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Henry N Higgs - One of the best experts on this subject based on the ideXlab platform.
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Regulation of INF2-mediated actin polymerization through site-specific lysine acetylation of actin itself.
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Mu A, Tak Shun Fung, Lisa M. Francomacaro, Thao Huynh, Tommi Kotila, Zdenek Svindrych, Henry N HiggsAbstract:INF2 is a formin protein that accelerates actin polymerization. A common mechanism for formin regulation is autoinhibition, through interaction between the N-terminal diaphanous inhibitory domain (DID) and C-terminal diaphanous autoregulatory domain (DAD). We recently showed that INF2 uses a variant of this mechanism that we term "facilitated autoinhibition," whereby a complex consisting of cyclase-associated protein (CAP) bound to lysine-acetylated actin (KAc-actin) is required for INF2 inhibition, in a manner requiring INF2-DID. Deacetylation of actin in the CAP/KAc-actin complex activates INF2. Here we use lysine-to-glutamine mutations as acetylmimetics to map the relevant lysines on actin for INF2 regulation, focusing on K50, K61, and K328. Biochemically, K50Q- and K61Q-actin, when bound to CAP2, inhibit full-length INF2 but not INF2 lacking DID. When not bound to CAP, these mutant actins polymerize similarly to WT-actin in the presence or absence of INF2, suggesting that the effect of the mutation is directly on INF2 regulation. In U2OS cells, K50Q- and K61Q-actin inhibit INF2-mediated actin polymerization when expressed at low levels. Direct-binding studies show that the CAP WH2 domain binds INF2-DID with submicromolar affinity but has weak affinity for actin monomers, while INF2-DAD binds CAP/K50Q-actin 5-fold better than CAP/WT-actin. Actin in complex with full-length CAP2 is predominately ATP-bound. These interactions suggest an inhibition model whereby CAP/KAc-actin serves as a bridge between INF2 DID and DAD. In U2OS cells, INF2 is 90-fold and 5-fold less abundant than CAP1 and CAP2, respectively, suggesting that there is sufficient CAP for full INF2 inhibition.
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Mice with mutant Inf2 show impaired podocyte and slit diaphragm integrity in response to protamine-induced kidney injury
Kidney international, 2016Co-Authors: Balajikarthick Subramanian, Henry N Higgs, Hua Sun, Paul Yan, Victoria T. Charoonratana, Fang Wang, Ka Man V Lai, David M. Valenzuela, Elizabeth J. Brown, Johannes SchlondorffAbstract:Mutations in the INF2 (inverted formin 2) gene, encoding a diaphanous formin family protein that regulates actin cytoskeleton dynamics, cause human focal segmental glomerulosclerosis (FSGS). INF2 interacts directly with certain other mammalian diaphanous formin proteins (mDia) that function as RhoA effector molecules. FSGS-causing INF2 mutations impair these interactions and disrupt the ability of INF2 to regulate Rho/Dia-mediated actin dynamics in vitro . However, the precise mechanisms by which INF2 regulates and INF2 mutations impair glomerular structure and function remain unknown. Here, we characterize an Inf2 R218Q point-mutant (knockin) mouse to help answer these questions. Knockin mice have no significant renal pathology or proteinuria at baseline despite diminished INF2 protein levels. INF2 mutant podocytes do show impaired reversal of protamine sulfate–induced foot process effacement by heparin sulfate perfusion. This is associated with persistent podocyte cytoplasmic aggregation, nephrin phosphorylation, and nephrin and podocin mislocalization, as well as impaired recovery of mDia membrane localization. These changes were partially mimicked in podocyte outgrowth cultures, in which podocytes from knockin mice show altered cellular protrusions compared to those from wild-type mice. Thus, in mice, normal INF2 function is not required for glomerular development but normal INF2 is required for regulation of the actin-based behaviors necessary for response to and/or recovery from injury.
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assembly and turnover of short actin filaments by the formin inf2 and profilin
Journal of Biological Chemistry, 2015Co-Authors: Pinar S Gurel, Bingquian Guo, Rui Shu, Dale F. Mierke, Henry N HiggsAbstract:INF2 (inverted formin 2) is a formin protein with unique biochemical effects on actin. In addition to the common formin ability to accelerate actin nucleation and elongation, INF2 can also sever filaments and accelerate their depolymerization. Although we understand key attributes of INF2-mediated severing, we do not understand the mechanism by which INF2 accelerates depolymerization subsequent to severing. Here, we show that INF2 can create short filaments (<60 nm) that continuously turn over actin subunits through a combination of barbed end elongation, severing, and WH2 motif-mediated depolymerization. This pseudo-steady state condition occurs whether starting from actin filaments or monomers. The rate-limiting step of the cycle is nucleotide exchange of ADP for ATP on actin monomers after release from the INF2/actin complex. Profilin addition has two effects: 1) to accelerate filament turnover 6-fold by accelerating nucleotide exchange and 2) to shift the equilibrium toward polymerization, resulting in longer filaments. In sum, our findings show that the combination of multiple interactions of INF2 with actin can work in concert to increase the ATP turnover rate of actin. Depending on the ratio of INF2:actin, this increased flux can result in rapid filament depolymerization or maintenance of short filaments. We also show that high concentrations of cytochalasin D accelerate ATP turnover by actin but through a different mechanism from that of INF2.
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nanostructured self assembly of inverted formin 2 inf2 and f actin inf2 complexes revealed by atomic force microscopy
Langmuir, 2014Co-Authors: Shivani Sharma, Pinar S Gurel, Henry N Higgs, Elena E Grintsevich, Emil Reisler, Jungreem Woo, James K. GimzewskiAbstract:Self-organization of cytoskeletal proteins such as actin and tubulin into filaments and microtubules is frequently assisted by the proteins binding to them. Formins are regulatory proteins that nucleate the formation of new filaments and are essential for a wide range of cellular functions. The vertebrate inverted formin 2 (INF2) has both actin filament nucleating and severing/depolymerizing activities connected to its ability to encircle actin filaments. Using atomic force microscopy, we report that a formin homology 2 (FH2) domain-containing construct of INF2 (INF2-FH1-FH2-C or INF2-FFC) self-assembles into nanoscale ringlike oligomeric structures in the absence of actin filaments, demonstrating an inherent ability to reorganize from a dimeric to an oligomeric state. A construct lacking the C-terminal region (INF2-FH1-FH2 or INF2-FF) also oligomerizes, confirming the dominant role of FH2-mediated interactions. Moreover, INF2-FFC domains were observed to organize into ringlike structures around single actin filaments. This is the first demonstration that formin FH2 domains can self-assemble into oligomers in the absence of filaments and has important implications for observing unaveraged decoration and/or remodeling of filaments by actin binding proteins.
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an actin dependent step in mitochondrial fission mediated by the er associated formin inf2
Science, 2013Co-Authors: Farida Korobova, Vinay Ramabhadran, Henry N HiggsAbstract:Mitochondrial fission is fundamentally important to cellular physiology. The dynamin-related protein Drp1 mediates fission, and interaction between mitochondrion and endoplasmic reticulum (ER) enhances fission. However, the mechanism for Drp1 recruitment to mitochondria is unclear, although previous results implicate actin involvement. Here, we found that actin polymerization through ER-localized inverted formin 2 (INF2) was required for efficient mitochondrial fission in mammalian cells. INF2 functioned upstream of Drp1. Actin filaments appeared to accumulate between mitochondria and INF2-enriched ER membranes at constriction sites. Thus, INF2-induced actin filaments may drive initial mitochondrial constriction, which allows Drp1-driven secondary constriction. Because INF2 mutations can lead to Charcot-Marie-Tooth disease, our results provide a potential cellular mechanism for this disease state.
Martin R. Pollak - One of the best experts on this subject based on the ideXlab platform.
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human kidney disease causing inf2 mutations perturb rho dia signaling in the glomerulus
EBioMedicine, 2014Co-Authors: Martin R. Pollak, Khaldoun I Alromaih, Calum A MacraeAbstract:Mutations in Inverted Formin 2 (INF2), a diaphanous formin family protein that regulates actin cytoskeleton dynamics, cause focal segmental glomerulosclerosis (FSGS) and Charcot–Marie–Tooth Disease (CMT) in humans. In addition to directly remodeling actin filaments in vitro, we have shown that INF2 regulates intracellular actin dynamics and actin dependent cellular behavior by opposing Rhoa/Dia signaling. As a step towards a better understanding of the human kidney disease, we wanted to explore the relevance of these findings to the in vivo situation. We used dose dependent knockdown of INF2 to first define an in vivo model and establish an overt glomerular phenotype in zebrafish. This simple assay was validated by rescue with wild type INF2 confirming the specificity of the findings. The edema, podocyte dysfunction, and an altered glomerular filtration barrier observed in the zebrafish pronephros correlate with mistrafficking of glomerular slit diaphragm proteins, defective slit-diaphragm signaling, and disinhibited diaphanous formin (mDia) activity. In contrast to wild-type human INF2, INF2 mutants associated with kidney disease fail to rescue the zINF2 morphant phenotype. Of particular interest, this INF2 knockdown phenotype is also rescued by loss of either RhoA or Dia2. This simple assay allows the demonstration that INF2 functions, at least in part, to modulate Dia-mediated Rho signaling, and that disease causing mutations specifically impair this regulatory function. These data support a model in which disease-associated diaphanous inhibitory domain (DID) mutants in INF2 interfere with its binding to and inhibition of Dia, leading to uncontrolled Rho/Dia signaling and perturbed actin dynamics. Methods to fine tune Rho signaling in the glomerulus may lead to new approaches to therapy in humans.
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Mutations in the INF2 gene account for a significant proportion of familial but not sporadic focal and segmental glomerulosclerosis
Kidney international, 2012Co-Authors: Moumita Barua, Hua Sun, Victoria T. Charoonratana, Elizabeth J. Brown, Giulio Genovese, Martin R. PollakAbstract:Mutations in the inverted formin 2 gene ( INF2 ) have recently been identified as the most common cause of autosomal dominant focal and segmental glomerulosclerosis (FSGS). To quantify the contribution of various genes contributing to FSGS, we sequenced INF2 where all mutations have previously been described (exons 2 to 5) in a total of 215 probands and 281 sporadic individuals with FSGS, along with other known genes accounting for autosomal dominant FSGS ( ACTN4 , TRPC6 , and CD2AP ) in 213 probands. Variants were classified as disease-causing if they altered the amino acid sequence and if they were not found in control samples and in families segregated with disease. Mutations in INF2 were found in a total of 20 of the 215 families (including those previously reported) in our cohort of autosomal dominant familial nephrotic syndrome or FSGS, thereby explaining disease in 9%. INF2 mutations were found in 2 of 281 individuals with sporadic FSGS. In contrast, ACTN4 - and TRPC6 -related diseases accounted for 3 and 2% of our familial cohort, respectively. INF2 -related disease showed variable penetrance, with onset of disease ranging widely from childhood to adulthood, and commonly leading to end-stage renal disease in the third and fourth decade of life. Thus, mutations in INF2 are a more common, although still a minor, monogenic cause of familial FSGS when compared with other known autosomal dominant genes associated with FSGS.
Alison A Bertuch - One of the best experts on this subject based on the ideXlab platform.
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The C-terminal extension unique to the long isoform of the shelterin component TIN2 enhances its interaction with TRF2 in a phosphorylation- and dyskeratosis congenita-cluster-dependent fashion
Molecular and cellular biology, 2018Co-Authors: Nya D Nelson, Lois M. Dodson, Laura Escudero, Ann T. Sukumar, Christopher L. Williams, Ivana Mihalek, Alessandro Baldan, Duncan Martin Baird, Alison A BertuchAbstract:TIN2 is central to the shelterin complex, linking the telomeric proteins TRF1 and TRF2 with TPP1/POT1. Mutations in TINF2, which encodes TIN2, that are found in dyskeratosis congenita (DC) result in very short telomeres and cluster in a region shared by the two TIN2 isoforms, TIN2S (short) and TIN2L (long). Here we show that TIN2L, but not TIN2S, is phosphorylated. TRF2 interacts more with TIN2L than TIN2S, and both the DC cluster and phosphorylation promote this enhanced interaction. The binding of TIN2L, but not TIN2S, is affected by TRF2-F120, which is also required for TRF2's interaction with end processing factors such as Apollo. Conversely, TRF1 interacts more with TIN2S than with TIN2L. A DC-associated mutation further reduces TIN2L-TRF1, but not TIN2S-TRF1, interaction. Cells overexpressing TIN2L or phosphomimetic TIN2L are permissive to telomere elongation, whereas cells overexpressing TIN2S or phosphodead TIN2L are not. Telomere lengths are unchanged in cell lines in which TIN2L expression has been eliminated by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated mutation. These results indicate that TIN2 isoforms are biochemically and functionally distinguishable and that shelterin composition could be fundamentally altered in patients with TINF2 mutations.
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three novel truncating TINF2 mutations causing severe dyskeratosis congenita in early childhood
Clinical Genetics, 2012Co-Authors: Ghadir Sasa, Albert Ribeszamora, Nya D Nelson, Alison A BertuchAbstract:Dyskeratosis congenita (DC) is a telomere biology disorder characterized by a mucocutaneous triad, aplastic anemia, and predisposition to cancer. Mutations in a narrow segment of TINF2 exon 6 have been recognized to cause often-severe DC that is either sporadic or autosomal dominant. We describe three children with very early presentations of DC, including one with the severe variant known as Revesz syndrome. Although most TINF2 mutations reported to date are missense changes, each of our patients carried a novel heterozygous nonsense or frameshift mutation, revealing a new 5' boundary to the affected gene segment in patients with DC. Examination of patient-derived lymphoblastoid cell lines revealed stable expression of the predicted truncated TIN2 proteins. In co-immunoprecipitation assays, the ability of a truncation mutant to interact with TRF1 was severely impaired, whereas the ability of the most common DC-associated mutant was much less affected. This suggests that the disruption of TIN2-TRF1 interaction may contribute to the severe clinical phenotype observed in the context of the TIN2 truncation mutation, but is unlikely to be the primary cause of telomere shortening associated with the more prevalent TIN2 missense mutations. Telomere flow-fluorescent in situ hybridization (FISH) analysis of one pedigree showed the dramatic effect a de novo nonsense TINF2 mutation had on telomere length in early development. These cases underscore the severe manifestations of truncating TINF2 mutations.
Inderjeet Dokal - One of the best experts on this subject based on the ideXlab platform.
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Differences in Disease Severity but Similar Telomere Lengths in Genetic Subgroups of Patients with Telomerase and Shelterin
2013Co-Authors: Tom J. Vulliamy, Richard Beswick, Upal Hossain, Charlotte Baqai, Michael J. Kirwan, Judith Marsh, A Walne, Inderjeet DokalAbstract:The bone marrow failure syndrome dyskeratosis congenita (DC) has been considered to be a disorder of telomere maintenance in which disease features arise due to accelerated shortening of telomeres. By screening core components of the telomerase and shelterin complexes in patients with DC and related bone marrow failure syndromes we have identified 24 novel mutations: 11 in the RNA component of telomerase (TERC), 8 in the reverse transcriptase component (TERT), 4in dyskerin (DKC1) and 1 in TRF1-interacting nuclear factor 2 (TINF2). This has prompted us to review these genetic subtypes in terms of telomere length, telomerase activity and clinical presentation among 194 genetically characterised index cases recruited onto the registry in London. While those with DKC1 and TINF2 mutations present at a younger age and have more disease features than those with TERC or TERT mutations, there is no difference in telomere length between these groups. There is no difference in the age of onset and numbers of disease features seen in those with TERC and TERT mutations despite the fact that the latter show higher levels of telomerase activity in vitro. The incidence of aplastic anaemia is greater in patients with TERC or TINF2 mutations compared to patients with DKC1 mutations, and cancer incidence is highest in patients with TERC mutations. These data are the first to provide robust comparisons between different genetic subtypes of telomerase and shelterin mutations (the ‘‘telomereopathies’’) and clearly demonstrate that disease severity is not explaine
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Differences in disease severity but similar telomere lengths in genetic subgroups of patients with telomerase and shelterin mutations.
PloS one, 2011Co-Authors: Tom Vulliamy, Amanda J Walne, Richard Beswick, Michael Kirwan, Upal Hossain, Charlotte Baqai, Anna Ratcliffe, Judith C. W. Marsh, Inderjeet DokalAbstract:The bone marrow failure syndrome dyskeratosis congenita (DC) has been considered to be a disorder of telomere maintenance in which disease features arise due to accelerated shortening of telomeres. By screening core components of the telomerase and shelterin complexes in patients with DC and related bone marrow failure syndromes we have identified 24 novel mutations: 11 in the RNA component of telomerase (TERC), 8 in the reverse transcriptase component (TERT), 4 in dyskerin (DKC1) and 1 in TRF1-interacting nuclear factor 2 (TINF2). This has prompted us to review these genetic subtypes in terms of telomere length, telomerase activity and clinical presentation among 194 genetically characterised index cases recruited onto the registry in London. While those with DKC1 and TINF2 mutations present at a younger age and have more disease features than those with TERC or TERT mutations, there is no difference in telomere length between these groups. There is no difference in the age of onset and numbers of disease features seen in those with TERC and TERT mutations despite the fact that the latter show higher levels of telomerase activity in vitro. The incidence of aplastic anaemia is greater in patients with TERC or TINF2 mutations compared to patients with DKC1 mutations, and cancer incidence is highest in patients with TERC mutations. These data are the first to provide robust comparisons between different genetic subtypes of telomerase and shelterin mutations (the “telomereopathies”) and clearly demonstrate that disease severity is not explained by telomere length alone.
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Telomere length measurement can distinguish pathogenic from non-pathogenic variants in the shelterin component, TIN2.
Clinical genetics, 2011Co-Authors: Tom Vulliamy, Amanda J Walne, Richard Beswick, Michael Kirwan, Upal Hossain, Inderjeet DokalAbstract:Dyskeratosis congenita (DC) is a heterogeneous disorder, both genetically and phenotypically (1). Clinical presentation is classically defined by a triad of mucocutaneous features: abnormal reticulate skin pigmentation, nail dystrophy and leukoplakia. Bone marrow failure and a spectrum of other somatic abnormalities are also commonly observed (2). In its severe form, DC overlaps with the Hoyeraal–Hreidarsson (HH) syndrome, characterized by immunodeficiency, cerebellar hypoplasia, microcephaly and growth retardation as well as aplastic anaemia (AA). Because of this wide range of phenotypes, a clinical diagnosis of DC can often be quite difficult, although we would usually define a DC patient as having at least two of the three mucocutaneous features with either evidence of bone marrow failure or two or more other somatic abnormalities (3). In some cases a genetic diagnosis can be made, based on the identification of a mutation in one of the seven genes (DKC1, TERC, TERT, NOP10, NHP2, TINF2 and C16orf57). Through genome-wide linkage analysis and candidate gene sequencing, the TINF2 gene was initially identified as the cause of DC in one family showing autosomal dominant inheritance of the disease (4). It has subsequently become clear that mutations in this gene usually arise de novo in sporadic cases, causing a relatively severe form of DC (4, 5). The gene encodes a core component of the shelterin complex – a group of proteins that interact to protect telomeres. This protein is called TIN2, the telomeric repeat binding factor 1 (TRF1)-interacting nuclear factor 2; it is an essential mediator of TRF1 function and acts as an important regulator of telomere length (6, 7). TIN2 acts as the central component of the shelterin complex, binding not only TRF1 but also TRF2, a second telomere DNA-binding protein (8) and TPP1, the TIN2-interacting protein (9). TIN2-anchored TPP1 plays a major role in the recruitment of telomerase to telomeres in human cells (10). TPP1 is also important for recruiting POT1 (protection of telomeres), which is the third DNA-binding protein of the shelterin complex. POT1 binds to telomeric single-stranded DNA, protecting chromosome ends from the DNA-damage response (11). A second larger isoform of TIN2 has recently been identified, and this appears to have a role in tethering telomeres to the nuclear matrix (12). Since our previous report in 2008 (5), we have been screening for TINF2 mutations in all patients referred to our DC registry with various forms of bone marrow failure. This has led to the identification of 16 new families with eight previously unreported variants. They show that the phenotype associated with TINF2 mutation is broader than previously thought, but also raise the question as to whether all of these novel variants are pathogenic.
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TINF2 mutations result in very short telomeres analysis of a large cohort of patients with dyskeratosis congenita and related bone marrow failure syndromes
Blood, 2008Co-Authors: Amanda J Walne, Tom Vulliamy, Richard Beswick, Michael Kirwan, Inderjeet DokalAbstract:Dyskeratosis congenita (DC) is a multisystem bone marrow failure syndrome characterized by a triad of mucocutaneous abnormalities and a predisposition to cancer. The genetic basis of DC remains unknown in more than 60% of patients. Mutations have been identified in components of the telomerase complex (dyskerin, TERC, TERT, NOP10, and NHP2), and recently in one component of the shelterin complex TIN2 (gene TINF2). To establish the role of TINF2 mutations, we screened DNA from 175 uncharacterised patients with DC as well as 244 patients with other bone marrow failure disorders. Heterozygous coding mutations were found in 33 of 175 previously uncharacterized DC index patients and 3 of 244 other patients. A total of 21 of the mutations affected amino acid 282, changing arginine to histidine (n = 14) or cysteine (n = 7). A total of 32 of 33 patients with DC with TINF2 mutations have severe disease, with most developing aplastic anaemia by the age of 10 years. Telomere lengths in patients with TINF2 mutations were the shortest compared with other DC subtypes, but TERC levels were normal. In this large series, TINF2 mutations account for approximately 11% of all DC, but they do not play a significant role in patients with related disorders. This study emphasises the role of defective telomere maintenance on human disease.
Blanche P Alter - One of the best experts on this subject based on the ideXlab platform.
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sequence analysis of the shelterin telomere protection complex genes in dyskeratosis congenita
Journal of Medical Genetics, 2011Co-Authors: Sharon A. Savage, Neelam Giri, Lea Jessop, Kristen Pike, Teri M Plona, Laurie Burdett, Blanche P AlterAbstract:Background Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterised by dystrophic nails, abnormal skin pigmentation and oral leukoplakia. Patients are at very high risk of cancer and other medical problems. They have exceedingly short telomeres for their age and approximately 60% have a germline mutation in a gene important in telomere biology ( DKC1 , TERC , TERT , TINF2 , NOP10 , or NHP2 ). The shelterin complex consists of six proteins encoded by TINF2 , ACD , POT1 , TERF1 , TERF2 and TERF2IP , which are essential for telomeric stability. TINF2 mutations are present in 11–25% of patients with DC. Methods Bi-directional sequence analysis was conducted of all exons, intron–exon boundaries and the proximal promoter of the other five shelterin genes to determine whether mutations in these genes were associated with DC. Sixteen mutation-negative patients, nine with DC and seven patients with short telomeres and bone marrow failure, were evaluated. Results Two variants were identified, ACD Ex1+189 G→A and TERF1 Ex9+59 G→A, which were each present in one patient and a healthy parent but absent in 364 controls. Three other variants were rare (<1%) but present in both patients and controls. Discussion These data suggest that except for TINF2 , mutations in shelterin genes are not a common cause of DC.
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TINF2 a component of the shelterin telomere protection complex is mutated in dyskeratosis congenita
American Journal of Human Genetics, 2008Co-Authors: Sharon A. Savage, Neelam Giri, Gabriela M. Baerlocher, Peter M Lansdorp, Blanche P AlterAbstract:Patients with dyskeratosis congenita (DC), a heterogeneous inherited bone marrow failure syndrome, have abnormalities in telomere biology, including very short telomeres and germline mutations in DKC1, TERC, TERT, or NOP10, but ∼60% of DC patients lack an identifiable mutation. With the very short telomere phenotype and a highly penetrant, rare disease model, a linkage scan was performed on a family with autosomal-dominant DC and no mutations in DKCI, TERC, or TERT. Evidence favoring linkage was found at 2p24 and 14q11.2, and this led to the identification of TINF2 (14q11.2) mutations, K280E, in the proband and her five affected relatives and TINF2 R282H in three additional unrelated DC probands, including one with Revesz syndrome; a fifth DC proband had a R282S mutation. TINF2 mutations were not present in unaffected relatives, DC probands with mutations in DKC1, TERC, or TERT or 298 control subjects. We demonstrate that a fifth gene, TINF2, is mutated in classical DC and, for the first time, in Revesz syndrome. This represents the first shelterin complex mutation linked to human disease and confirms the role of very short telomeres as a diagnostic test for DC.
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TINF2 , a Component of the Shelterin Telomere Protection Complex, Is Mutated in Dyskeratosis Congenita.
Blood, 2007Co-Authors: Sharon A. Savage, Neelam Giri, Gabriela M. Baerlocher, Peter M Lansdorp, Nick Orr, Blanche P AlterAbstract:Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by the triad of abnormal nails, lacey reticular skin pigmentation, and oral leukoplakia. Patients with DC are at high risk of developing aplastic anemia, myelodysplastic syndrome and leukemia. Diagnosis of DC is challenging due to variability of the triad and heterogeneous clinical findings such as pulmonary and liver disease, avascular necrosis, esophageal or urethral stenosis and development delay. The unifying feature in DC is exceedingly short telomere lengths and defects in telomere biology. Gene mutations have been identified in DKC1 (X-linked), TERC and TERT (dominant, AD) and NOP10 (recessive), but approximately 60% of DC patients lack a known mutation. We identified a non-consanguineous family with AD DC and no mutations in DKCI, TERC or TERT . The first DC cases were monozygotic twin brothers (now deceased). Telomere length was determined by flow-FISH on the twins’ children (6 affected, 4 unaffected), wives, their 8 siblings and parents. Despite variable clinical phenotypes, the 6 affected individuals (1 female, 5 male) all had very short telomere lengths ( st %ile for age). Unaffected relatives had normal telomere lengths. A single nucleotide polymorphism (SNP) genome-wide linkage screen (Human Linkage IVb, Illumina, Inc) was conducted using telomere length st %ile as the affected phenotype. SNPLINK was used to remove SNPs in linkage disequilibrium (D′=0.7, R 2 =0.4). Data were analyzed with GeneHunter under a parametric, AD, rare, highly-penetrant disease model. Evidence favoring linkage was found in a 17 megabase (Mb) region on chromosome 2p and a 3.1 Mb region on chromosome14q (LOD score=2.62 at both sites). Bi-directional sequence analysis of the two best candidate genes, DDX1 (2p) and TINF2 (14q) was conducted to identify mutations. A novel mutation, K280E, in TINF2 (protein name TIN2) was identified in the 6 living, affected family members but not in the 8 unaffected relatives, suggesting inheritance from the affected fathers. There were no mutations in DDX1. TINF2 was sequenced in 8 additional, unrelated DC probands without DKCI, TERC or TERT mutations and 7 with known mutations. An R282H mutation was present in 3 unrelated DC probands (1 Hoyeraal-Hreidarsson, 1 Revesz Syndrome). Another DC patient had an R282S mutation. TINF2 mutations were not present in unaffected relatives, DC probands with mutations in DKC1, TERC or TERT, or 298 controls. The mutation prevalence in DC probands represented in our cohort of patients with DC are DKC1 (18.8%), TERC (18.8%), TERT (6.2%), and TINF2 (31.2%). As a component of shelterin, the protein complex that stabilizes telomeres, TIN2 is highly evolutionarily conserved. It serves as a bridge between the three primary telomere DNA-binding proteins, TRF1, TRF2 and POT1 (via TPP1). In silico analyses predict that K280E, R282H and R282S are deleterious mutations. Functional studies are underway to characterize possible effects of these mutations on shelterin protein interactions. By focusing on telomere length as the affected phenotype, instead of the heterogeneous clinical features present in DC patients, we identified mutations in TINF2 and further validated telomere length as a diagnostic test for DC. This study demonstrates that TINF2 is the 5th gene mutated in DC, the 1 st in Revesz Syndrome, and the 1 st shelterin complex gene mutated in human disease.