell lines, at partially suppressing the defects resulting from mutations in their cognate mt-tRNAs. We now show that leucyl tRNA synthetase is able to partially rescue defects caused by mutations in non-cognate mt-tRNAs. Further, a C terminal peptide alone can enter mitochondria and interact with the same spectrum of mt-tRNAs as the entire synthetase, in intact cells. These data support the possibility that a small peptide could correct at least the biochemical defect associated with many mt-tRNA mutations, inferring a novel therapy for these disorders.

Prevention by metformin of alterations induced by chronic exposure to high glucose in human islet beta cells is associated with preserved ATP/ADP ratio

AimWe have explored whether the insulin secretory defects induced by glucotoxicity in human pancreatic islets could be prevented by metformin and investigated some of the possible mechanisms involved. MethodsHuman pancreatic islets and INS-1E cells were cultured for 24h with or without high glucose (16.7mM) concentration in the presence or absence of therapeutical concentration of metformin and then glucose-stimulated insulin release, adenine nucleotide levels and mitochondrial complex I and II activities were measured. Islet ultrastructure was analyzed by electron microscopy. ResultsCompared to control islets, human islets cultured with high glucose showed a reduced glucose-stimulated insulin secretion that was associated with lower ATP levels and a lower ATP/ADP ratio. These functional and biochemical defects were significantly prevented by the presence of metformin in the culture medium, that was also able to significantly inhibit the activity of mitochondrial complex I especially in beta cells exposed to high glucose. Ultrastructural observations showed that mitochondrial volume density was significantly increased in high glucose cultured islets. The critical involvement of mitochondria was further supported by the observation of remarkably swollen organelles with dispersed matrix and fragmented cristae. Metformin was able to efficiently prevent the appearance of all these ultrastructural alterations in human islets exposed to high glucose. ConclusionsOur results show that the functional, biochemical and ultrastructural abnormalities observed in human islet cells exposed to glucotoxic condition can be significantly prevented by metformin, further highlighting a direct beneficial effect of this drug on the insulin secreting human pancreatic beta cells.

Dysmorphology of inborn errors of metabolism.

As we discover the molecular mechanism of disorders, eventually all dysmorphic syndromes will ultimately be considered biochemical defects. An overview on the recognition and classification of dysmorphic features will be provided. Categories of inborn errors of metabolism associated with dysmorphic manifestations will be discussed. For e.g. abnormal eye findings are an important clue to the diagnosisin galactosemia, cystinosis, Lowe syndrome, and homocystinuria. Unusual kinky hair is seen in Menkes disease. Skin findings typically lead to the diagnosis in Fabry, Hunter and steroid sulphatase deficiency. Infants who have peroxisomal disorders (Zellweger) pyruvate dehydrogenase deficiency, cholesterol biosynthetic disorders (Smith-Lemli-Opitz), multipleacyl-CoA dehydrogenase deficiency (glutaric aciduria type II), infants of mothers with phenylketonuria have striking facial dysmorphism and structural anomalies at birth. In other disorders dysmorphic features may not be present at birth but may develop with age at varying rates such as in lysosomal storage disorders (mucopolysaccharidoses, oligosaccharidoses), congenital disorders of glycosylation and mitochondrial disorders. Salient clinical features, biochemical defects, molecular basis, and diagnostic strategies will be discussed to permit early treatment of these disorders.

Streptozotocin-induced diabetes prolongs twitch duration without affecting the energetics of isolated ventricular trabeculae.

BackgroundDiabetes induces numerous electrical, ionic and biochemical defects in the heart. A general feature of diabetic myocardium is its low rate of activity, commonly characterised by prolonged twitch duration. This diabetes-induced mechanical change, however, seems to have no effect on contractile performance (i.e., force production) at the tissue level. Hence, we hypothesise that diabetes has no effect on either myocardial work output or heat production and, consequently, the dependence of myocardial efficiency on afterload of diabetic tissue is the same as that of healthy tissue.MethodsWe used isolated left ventricular trabeculae (streptozotocin-induced diabetes versus control) as our experimental tissue preparations. We measured a number of indices of mechanical (stress production, twitch duration, extent of shortening, shortening velocity, shortening power, stiffness, and work output) and energetic (heat production, change of enthalpy, and efficiency) performance. We calculated efficiency as the ratio of work output to change of enthalpy (the sum of work and heat).ResultsConsistent with literature results, we showed that peak twitch stress of diabetic tissue was normal despite suffering prolonged duration. We report, for the first time, the effect of diabetes on mechanoenergetic performance. We found that the indices of performance listed above were unaffected by diabetes. Hence, since neither work output nor change of enthalpy was affected, the efficiency-afterload relation of diabetic tissue was unaffected, as hypothesised.ConclusionsDiabetes prolongs twitch duration without having an effect on work output or heat production, and hence efficiency, of isolated ventricular trabeculae. Collectively, our results, arising from isolated trabeculae, reconcile the discrepancy between the mechanical performance of the whole heart and its tissues.

Involvement of Interleukin-1β, cyclooxygenase-2, and hypoxia-inducible factor -1α in idiopathic asthenozoospermia

Asthenozoospermia may result from different biochemical and functional defects. In particular, it has been demonstrated that reactive oxygen species (ROS) have a role in its pathogenesis. These reactive molecules may mediate the biological effects of cytokines, acting as intracellular signals. Human sperm cells are able to produce biologically active interleukin-1β (IL-1β), which stimulates cyclooxygenase-2 (COX-2) in various cells and tissues, including human reproductive tissues, even if its effects on male reproductive biology are largely unknown. COX-2 is the inducible isoform of the key enzyme in the biosynthesis of prostaglandins, and it could be also induced by hypoxia-inducible factor -1α (HIF -1α), which regulates many genes important for oxygen homeostasis. It has been recently proposed an involvement of COX-2 in male fertility disorders. In this scenario, our aim was to evaluate sperm parameters, as well as the expression of IL-1β, COX-2, and HIF -1α in spermatozoa isolated from normospermic fertile donors and asthenozoospermic infertile patients, by means of immunohistochemical analysis and enzyme-linked immunosorbent assay (ELISA). Sperm cells obtained from the asthenozoospermic group showed a higher expression of IL-1β, COX-2, and HIF-1α, compared with those from normospermic fertile subjects. Our results can lead us to speculate that the increased expression of these substances may influence the kinetic characteristics of spermatozoa. Nevertheless, further studies are in progress in order to assess whether these bioactive mediators may represent targets for future therapeutic strategies.

What Can We Learn From Clinical Trials of Exon Skipping for DMD?

On 20 September 2013, GlaxoSmithKline (GSK) and Prosensa announced that GSK's Phase III clinical trial ({"type":"clinical-trial","attrs":{"text":"NCT01254019","term_id":"NCT01254019"}}NCT01254019) of Drisapersen, an exon skipping drug for Duchenne muscular dystrophy (DMD), failed to meet the primary endpoint of a statistically significant improvement in the 6 Minute Walking Distance Test (6MWT) compared to placebo.1 On 12 November 2013, Sarepta Therapeutics announced that the US Food and Drug Administration (FDA) has considered its application for accelerated approval of Eteplirsen as a DMD drug to be premature.2 The news came as a great disappointment to the scientific community and more specifically to the families and foundations that had followed the trail of research articles and press announcements. Moreover, the clinical trial results and the FDA's view of the relationship between the dystrophin biomarker and functional endpoint of 6MWT in clinic will have a far reaching impact beyond exon skipping therapy in DMD.

Common and Novel TMEM70 Mutations in a Cohort of Italian Patients with Mitochondrial Encephalocardiomyopathy.

ATP synthase or complex V (cV) of the oxidative phosphorylation system is responsible for the production of ATP, dissipating the electrochemical gradient generated by the mitochondrial respiratory chain. In addition to maternally transmitted cV dysfunction caused by mutations in mtDNA genes (MT-ATP6 or MT-ATP8), encoding cV subunits, recessive mutations in the nuclear TMEM70 are the most frequent cause of ATP synthase deficiency.We report on a cohort of ten Italian patients presenting with neonatal lactic acidosis, respiratory distress, hypotonia, cardiomyopathy and psychomotor delay and harbouring mutations in TMEM70, including the common splice mutation and four novel variants. TMEM70 protein was virtually absent in all tested TMEM70 patients' specimens.The exact function of TMEM70 is not known, but it is considered to impact on cV assembly since TMEM70 mutations have been associated with isolated cV activity reduction. We detected a clear cV biochemical defect in TMEM70 patients' fibroblasts, whereas the assay was not reliable in frozen muscle. Nevertheless, the evaluation of the amount of holocomplexes in patients with TMEM70 mutations showed a nearly absent cV in muscles and a strong decrease of cV with accumulation of sub-assembly species in fibroblasts. In our cohort we found not only cV deficiencies but also impairment of other OXPHOS complexes. By ultrastructural analysis of muscle tissue from one patient with isolated cV deficiency, we found a severely impaired mitochondrial morphology with loss of the cristae. These findings indicate that cV impairment could indirectly alter other respiratory chain complex activities by disrupting the mitochondrial cristae structure.

Skeletal Muscle Myopathy Mutations in Tropomyosin Gene TPM3 affect Thin Filament Transitions between the Inactive and Active States

It has recently been discovered that nemaline myopathy and cap myopathy can be caused by mutations in β-tropomyosin (TPM2 gene) and that a third skeletal muscle myopathy, congenital fibre-type disproportion, is predominantly due to mutations in slow α-tropomyosin (TPM3 gene). We have expressed tropomyosin TPM3 mutations of residues R90, R167 and R244 in a baculovirus-Sf9 insect cells system and studied reconstituted actin-tropomyiosin mutant filaments biochemical properties. We analysed the effects of these TPM3 mutations on the biochemical properties of tropomyosin namely secondary structure, actin binding and the equilibrium constants of transitions between the ‘open’, ‘closed’ and ‘blocked’ states of thin filament and the cooperativity of these transitions. We also assessed their functional effects on the actomyosin ATPase. We found that TPM3 mutations did not affect secondary structure of tropomyosin and its interaction with actin. In contrast these mutations affected troponin-tropomyosin inhibition and activation of actomyosin ATPase. TPM3 mutations also affected the Blocked-closed transitions in the cooperative allosteric mechanism of regulation of the actomyosin complex. These findings are in agreement with recent structural studies of tropomyosin bound to actin in the closed and blocked conformations. We propose that destabilisation of the actin-tropomyosin interface by charge change in the blocked state may represent the underlying biochemical defect for the onset of skeletal muscle myopathies linked to these mutations.

Administration of deoxyribonucleosides or inhibition of their catabolism as a pharmacological approach for mitochondrial DNA depletion syndrome

Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is characterized by a reduction in mtDNA copy number and consequent mitochondrial dysfunction in affected tissues. A subgroup of MDS is caused by mutations in genes that disrupt deoxyribonucleotide metabolism, which ultimately leads to limited availability of one or several deoxyribonucleoside triphosphates (dNTPs), and subsequent mtDNA depletion. Here, using in vitro experimental approaches (primary cell culture of deoxyguanosine kinase-deficient cells and thymidine-induced mtDNA depletion in culture as a model of mitochondrial neurogastrointestinal encephalomyopathy, MNGIE), we show that supplements of those deoxyribonucleosides (dNs) involved in each biochemical defect (deoxyguanosine or deoxycytidine, dCtd) prevents mtDNA copy number reduction. Similar effects can be obtained by specific inhibition of dN catabolism using tetrahydrouridine (THU; inhibitor of cytidine deaminase) or immucillin H (inhibitor of purine nucleoside phosphorylase). In addition, using an MNGIE animal model, we provide evidence that mitochondrial dNTP content can be modulated in vivo by systemic administration of dCtd or THU. In spite of the severity associated with diseases due to defects in mtDNA replication, there are currently no effective therapeutic options available. Only in the case of MNGIE, allogeneic hematopoietic stem cell transplantation has proven efficient as a long-term therapeutic strategy. We propose increasing cellular availability of the deficient dNTP precursor by direct administration of the dN or inhibition of its catabolism, as a potential treatment for mtDNA depletion syndrome caused by defects in dNTP metabolism.

Metabolic myopathies.

The metabolic myopathies result from inborn errors of metabolism affecting intracellular energy production due to defects in glycogen, lipid, adenine nucleotides, and mitochondrial metabolism. This article provides an overview of the most common metabolic myopathies. Our knowledge of metabolic myopathies has expanded rapidly in recent years, providing us with major advances in the detection of genetic and biochemical defects. New and improved diagnostic tools are now available for some of these disorders, and targeted therapies for specific biochemical deficits have been developed (ie, enzyme replacement therapy for acid maltase deficiency). The diagnostic approach for patients with suspected metabolic myopathy should start with the recognition of a static or dynamic pattern (fixed versus exercise-induced weakness). Individual presentations vary according to age of onset and the severity of each particular biochemical dysfunction. Additional clinical clues include the presence of multisystem disease, family history, and laboratory characteristics. Appropriate investigations, timely treatment, and genetic counseling are discussed for the most common conditions.

Mutation for Nonsyndromic Mental Retardation in the trans-2-Enoyl-CoA Reductase TER Gene Involved in Fatty Acid Elongation Impairs the Enzyme Activity and Stability, Leading to Change in Sphingolipid Profile

Very long-chain fatty acids (VLCFAs, chain length >C20) exist in tissues throughout the body and are synthesized by repetition of the fatty acid (FA) elongation cycle composed of four successive enzymatic reactions. In mammals, the TER gene is the only gene encoding trans-2-enoyl-CoA reductase, which catalyzes the fourth reaction in the FA elongation cycle. The TER P182L mutation is the pathogenic mutation for nonsyndromic mental retardation. This mutation substitutes a leucine for a proline residue at amino acid 182 in the TER enzyme. Currently, the mechanism by which the TER P182L mutation causes nonsyndromic mental retardation is unknown. To understand the effect of this mutation on the TER enzyme and VLCFA synthesis, we have biochemically characterized the TER P182L mutant enzyme using yeast and mammalian cells transfected with the TER P182L mutant gene and analyzed the FA elongation cycle in the B-lymphoblastoid cell line with the homozygous TER P182L mutation (TER(P182L/P182L) B-lymphoblastoid cell line). We have found that TER P182L mutant enzyme exhibits reduced trans-2-enoyl-CoA reductase activity and protein stability, thereby impairing VLCFA synthesis and, in turn, altering the sphingolipid profile (i.e. decreased level of C24 sphingomyelin and C24 ceramide) in the TER(P182L/P182L) B-lymphoblastoid cell line. We have also found that in addition to the TER enzyme-catalyzed fourth reaction, the third reaction in the FA elongation cycle is affected by the TER P182L mutation. These findings provide new insight into the biochemical defects associated with this genetic mutation.

Tmem14c Plays An Essential Role In Mitochondrial Heme Metabolism

Red cells synthesize large amounts of heme during terminal differentiation. Central to this process is the transport and trafficking of heme synthesis intermediates within the cell. Despite the importance of transport during heme synthesis, the molecules involved in this process are largely unknown. In a screen for genes that are upregulated during erythroid terminal differentiation, we identified Tmem14c, a predicted multi-pass transmembrane protein as an essential component of the porphyrin metabolism pathway.Here, we report that Tmem14c facilitates the synthesis of mitochondrial protoporphyrin IX from coproporphyrinogen III and is thus required for heme synthesis. Tmem14c is a mitochondrial inner-membrane protein enriched in vertebrate hematopoietic tissues and is required for terminal erythropoiesis. Tmem14c gene-trap mouse embryos are severely anemic and mostly die by E13.5 (Fig. A). Fetal liver erythroid cells derived from gene-trap embryos experience maturation arrest. shRNA silencing of Tmem14c in Friend murine erythroleukemia (MEL) cells results in a significant decrease in de-novo heme synthesis. The biochemical defect is due to a decrease in mitochondrial protoporphyrin IX synthesis, while cytoplasmic porphyrin levels remain normal (Fig. B). The heme synthesis defect in Tmem14c-silenced MEL cells is complemented with a protoporphyrin IX analog. These data show the role of Tmem14c in regulating the terminal steps in mitochondrial porphyrin trafficking. [Display omitted] Our findings collectively demonstrate that Tmem14c is required for the transport of mitochondrial porphyrins in developing erythroid cells. Due to its inner-mitochondrial localization and its relative proximity to heme synthetic enzymes coproporphyrinogen oxidase and protoporphyrinogen oxidase (Rhee et al., 2013 Science), Tmem14c can function as a molecular adaptor that facilitates the interaction of proteins involved in porphyrin transport, or as a protoporphyrinogen IX transporter (Fig. C). The identification of Tmem14c as an essential regulator of porphyrin transport and heme synthesis provides a novel genetic tool for exploring erythropoiesis and disorders of heme synthesis such as porphyria and anemia. Disclosures:No relevant conflicts of interest to declare.

TIN2 Mutations In Dyskeratosis Congenita Cause Telomere Shortening In Induced Pluripotent Stem Cells Through Potent Dominant Negative Inhibition Of Telomerase

Dyskeratosis congenita (DC) is a bone marrow failure syndrome characterized by widespread defects in diverse tissues and a strong predisposition to cancer. DC is caused by germline mutations in genes controlling maintenance of telomeres, nucleoprotein caps that protect chromosome ends. Mutations in components of the telomerase enzyme comprise a large share of cases, including in TERT, TERC, dyskerin, TCAB1, NOP10 and NHP2. These mutations compromise telomerase function leading to telomere shortening, which in turn impairs stem cell function. We previously created patient-derived iPS cells from patients with mutations in TERT, dyskerin or TCAB1 and analyzed these cells to understand the biochemical defects in the telomerase pathway. In each case we found a unique mechanism underlying these telomerase defects, including: reduced catalytic function (TERT mutations), impaired telomerase assembly (dyskerin mutations) and mislocalization of the enzyme to nucleoli (TCAB1 mutations). A six-member protein complex – shelterin - is essential for proper function of telomeres. Despite the critical importance of shelterin proteins in telomere regulation, only a single telomere binding protein – TIN2 – is mutated in DC. However, how these mutations compromise telomere maintenance remains poorly understood. TIN2 mutations occur in a common, autosomal dominant form of DC, presenting in early life, with particularly severe clinical manifestations and poor outcomes. Mutations in the TIN2 gene are clustered in exon 6a, which corresponds to a protein domain of unknown function. To understand how TIN2 mutations impair telomere maintenance and cause DC, we reprogrammed fibroblasts from patients with TIN2 mutations to iPS cells. We succeeded in generating pluripotent iPS cells from a patient with a frame shift mutation at position 284 of the protein. TIN2-mutant iPS cells expressed all the markers of wild-type iPS cells and human ES cells and could be differentiated to all three germ cell layers in culture. With reprogramming from fibroblasts to iPS cells, telomerase is upregulated and causes telomere elongation in wild-type cells. In analyzing telomeres from TIN2-mutant iPS cells, we found that telomere elongation was abrogated. Instead of telomere elongation, TIN2-mutant iPS cells showed telomere shortening with reprogramming and during passage in cell culture. After extended cell passage, TIN2-mutant iPS cells lost the ability to self-renew and differentiated, concomitant with the activation of the telomere surveillance checkpoint p53. To better understand how TIN2 mutant proteins interfere with telomere maintenance, we overexpressed GFP, wild-type TIN2, or TIN2 truncation mutants from DC patients into human, telomerase-positive cancer cells. Genomic DNA was collected from these cells during passage and analyzed for telomere lengths by Southern blot. Expression of GFP or wild-type TIN2 had no effect on telomere lengths, which were stably maintained during the experiment. In marked contrast, expression of the TIN2 truncation mutants from DC patients led to progressive and dramatic telomere shortening with cell passage. Together, these data in patient-derived iPS cells and in human cancer cells suggest that TIN2 mutants inhibit the action of telomerase at telomeres. These results constitute a new molecular mechanism at play in DC and yield new insight into one of the most common forms of DC. Disclosures:No relevant conflicts of interest to declare.

Lysosome dysfunction in the pathogenesis of kidney diseases

The lysosome, an organelle central to macromolecule degradation and recycling, plays a pivotal role in normal cell processes, ranging from autophagy to redox regulation. Not surprisingly, lysosomes are an integral part of the renal epithelial molecular machinery that facilitates normal renal physiology. Two inherited diseases that manifest as kidney dysfunction are Fabry's disease and cystinosis, each of which is caused by a primary biochemical defect at the lysosome resulting from loss-of-function mutations in genes that encode lysosomal proteins. The functions of the lysosomes in the kidney and how lysosomal dysfunction might contribute to Fabry's disease and cystinosis are discussed. Unlike most other pediatric renal diseases, therapies are available for Fabry's disease and cystinosis, but require early diagnosis. Recent analysis of ceroid neuronal lipofuscinosis type 3 (Cln3) null mice, a mouse model of lysosomal disease that is primarily associated with neurological deficits, revealed renal functional abnormalities. As current and future therapeutics increase the life-span of those suffering from diseases like neuronal ceroid lipofuscinosis, it remains a distinct possibility that many more lysosomal disorders that primarily manifest as infant and juvenile neurodegenerative diseases may also include renal disease phenotypes.