Etiological point mutations in the hereditary multiple exostoses gene EXT1: a functional analysis of heparan sulfate polymerase activity.
Department of Microbiology and Immunology, University of British Columbia, 6174 University Boulevard, Vancouver, British Columbia, V6T 1Z3, Canada.
Hereditary multiple exostoses (HME), a dominantly inherited genetic disorder characterized by multiple cartilaginous tumors, is caused by mutations in members of the EXT gene family, EXT1 or EXT2. The corresponding gene products, exostosin-1 (EXT1) and exostosin-2 (EXT2), are type II transmembrane glycoproteins which form a Golgi-localized heterooligomeric complex that catalyzes the polymerization of heparan sulfate (HS). Although the majority of the etiological mutations in EXT are splice-site, frameshift, or nonsense mutations that result in premature termination, 12 missense mutations have also been identified. Furthermore, two of the reported etiological missense mutations (G339D and R340C) have been previously shown to abrogate HS biosynthesis (McCormick et al. 1998). Here, a functional assay that detects HS expression on the cell surface of an EXT1-deficient cell line was used to test the remaining missense mutant exostosin proteins for their ability to rescue HS biosynthesis in vivo. Our results show that EXT1 mutants bearing six of these missense mutations (D164H, R280G/S, and R340S/H/L) are also defective in HS expression, but surprisingly, four (Q27K, N316S, A486V, and P496L) are phenotypically indistinguishable from wild-type EXT1. Three of these four "active" mutations affect amino acids that are not conserved among vertebrates and invertebrates, whereas all of the HS-biosynthesis null mutations affect only conserved amino acids. Further, substitution or deletion of each of these four residues does not abrogate HS biosynthesis. Taken together, these results indicate that several of the reported etiological mutant EXT forms retain the ability to synthesize and express HS on the cell surface. The corresponding missense mutations may therefore represent rare genetic polymorphisms in the EXT1 gene or may interfere with as yet undefined functions of EXT1 that are involved in HME pathogenesis.
Genetic models of osteochondroma onset and neoplastic progression: evidence for mechanisms alternative to EXT genes inactivation.
Department of Medical Genetics and Skeletal Rare Diseases, Rizzoli Orthopaedic Institute, Bologna, Italy.
Osteochondroma, the most common benign bone tumor, may occur as a sporadic lesion or as multiple neoplasms in the context of multiple osteochondromas syndrome. The most severe complication is malignant transformation into peripheral secondary chondrosarcoma. Although both benign conditions have been linked to defects in EXT1 or EXT2 genes, contradictory reports are present in the literature regarding the requirement of their biallelic inactivation for osteochondroma development. A major limitation of these studies is represented by the small number of samples available for the screening. Taking advantage of a large series of tissues, our aim was to contribute to the definition of a genetic model for osteochondromas onset and transformation. EXT genes point mutations and big deletions were analyzed in 64 tissue samples. A double hit was found in 5 out of 35 hereditary cases, 6 out of 16 chondrosarcomas and 2 recurrences; none of the 11 sporadic osteochondromas showed two somatic mutations. Our results clearly indicate that, in most cases, biallelic inactivation of EXT genes does not account for osteochondromas formation; this mechanism should be regarded as a common feature for hereditary osteochondromas transformation and as an event that occurs later in tumor progression of solitary cases. These findings suggest that mechanisms alternative to EXT genetic alteration likely have a role in osteochondromas pathogenesis.
Kevin B Jones, Virginia Piombo, Charles Searby, Gail Kurriger, Baoli Yang, Florian Grabellus, Peter J Roughley, Jose A Morcuende, Joseph A Buckwalter, Mario R Capecchi, Andrea Vortkamp, Val C Sheffield
Department of Orthopaedics, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA.
We report a mouse model of multiple osteochondromas (MO), an autosomal dominant disease in humans, also known as multiple hereditary exostoses (MHE or HME) and characterized by the formation of cartilage-capped osseous growths projecting from the metaphyses of endochondral bones. The pathogenesis of these osteochondromas has remained unclear. Mice heterozygous for Ext1 or Ext2, modeling the human genotypes that cause MO, occasionally develop solitary osteochondroma-like structures on ribs [Lin et al.(2000) Dev Biol 224(2):299-311; Stickens et al.(2005) Development 132(22):5055-5068]. Rather than model the germ-line genotype, we modeled the chimeric tissue genotype of somatic loss of heterozygosity (LOH), by conditionally inactivating Ext1 via head-to-head loxP sites and temporally controlled Cre-recombinase in chondrocytes. These mice faithfully recapitulate the human phenotype of multiple metaphyseal osteochondromas. We also confirm homozygous disruption of Ext1 in osteochondroma chondrocytes and their origin in proliferating physeal chondrocytes. These results explain prior modeling failures with the necessity for somatic LOH in a developmentally regulated cell type.
Hereditary multiple exostoses and juvenile colon carcinoma: A case with a common genetic background?
Giacomo Pata, Riccardo Nascimbeni, Diego Di Lorenzo, Maria Gervasi, Vincenzo Villanacci, Bruno Salerni
Department of Medical & Surgical Sciences, 1st Division of General Surgery, University of Brescia, 25124 Brescia, Italy. firstname.lastname@example.org
A case of obstructing colon cancer is described in a 31-year-old patient affected by hereditary multiple exostoses. The association of these two rare conditions, which has never been described previously, and their early onset prompt us to discuss the clinical and genetic elements of a potential common pathogenic scenario.
Department of Orthopaedic Surgery, Kaplan Medical Center, P.O. Box 1, Rehovot, 76100, Israel, email@example.com.
BACKGROUND Hereditary multiple exostoses (HME) is a genetic disorder that causes limb deformities due to disturbance at the growth plates. MATERIALS AND METHODS Six adolescents, whith symptomatic valgus deformity at the ankle and knee (seven affected legs) underwent correction procedures using the Ilizarov apparatus. In 5 legs, a bifocal Ilizarov apparatus was used, whereas in 2 legs the use of a monofocal apparatus was sufficient. RESULTS Correction of the mechanical axis was achieved in all cases, and limb length discrepancy was equalized in the 3 cases that underwent limb elogation. The average knee and ankle corrections were 15 degrees and 18 degrees , respectively. The average time from application to removal of the Ilizarove apparatus was 4.6 months. No major complication occurred. CONCLUSIONS The use of the Ilizarov method in adolescents with HME enables successful simultaneous correction of multiplanar, multifocal complex limb deformities.
Barbara Leube, Karin Hardt, Sebastian Portier, Bettina Westhoff, Marcus Jäger, Rüdiger Krauspe, Brigitte Royer-Pokora
Institute of Human Genetics, Heinrich-Heine University Duesseldorf, Duesseldorf, Germany.
Multiple osteochondromas (MO) is an autosomal-dominant inherited disorder. The two genes responsible (EXT1 and EXT2) have been identified. We investigated 12 MO families for phenotype details and the genetic basis by cosegregation and mutation analysis (seven novel pathogenic mutations [five frameshift, one splice site, and one gross deletion] and one novel missense polymorphism). We found EXT1 to be responsible in seven families (19 affected members) and EXT2 in four families (17 affected members). One family remains undetermined. We found a tendency to a more severe phenotype in EXT1 families. As a novel finding, we could identify a single parameter (ulna/height ratio) that separates EXT1 family from EXT2 family in our series.
Mol Biol Rep. 2009 Apr ;36 (4):661-7 18330718
Genetic analysis of hereditary multiple exostoses in Tunisian families: a novel frame-shift mutation in the EXT1 gene.
Sana Sfar, Abderrazak Abid, Wijden Mahfoudh, Houyem Ouragini, Farah Ouechtati, Sonia Abdelhak, Lotfi Chouchane
Department of Molecular Immuno-Oncology, Faculty of Medicine, University of Monastir, Monastir, Tunisia.
Hereditary multiple exostoses (HME) is an autosomal dominant orthopaedic disorder most frequently caused by mutations in the EXT1 gene. The aim of the present study is to determine the underlying molecular defect of HME in two multigenerational Tunisian families with 21 affected members and to examine the degree of intrafamilial variability. Linkage analysis was performed using three microsatellite markers encompassing the EXT1 locus and mutation screening was carried out by direct sequencing. In family 1, evidence for linkage to EXT1 was obtained on the basis of a maximum LOD score of 4.26 at theta = 0.00 with D8S1694 marker. Sequencing of the EXT1 revealed a heterozygous G > T transversion (c.1019G>T) in exon 2, leading to a missense mutation at the codon 340 (p.Arg340Leu). In family 2 we identified a novel heterozygous 1 bp deletion in the exon 1 (c.529_531delA) leading to a premature codon stop and truncated EXT1 protein expression (p.Lys177LysfsX15). This mutation was associated with the evidence of an intrafamilial clinical variability and considered to be a novel disease-causing mutation in the EXT1 gene. These findings provide additional support for the involvement of EXT1 gene in the HME disease.
A combined analytical approach reveals novel EXT1/2 gene mutations in a large cohort of Italian multiple osteochondromas patients.
Emanuela Signori, Emanuela Massi, Maria Giovanna Matera, Monica Poscente, Carolina Gravina, Gianluca Falcone, Michele Attilio Rosa, Monica Rinaldi, Wim Wuyts, Davide Seripa, Bruno Dallapiccola, Vito Michele Fazio
Laboratory of Molecular Medicine and Biotechnology, University Campus Bio-Medico School of Medicine and Institute of Neurobiology and Molecular Medicine, CNR, Rome, Italy. firstname.lastname@example.org
Multiple osteochondromas (MO), also known as hereditary multiple exostoses (HME), is one of the most common hereditary musculoskeletal diseases in Caucasians (1/50,000) with wide clinical variability and genetic heterogeneity. Two genes have thus far been identified as causing the disease, namely EXT1 and EXT2. Various methods to detect mutations in the EXT genes have been used. Here a cohort of 100 MO patients belonging to unrelated Italian families have been analyzed by single-strand conformation polymorphism (SSCP) analysis or by denaturing high performance liquid chromatography (DHPLC). However, neither of these techniques can detect deletions or duplications of entire exons. Families that were negative at SSCP/DHPLC analysis underwent two-color multiple ligation-dependent probe amplification (MLPA) analysis. By these complementary techniques mutation detection was significantly improved and 26 novel mutations have been revealed as well as 18 previously described mutations to give a total of 44 different mutations. Thus we can conclude that combining MLPA with DHPLC in point-mutations negative MO families, the detection of mutations in EXT genes can significantly improve the identification of both point-mutations and mid-size rearrangements. More important, we were able to characterize all those patients who were negative at the first PCR-based method screening.
Decreased EXT expression and intracellular accumulation of heparan sulphate proteoglycan in osteochondromas and peripheral chondrosarcomas.
L Hameetman, G David, A Yavas, Sj White, Ahm Taminiau, A-M Cleton-Jansen, Pcw Hogendoorn, Jvmg Bovée
Department of Pathology, Leiden University Medical Centre, Leiden, The Netherlands.
Mutational inactivation of EXT1 or EXT2 is the cause of hereditary multiple osteochondromas. These genes function in heparan sulphate proteoglycan (HSPG) biosynthesis in the Golgi apparatus. Loss of heterozygosity of the EXT1 locus at 8q24 is frequently found in solitary osteochondromas, whereas somatic mutations are rarely found. We investigated the expression of EXT1 and EXT2 (quantitative RT-PCR) and of different HSPGs (immunohistochemistry) in solitary and hereditary osteochondromas and in cases with malignant progression to secondary peripheral chondrosarcoma, in relation to possible mutations and promoter methylation. The mutation status of patients with multiple osteochondromas correlated with decreased EXT1 or EXT2 expression found in their resected tumours. We could not show somatic point mutations or promoter hypermethylation in 17 solitary tumours; however, EXT1 expression was decreased in 15 cases, whereas EXT2 was not. Intracellular accumulation of syndecan-2 and heparan sulphate-bearing isoforms of CD44 (CD44v3) was found in most tumours, which concentrated in the Golgi apparatus as shown by confocal microscopy. This contrasted with the extracellular expression found in normal growth plates. In conclusion, mutational inactivation of either EXT1 or EXT2 leads to loss of mRNA expression of the corresponding gene. We hypothesize that loss of EXT expression disrupts the function of the EXT1/2 complex in HSPG biosynthesis, resulting in the intracellular accumulation of HSPG core proteins that we found in these tumours. Copyright (c) 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Novel EXT1 and EXT2 mutations identified by DHPLC in Italian patients with multiple osteochondromas.
Elena Pedrini, Alessandro De Luca, Enza Maria Valente, Veronica Maini, Silvia Capponcelli, Marina Mordenti, Rita Mingarelli, Luca Sangiorgi, Bruno Dallapiccola
Modulo di Familiarità e Genetica, Lab. Ricerca Oncologica, Istituti Ortopedici Rizzoli, Bologna, Italy.
We describe the results of an optimised DHPLC-based mutation screening of the EXT1 and EXT2 genes in Italian patients affected by multiple osteochondromas [MO; also referred to as hereditary multiple exostoses (HME) in the literature], using a multistep approach. We first analysed 36 unrelated probands for EXT1 mutations by DHPLC analysis and subsequent direct sequencing of all samples with abnormal elution profile. Negative cases were then screened for EXT2 mutations using the same approach. In patients who tested normal at DHPLC screening, all EXT1 and EXT2 exons and splice-site junctions were directly sequenced. In 7 informative families, we also performed a pre-screening linkage analysis to selectively focus the DHPLC testing on the EXT1 or EXT2 gene. We detected 31 MO-related mutations, of which 23 (74%) were novel. Seven polymorphisms were also found. Twenty-four mutations (77%) were found in EXT1 and 7 (23%) in EXT2. No disease-causing mutations were detected in five of 36 patients, with a mutation frequency of 86%. According with previous studies, most mutations (90%) are loss of function. Neither false positive nor false negative results were obtained. This multistep method can be considered a fast and reliable diagnostic strategy for the detection of EXT1/2 mutations, with excellent sensitivity and specificity.
Manuel C Lemos, Peter Kotanko, Paul T Christie, Brian Harding, Theodora Javor, Christine Smith, Richard Eastell, Rajesh V Thakker
Academic Endocrine Unit, Nuffield Department of Clinical Medicine, University of Oxford, Oxford Center for Diabetes, Endocrinology, and Metabolism, Churchill Hospital, Headington, Oxford OX3 7LJ, United Kingdom.
CONTEXT Hereditary multiple exostosis (HME) is an autosomal dominant disorder characterized by the development of benign cartilage-capped tumors at the juxta-epiphyseal regions of long bones. HME is usually caused by mutations of EXT1 or EXT2. OBJECTIVE The objective of this study was to investigate a three-generation Austrian kindred with HME for EXT1 and EXT2 mutations and for abnormalities of bone mineral density (BMD). METHODS DNA sequence and mRNA analyses were used to identify the mutation and its associated consequences. Serum biochemical and radiological investigations assessed bone metabolism and BMD. RESULTS HME-affected members had a lower femoral neck BMD compared with nonaffected members (z-scores,-2.98 vs.-1.30; P = 0.011), and in those less than 30 yr of age, the lumbar spine BMD was also low (z-scores,-2.68 vs.-1.42; P = 0.005). However, they had normal mobility and normal serum concentrations of calcium, phosphate, alkaline phosphatase activity, creatinine, PTH, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, osteocalcin, and beta-crosslaps. DNA sequence analysis of EXT1 revealed a heterozygous g-->c transversion that altered the invariant ag dinucleotide of the intron 8 acceptor splice site. RT-PCR analysis using lymphoblastoid RNA showed that the mutation resulted in skipping of exon 9 with a premature termination at codon 599. DNA sequence abnormalities of the osteoprotegerin gene, which is in close proximity to the EXT1 gene, were not detected. CONCLUSIONS A novel heterozygous acceptor splice site mutation of EXT1 results in HME that is associated with a low peak bone mass, indicating a possible additional role for EXT1 in bone biology and in regulating BMD.
Other papers by authors:
The link between heparan sulfate and hereditary bone disease: finding a function for the EXT family of putative tumor suppressor proteins.
Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada.
Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada V6T 1Z3.
To gain entry into the host, viruses use host cell surface molecules that normally serve as receptors for other ligands. Herpes simplex virus type 1 (HSV-1) uses heparan sulphate (HS) glycosaminoglycans (GAGs) as receptors for initial attachment to the host cell surface. HS GAGs are both ubiquitous and structurally diverse, and normally serve as critical mediators of interactions between the cell and the extracellular environment. We have used the HS binding ability of HSV-1 to identify the function of a cellular gene, EXT1, which is involved in HS polymerisation. Cellular factors that affect virus growth and replication are often key regulators of the cell cycle and EXT1 is no different-humans with inherited mutations in EXT1 have developmental defects that lead to bone tumours (hereditary multiple exostoses, HME) and sometimes chondrosarcomas. Thus, as a result of using HSV-1 as a molecular probe, a functionally orphaned disease gene now has a defined function. These findings highlight the utility of viruses for investigating important cellular processes.
The putative tumor suppressors EXT1 and EXT2 form a stable complex that accumulates in the Golgi apparatus and catalyzes the synthesis of heparan sulfate.
Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
Hereditary multiple exostoses, a dominantly inherited genetic disorder characterized by multiple cartilaginous tumors, is caused by mutations in members of the EXT gene family, EXT1 or EXT2. The proteins encoded by these genes, EXT1 and EXT2, are endoplasmic reticulum-localized type II transmembrane glycoproteins that possess or are tightly associated with glycosyltransferase activities involved in the polymerization of heparan sulfate. Here, by testing a cell line with a specific defect in EXT1 in in vivo and in vitro assays, we show that EXT2 does not harbor significant glycosyltransferase activity in the absence of EXT1. Instead, it appears that EXT1 and EXT2 form a hetero-oligomeric complex in vivo that leads to the accumulation of both proteins in the Golgi apparatus. Remarkably, the Golgi-localized EXT1/EXT2 complex possesses substantially higher glycosyltransferase activity than EXT1 or EXT2 alone, which suggests that the complex represents the biologically relevant form of the enzyme(s). These findings provide a rationale to explain how inherited mutations in either of the two EXT genes can cause loss of activity, resulting in hereditary multiple exostoses.
Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada V6T 1Z3.
Bone development is a highly regulated process sensitive to a wide variety of hormones, inflammatory mediators and growth factors. One of the most common hereditary skeletal dysplasias, hereditary multiple exostoses (HME), is an autosomal dominant disorder characterized by skeletal malformations that manifest as bony, benign tumours near the end of long bones. HME is usually caused by defects in either one of two genes, EXT1 and EXT2, which encode enzymes that catalyse the biosynthesis of heparan sulphate, an important component of the extracellular matrix. Thus, HME-linked bone tumours, like many other skeletal dysplasias, probably result from disruptions in cell surface architecture. However, despite the recent success in unravelling functions for several members of the EXT gene family, significant challenges remain before this knowledge can be used to develop new approaches for the diagnosis and treatment of disease.
Biosynthesis of the linkage region of glycosaminoglycans: cloning and activity of galactosyltransferase II, the sixth member of the beta 1,3-galactosyltransferase family (beta 3GalT6).
Department of Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California, San Diego, La Jolla, California 92093-0687, USA.
A family of five beta1,3-galactosyltransferases has been characterized that catalyze the formation of Galbeta1,3GlcNAcbeta and Galbeta1,3GalNAcbeta linkages present in glycoproteins and glycolipids (beta3GalT1,-2,-3,-4, and -5). We now report a new member of the family (beta3GalT6), involved in glycosaminoglycan biosynthesis. The human and mouse genes were located on chromosomes 1p36.3 and 4E2, respectively, and homologs are found in Drosophila melanogaster and Caenorhabditis elegans. Unlike other members of the family, beta3GalT6 showed a broad mRNA expression pattern by Northern blot analysis. Although a high degree of homology across several subdomains exists among other members of the beta3-galactosyltransferase family, recombinant enzyme did not utilize glucosamine- or galactosamine-containing acceptors. Instead, the enzyme transferred galactose from UDP-galactose to acceptors containing a terminal beta-linked galactose residue. This product, Galbeta1,3Galbeta is found in the linkage region of heparan sulfate and chondroitin sulfate (GlcAbeta1,3Galbeta1,3Galbeta1,4Xylbeta-O-Ser), indicating that beta3GalT6 is the so-called galactosyltransferase II involved in glycosaminoglycan biosynthesis. Its identity was confirmed in vivo by siRNA-mediated inhibition of glycosaminoglycan synthesis in HeLa S3 cells. Its localization in the medial Golgi indicates that this is the major site for assembly of the linkage region.
Cloning, Golgi localization, and enzyme activity of the full-length heparin/heparan sulfate-glucuronic acid C5-epimerase.
Department of Cellular Medicine, Glycobiology Research and Training Center, University of California, San Diego, La Jolla, California 92093-0687, USA.
While studying the cellular localization and activity of enzymes involved in heparan sulfate biosynthesis, we discovered that the published sequence for the glucuronic acid C5-epimerase responsible for the interconversion of d-glucuronic acid and l-iduronic acid residues encodes a truncated protein. Genome analysis and 5'-rapid amplification of cDNA ends was used to clone the full-length cDNA from a mouse mastocytoma cell line. The extended cDNA encodes for an additional 174 amino acids at the amino terminus of the protein. The murine sequence is 95% identical to the human epimerase identified from genomic sequences and fits with the general size and structure of the gene from Drosophila melanogaster and Caenorhabditis elegans. Full-length epimerase is predicted to have a type II transmembrane topology with a 17-amino acid transmembrane domain and an 11-amino acid cytoplasmic tail. An assay with increased sensitivity was devised that detects enzyme activity in extracts prepared from cultured cells and in recombinant proteins. Unlike other enzymes involved in glycosaminoglycan biosynthesis, the addition of a c-myc tag or green fluorescent protein to the highly conserved COOH-terminal portion of the protein inhibits its activity. The amino-terminally truncated epimerase does not localize to any cellular compartment, whereas the full-length enzyme is in the Golgi, where heparan sulfate synthesis is thought to occur.
The putative tumor suppressors EXT1 and EXT2 are glycosyltransferases required for the biosynthesis of heparan sulfate.
Department of Medical Biochemistry and Microbiology, Uppsala University, The Biomedical Center, Box 575, S-751 23 Uppsala, Sweden. Thomas.Lind@medkem.uu.se
Hereditary multiple exostoses, characterized by multiple cartilaginous tumors, is ascribed to mutations at three distinct loci, denoted EXT1-3. Here, we report the purification of a protein from bovine serum that harbored the D-glucuronyl (GlcA) and N-acetyl-D-glucosaminyl (GlcNAc) transferase activities required for biosynthesis of the glycosaminoglycan, heparan sulfate (HS). This protein was identified as EXT2. Expression of EXT2 yielded a protein with both glycosyltransferase activities. Moreover, EXT1, previously found to rescue defective HS biosynthesis (McCormick, C., Leduc, Y., Martindale, D., Mattison, K., Esford, L. E., Dyer, A. P., and Tufaro, F.(1998) Nat. Genet. 19, 158-161), was shown to elevate the low GlcA and GlcNAc transferase levels of mutant cells. Thus at least two members of the EXT family of tumor suppressors encode glycosyltransferases involved in the chain elongation step of HS biosynthesis.
Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by the formation of cartilage-capped tumours (exostoses) that develop from the growth plate of endochondral bone. This condition can lead to skeletal abnormalities, short stature and malignant transformation of exostoses to chondrosarcomas or osteosarcomas. Linkage analyses have identified three different genes for HME, EXT1 on 8q24.1, EXT2 on 11p11-13 and EXT3 on 19p (refs 6-9). Most HME cases have been attributed to missense or frameshift mutations in these tumour-supressor genes, whose functions have remained obscure. Here, we show that EXT1 is an ER-resident type II transmembrane glycoprotein whose expression in cells results in the alteration of the synthesis and display of cell surface heparan sulfate glycosaminoglycans (GAGs). Two EXT1 variants containing aetiologic missense mutations failed to alter cell-surface glycosaminoglycans, despite retaining their ER-localization.
Department of Medicine, University of British Columbia, Vancouver, Canada.
In the majority of cases, the mechanism underlying the resistance to acyclovir (ACV) of herpes simplex viruses (HSVs) is thymidine kinase (TK) deficiency. Plaque isolates from eight ACV-resistant (ACVr) clinical isolates from AIDS patients, of which five reactivated, were sequenced to determine the genetic lesion within the tk gene conferring resistance and whether this may have correlated with reactivation potential. Mutations were clustered within two homopolymer nucleotide stretches. Three plaque isolates (1737-14, 90-150-3, and 89-650-5) had insertion mutations within a stretch of 7 guanosines, while two isolates (89-063-1 and 89-353-1) had frameshift mutations within a stretch of 6 cytosines (a deletion and an insertion, respectively). Mutations resulted in premature termination codons, and the predicted 28- and 32-kDa truncated TK products were detected by Western blot analysis of virus-infected cell extracts. The repair of one homopolymer frameshift mutation (in isolate 1737-14) restored TK activity, demonstrating that this mutation is the basis of TK deficiency. Of the five reactivated isolates, four were TK deficient and contained frameshift mutations while the fifth retained TK activity because of its altered-TK or Pol- phenotype. These data demonstrate that the majority of ACVr clinical isolates contain frameshift mutations within two long homopolymer nucleotide stretches which function as hot spots within the HSV tk gene and produce nonfunctional, truncated TK proteins.
R Fallows, K McCoy, J Hertza, E Klosson, B Estes, I Stroescu, Cm Salinas, A Stringer, S Aronson, W Macallister, A Spurgin, M Morriss, P Glasier, P Stavinoha, A Houshyarnejad, J Jacobus, M Norman, S Peery, M Mattingly, T Pennuto, C Anderson-Hanley, A Miele, M Dunnam, M Edwards, S O'Bryant, L Johnson, R Barber, A Inscore, J Kegel, A Kozlovsky, B Tarantino, A Goldberg, J Herrera-Pino, N Jubiz-Bassi, K Rashid, Y Noniyeva, K Vo, V Stephens, R Gomez, C Sanders, M Kovacs, B Walton, M Schmitter-Edgecombe, C Parsey, D Cook, S Woods, M Weinborn, A Velnoweth, A Rooney, R Bucks, C Adalio, S White, J Blair, B Barber, S Marcy, J Boseck, C McCormick, A Davis, K Berry, E Koehn, N Tiberi, B Gelder, B Brooks, E Sherman, Mi Garcia, R Robillard, J Gunner, J Lynch, R McCaffrey, J Hamilton, K Froming, D Nemeth, A Steger, P Lebby, J Harrison, A Mounoutoua, J Preiss, A Brimager, E Gates, J Chang, H Cisneros, J Long, V Petrauskas, J Casey, E Picard, M Rodriguez, F Fonseca, C Golden, M Semrud-Clikeman, J Goldenring Fine, J Bledsoe, N Thaler, D Allen, D Bello, N Wood, L Etcoff, L Thede, J Oraker, F Gibson, L Stanford, S Gray, L Vroman, T Taylor, K Seydel, A Bure-Reyes, J Stewart, I Tourgeman, Y Demsky, W Burns, K Burns, C Calderon, C Neblina, L San Miguel Montes, A Strutt, B Scott, P Armstrong, C Booth, K Blackstone, D Moore, B Gouaux, R Ellis, J Atkinson, I Grant, L Brennan, M Schultheis, H Hurtig, D Weintraub, J Duda, P Moberg, D Chute, A Siderowf, N Brescian, C Gass, R Brewster, T King, R Morris, N Krawiecki, D Dinishak, G Richardson, M Knight, R Fallows, S Garcia, G Strain, M Devlin, R Cohen, R Paul, R Crosby, J Mitchell, J Gunstad, L Hancock, J Bruce, B Roberg, S Lynch, E Varnadore, W Schiff, Rs Kaufman, E Rinehardt, M Schoenberg, Y Rosado, S Velamuri, M Leblanc, P Pimental, S Lynch-Chee, D Broshek, P Lyons, J McKeever, C Morse, J Ang, T Leist, J Tracy, E Morgan, W Perry, S Letendre, M Musso, G Jones, B Hill, D Proto, A Barker, W Gouvier, K Nersesova, M Drexler, E Cherkasova, M Sakamoto, T Marcotte, R Hilsabeck, M Carlson, F Barakat, T Hassanein, K Shevchik, W McCaw, B Schrock, M Smith, D Moser, J Mills, E Epping, J Paulsen, M Somogie, F Bryan, L Buscher, J Tyrer, A Stabler, J Thelen, C Lovelace, D Graves, B Greenberg, L Harder, M Szczebak, M Glisky, D Ukueberuwa, P Arnett, L Vahter, M Ennok, K Päll, K Gross-Paju, G Vargas, J Medaglia, N Chiaravalloti, C Zakrzewski, F Hillary, A Andrews, K Belloni, J Nicewander, D Miller, S Johnson, Z David, E Weideman, D Lawson, E Currier, J Morton, J Robinson, R Pella, M Vertinski, D Heisler, B Park, S Barney, N Kucukboyaci, H Girard, N Kemmotsu, C Cheng, J Kuperman, C McDonald, C Carroll, A Odland, L Miller, W Mittenberg, D Coalson, D Wahlstrom, S Raiford, J Holdnack, E Gardner, N Dasher, B Fowler, P Vik, M Grajewski, M Lamar, D Penney, R Davis, L Korthauer, D Libon, A Kumar, G Iverson, G Chelune, C Hunter, E Zimmerman, R Klein, N Prathiba, A Hopewell, D Cooper, J Kennedy, M Long, J Moses Jr, J Lutz, R Dean, J Miller, B Axelrod, S Van Dyke, L Rapport, C Schutte, R Hanks, J O'Rourke, S Bowden, R Romero, R Hulkonen, M Boivin, P Bangirana, C John, E Shapiro, A Slonaker, L Pass, J Smigielski, J Biernacka, J Geske, D Hall-Flavin, L Loukianova, T Schneekloth, O Abulseoud, D Mrazek, V Karpyak, J Terranova, E Safko, D Zink, A Puente, H Ames, J Lepage, K Knee, T Cummings, F Webbe, E Shepherd, J Marcinak, M Diaz-Santos, D Seichepine, K Sullivan, S Neargarder, A Cronin-Golomb, E Franchow, Y Suchy, M Kraybill, A Holland, S Newton, D Hinson, A Smith, M Coe, J Carmona, D Harrison, L Hyer, M Atkinson, J Dalibwala, C Yeager, C Scott, K Jacobson, K Olson, R Kaufman, A Sartori, O Clay, F Ovalle, R Rothman, M Crowe, A Schmid, L Horne, G Horn, B Johnson-Markve, P Gorman, J Tam, C McAlister, M Wagner, L Brenner, A Walker, L Armstrong, E Inman, J Grimmett, A Cornelius, M Willingham, L Restrepo, J Bolanos, F Patel, J Rice, M Dougherty, V Sharma, P Martin, E Bradley, C Lockwood, J Poole, T Brickell, R Lange, L French, L Chao, S Klein, G Warner, K Donnelly, J Donnelly, J Kittleson, C Bradshaw, M Alt, D England, R Denney, J Meyers, J Evans, C Kennedy, J Moore, A Fedor, M Spitznagel, M Ferland, Nunez K Guerrero, P Davidson, B Collins, S Marshall, G Samper, S Ibarra, D Parrott, F Steffen, S Backhaus, C Karver, S Wade, H Taylor, T Brown, M Kirkwood, T Stancin, K Krishnan, C Culver, A Arenivas, C Bosworth, E Shokri-Kojori, R Diaz-Arrastia, Plata C Marquez de la, B Ivins, K Marshall, K Schwab, G Parkinson, A Bhagwat, J Lichtenstein, Z Adams-Deutsch, J Fleischer, K Goldberg, M Ehrler, A Hull, C Sullivan, D Schuster, K Al-Khalil, A Myers, S Ireland, E Simco, E Palmer, K Piecora, N Mroczek, A Snyder, A Rabinowitz, P Schatz, N Cameron, P Stolberg, J Hart, W Jones, J Mayfield, S Edmed, R Vanderploeg, M Silva, C Vaughan, E McGuire, E Gerst, S Fricke, J Vanmeter, J Newman, G Gioia, A Wahlberg, S Zelonis, A Chatterjee, S Smith, E Whipple, L Mace, K Manning, J Wilk, R Herrell, C Hoge, K Zakzanis, S Yu, E Jeffay, A Zimmer, M Adler, J Holster, S Schleicher-Dilks, S Arffa, J Thornton, A Canas, C Sevadjian, A Fournier, D Maricle, J Donders, T Larsen, J Gidley Larson, J Sheehan, K Higgins, S Rolin, K Dunham, S Akeson, A Horton, C Reynolds, L Jordan, S Gonzalez, S Heaton, T Olivier, S West, L Prinzi, J Robbins, B Bruzinski, C Riccio, A Blakely, M Yoon, J Pearlson, Larson Jc Gidley, H Carlson, I Gaxiola-Valdez, X Wei, C Beaulieu, W Hader, A Kirton, K Barlow, M Hrabok, I Mohamed, S Wiebe, K Smith, A Ailion, M Ivanisevic, S Thorgusen, D Bowman, K Walsh, F Mitchell, G Jill, P Iris, K Ross, A Madan-Swain, P Isquith, D Webber, N Defilippis, M Collins, F Hill, R Weber, A Johnson, C Wiley, T Burns, D Ritchie, A Stevens, L Hartlage, B Williams, E Weidemann, G Demakis, J Avila, J Razani, S Burkhart, W Adams, J Hall, P Grammas, G Gong, K Hargrave, S Mattevada, H Vo, Rc Barber, J Davis, K O'Connor, T Rehm-Hamilton, D Ploetz, M Rohling, E Potter, D Loewenstein, R Duara, M Hernandez Finch, M Cannon, S McGregor, D Reitman, J Rey, D Scarisbrick, H Whoolery, P Vekaria, L Whittington, A Gremillion, S Amirthavasagam, D Umuhoza, G Strauss, D Knatz-Bello, J Bell, D Frisch, D McIntosh, K Kjernisted, A Young, T Kiely, C Tai, A Schatzberg, J Keller, E Rhodes, O Ajilore, A Zhang, E Ringdahl, G Sutton, A Turner, J Snyder, R Verbiest, E Walkenhorst, S Crowe, A August-Fedio, J Sexton, S Cummings, K Brown, P Fedio, A Grigorovich, J Fish, M Gomez, L Leach, H Lloyd, M Nichols, M Goldberg, T Novakovic-Agopian, A Chen, G Abrams, A Rossi, D Binder, J Muir, G Carlin, M Murphy, R McKim, R Fitsimmons, M D'Esposito, A Vernon, R Frank, P Zurita Ona, E Freitag, E Weber, E Kellogg, M Basso, B Dyer, M Daniel, P Michael, R Fontanetta, A Stripling, C Corsun-Ascher, M Legaretta, E Van Ness, K Noll, D Denney, A Wiechman, T Stephanie, L Lacritz, M Padua, K Sandhu, J Sordahl, J Anderson, V Wheaton, K Berggren, D Cheung, H Luber, J Loftis, M Huckans, T Bennett, C Dawson, H Soper, K Carter, A Hester, W Ringe, J Spence, M Posamentier, R Haley, R Curiel, E Hu, S Jordan, V Goetsch, S Small, Y Mansoor, E Homer-Smith, J Moses, J Perle, V Patt, A Minassian, S Polott, K Mulligan, K Shaneyfelt, J Wall, J Thompson, V Compono, L Trettin, J Tsou, B Waldron-Perrine, H Tree, R Spencer, A McGuire, S Na, P Pangilinan, L Bieliauskas, S You, K An, C Biddle, R Fazio, K Willett, M O'Grady, K Bresnan, D Erlanger, R Seegmiller, T Kaushik, A Krol, T McHugh, J Deright, R Jorgensen, L Lewandowski, S Ortigue, J Etherton, C Green, H Snead, J Kirk, A Connery, Misialek L Hanson, W Myers, C Peck, R Schroeder, B Boatwright, R Heinrichs, L Baade, M Womble, J Shenesey, E Vondran, B Webster, C Brockman, A Burgess, T White, J Gold, A Vincent, T Roebuck-Spencer, A Bowles, K Gilliland, A Watts, F Ahmed, A Yon, B Gordon
Objective: The seminal paper on cerebellar cognitive affective syndrome by Schmahmann and Sherman (1998), and subsequent studies, has expanded our understanding of the role of the cerebellum beyond motor functioning to psychological and cognitive functioning. However, many of these studies have examined patients between 1 week and 5 years post-injury and have tended to exclude patients with prior neurological injuries. Thus, the objective of this case study was to examine cerebellar injury in the context of remote traumatic brain injury (TBI) and describe the long-term cognitive, psychological, and psychosocial sequelae of injury in a 33-year-old, right-handed, Caucasian veteran (S.M.). Method: At age 23, S.M. was referred for neuroimaging by psychiatry due to concern that a TBI from age 16 was the cause of recent onset aggressive behavior. Multiple neuroimaging studies showed no neuroanatomical sequelae of TBI, but revealed a right cerebellar arteriovenous malformation (AVM). Embolization resulted in >50% removal of the AVM, but uncovered an intranidal aneurysm. Repeat neuroimaging revealed a large hemorrhage within the cerebellum with the mass effect and hydrocephalus; subsequent treatment resulted in a complicated 5-month hospital stay. Results: Neuropsychological evaluation conducted 10 years after injury revealed deficits in basic attention, working memory, and information processing speed with relatively intact executive functioning and memory. Physical deficits, including ataxia, dysarthria, and spasticity, and psychological difficulties, including impulsivity and low frustration tolerance, were more prominent and caused significant psychosocial distress, impacting interpersonal relationships. Conclusions: This case highlights the cognitive residual of cerebellar injury and the potential long-term impact on psychological and social functioning.
Latest similar papers:
Tiling resolution array-CGH shows that somatic mosaic deletion of the EXT gene is causative in EXT gene mutation negative multiple osteochondromas patients.
Károly Szuhai, Ivy Jennes, Danielle de Jong, Judith V M G Bovée, Malgorzata Wiweger, Wim Wuyts, Pancras C W Hogendoorn
Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands. email@example.com
Multiple osteochondromas (MO) is a hereditary skeletal disorder characterized by the presence of cartilage capped bony outgrowths at bone surface. Causative mutations in EXT1 or EXT2 genes have been described in 85-90 % of MO cases. However, in about 10-15 % of the MO cases, genomic alterations can not be detected, implying the potential role of other alterations. We have designed a custom-made Agilent oligonucleotide-based microarray, containing 44,000 probes, with tiling coverage of EXT1/2 genes and addition of 68 genes involved in heparan sulfate biosynthesis and other related pathways. Out of the 17 patient samples with previously undetected mutations, a low level of deletion of the EXT1 gene in about 10-15% of the blood cells was detected in two patients and mosaic deletion of the EXT2 was detected in one patient. Here we show that for the first time somatic mosaicism with large genomic deletions as the underlying mechanism in MO formation was identified. We propose that the existence of mosaic mutations and not alterations of other heparan sulfate biosynthesis related genes play a significant role in the development of MO in patients who are tested negative for mutations in Exostosins.
A mouse model of chondrocyte-specific somatic mutation reveals a role for Ext1 loss of heterozygosity in multiple hereditary exostoses.
Sanford Children's Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA.
Multiple hereditary exostoses (MHE) is one of the most common skeletal dysplasias, exhibiting the formation of multiple cartilage-capped bony protrusions (osteochondroma) and characteristic bone deformities. Individuals with MHE carry heterozygous loss-of-function mutations in Ext1 or Ext2, genes which together encode an enzyme essential for heparan sulfate synthesis. Despite the identification of causative genes, the pathogenesis of MHE remains unclear, especially with regard to whether osteochondroma results from loss of heterozygosity of the Ext genes. Hampering elucidation of the pathogenic mechanism of MHE, both Ext1(+/-) and Ext2(+/-) heterozygous mutant mice, which mimic the genetic status of human MHE, are highly resistant to osteochondroma formation, especially in long bones. To address these issues, we created a mouse model in which Ext1 is stochastically inactivated in a chondrocyte-specific manner. We show that these mice develop multiple osteochondromas and characteristic bone deformities in a pattern and a frequency that are almost identical to those of human MHE, suggesting a role for Ext1 LOH in MHE. Surprisingly, however, genotyping and fate mapping analyses reveal that chondrocytes constituting osteochondromas are mixtures of mutant and wild-type cells. Moreover, osteochondromas do not possess many typical neoplastic properties. Together, our results suggest that inactivation of Ext1 in a small fraction of chondrocytes is sufficient for the development of osteochondromas and other skeletal defects associated with MHE. Because the observed osteochondromas in our mouse model do not arise from clonal growth of chondrocytes, they cannot be considered true neoplasms.
DNA Cell Biol. 2010 Mar 10;: 20218897
Functional Characterization of Single-Nucleotide Polymorphisms in the Human Undifferentiated Embryonic-Cell Transcription Factor 1 Gene.
Developmental Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen , Haren, The Netherlands .
Single-nucleotide polymorphisms (SNPs) are single-nucleotide sequence variations between individuals. Two missense SNPs are present in the human undifferentiated embryonic-cell transcription factor 1 (UTF1) gene and their consequences for UTF1 function are investigated in this study. Expression of the UTF1 gene is restricted to pluripotent cells and UTF1 is a chromatin-associated protein with core histone-like properties. UTF1 further acts as a transcriptional repressor and is required for proper differentiation of pluripotent cells. Two missense mutations in UTF1 are reported: rs11599284, which results in a glycine to an arginine change at amino acid 73, and rs4480453, resulting in a leucine to methionine change at amino acid 275. To study the effects of these two SNPs, P19CL6 mouse embryonic carcinoma cells stably expressing eGFP-hUTF1 constructs containing either one or both SNPs were generated. The single and double SNPs did not alter the localization or transcriptional repressor activity of the protein. Further, the single SNPs did not alter the chromatin association and mobility of hUTF1. However, the double mutant, G73R/L275M, demonstrated a decreased chromatin association, indicating a degree of protein malfunction.
Identification of four novel EXT1 and EXT2 mutations in five Chinese pedigrees with hereditary multiple exostoses.
Department of Pediatric Orthopedics, Shanghai Children's Medical Center, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China.
Hereditary multiple exostoses (HME) is an autosomal dominant skeletal disorder most frequently caused by the EXT1 and EXT2 gene mutations resulting in reduction or absence of heparan sulfate (HS) in the exostotic cartilage cap. In this study, we investigated the molecular defects in five Chinese pedigrees with HME by direct sequencing analysis. Two novel EXT1 gene mutations and two novel EXT2 gene mutations were identified in two and three pedigrees, respectively. Of the four mutations identified, the c.651-664delinsTTT and c.680delG mutations in the exon 1 of EXT1 gene would cause frameshift (K218fs and R227fs) and introduce premature stop codon at amino acid site 220 and 251, respectively. The two missense mutations of c.398T > G in exon 2 and c.1016G > A in exon 6 of EXT2 gene result in the Leu133Arg and Cys339Tyr substitution, respectively. As HME is caused by defects in HS synthesis that is a complex process and not fully understood, these naturally occurring EXT mutations may provide important clues to future studies elucidating how EXT proteins contribute to HS biosynthesis.
Identification of a novel point mutation in ENT1 that confers resistance to Ara-C in human T cell leukemia CCRF-CEM cells.
Department of Pharmacology, 4009 Genetic Medicine Bldg., 120 Mason Farm Rd. CB# 7365, University of North Carolina @ Chapel Hill, Chapel Hill, NC 27599-7365, United States.
The genetic basis for the Ara-C resistance of CCRF-CEM Ara-C/8C leukemia cells was investigated. DNA sequencing revealed that these cells expressed an equilibrative nucleoside transporter 1 (ENT1) with a single missense mutation resulting in glycine to arginine replacement (G24R). To test the importance of this residue, additional G24 mutants were created and examined for [3H]-uridine and [3H]-Ara-C uptake. Both a G24E and G24A mutant showed reduced ENT1-dependent activity. An EGFP-tagged G24R ENT1 displayed plasma membrane localization even though it was unable to bind [3H]-NBMPR, an ENT1-specific inhibitor. These results define G24 as critical amino acid for ENT1 nucleoside uptake and suggest that mutations in TM1 may provide a mechanism for Ara-C resistance in CCRF-CEM Ara-C/8C cells.
Functional analysis of pancreatitis-associated missense mutations in the pancreatic secretory trypsin inhibitor (SPINK1) gene.
 1INSERM, U613, Brest, France  2Faculté de Médecine de Brest et des Sciences de la Santé, Université de Bretagne Occidentale, Brest, France.
Variations in the SPINK1 gene (encoding pancreatic secretory trypsin inhibitor (PSTI)) are associated with chronic pancreatitis. We have recently determined the functional consequences of three missense mutations that occurred within the signal peptide sequence of PSTI by Western blotting analysis of wild-type and mutant PSTI expressed in Chinese hamster ovary cells. Here, this approach was extended to analyze seven missense mutations (p.N34S, p.G48E, p.D50E, p.Y54H, p.P55S, p.R65Q and p.R67C) occurring within the mature peptide of PSTI. This analysis enabled us to classify these missense mutations into three categories. The first category comprises the p.N34S and p.P55S polymorphisms, both of which occur in evolutionarily non-conserved residues, involve amino-acid substitutions with similar physicochemical properties, and do not cause any significant reduction in terms of PSTI mature peptide expression. The second category contains only the p.R65Q missense mutation, which occurs in a well-conserved residue, involves the substitution of a positively charged amino acid by a non-charged one, and causes a approximately 60% reduction of protein expression. The third category comprises p.G48E, p.D50E, p.Y54H, and p.R67C, all of which occur in strictly conserved residues, involve charged amino acids, and cause complete or nearly complete loss of PSTI expression. Having excluded the possibility that the reduced protein expression may have resulted from reduced transcription or unstable mRNA, we surmise that these missense mutations probably cause intracellular retention of their respective mutant proteins. This is suggestive of a potential unifying pathological mechanism underlying both the signal peptide and mature peptide mutations.European Journal of Human Genetics advance online publication, 13 June 2007; doi:10.1038/sj.ejhg.5201873.
H Okada, T Yamazaki, A Takagi, T Murate, K Yamamoto, J Takamatsu, T Matsushita, T Naoe, S Kunishima, M Hamaguchi, H Saito, T Kojima
Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan. firstname.lastname@example.org
OBJECTIVE To elucidate the molecular consequences of hereditary protein S (PS) deficiency, we investigated the in vitro synthesis of the PS missense mutants in COS-1 cells and their activated protein C (APC) cofactor activities. PATIENTS Four patients with quantitative PS deficiency suffering from venous thrombosis were examined. RESULTS We identified three distinct novel missense mutations, R275C, P375Q and D455Y, and two previously reported missense mutations, C80Y and R314H. The P375Q and D455Y mutations were found in one patient and observed to be in linkage on the same allele. The R314H mutant showed the lowest level of expression (32.7%), and the C80Y, P375Q + D455Y, and R275C mutants exhibited a moderate impairment of expression, that is, 43.8%, 49.5%, and 72.3% of the wild type, respectively. Furthermore, pulse-chase experiments demonstrated that all mutants showed impaired secretion and longer half-lives in the cells than the wild type PS. In the APC cofactor assays, the C80Y mutant showed no cofactor activity, and the R275C mutant showed reduced activity, 62.3% of the wild type PS, whereas the R314H and P375Q + D455Y mutants exhibited normal cofactor activity. CONCLUSION These data indicate that the C80Y and R275C mutations affect the secretion and function of the PS molecule, and that the R314H and P375Q + D455Y mutations are responsible for only secretion defects, causing the phenotype of quantitative PS deficiency observed in the patients.
Enza Maria Valente, Patrick M Abou-Sleiman, Viviana Caputo, Miratul M K Muqit, Kirsten Harvey, Suzana Gispert, Zeeshan Ali, Domenico Del Turco, Anna Rita Bentivoglio, Daniel G Healy, Alberto Albanese, Robert Nussbaum, Rafael González-Maldonado, Thomas Deller, Sergio Salvi, Pietro Cortelli, William P Gilks, David S Latchman, Robert J Harvey, Bruno Dallapiccola, Georg Auburger, Nicholas W Wood
CSS IRCCS, Mendel Institute, viale Regina Margherita 261, 00198 Rome, Italy.
Parkinson's disease (PD) is a neurodegenerative disorder characterized by degeneration of dopaminergic neurons in the substantia nigra. We previously mapped a locus for a rare familial form of PD to chromosome 1p36 (PARK6). Here we show that mutations in PINK1 (PTEN-induced kinase 1) are associated with PARK6. We have identified two homozygous mutations affecting the PINK1 kinase domain in three consanguineous PARK6 families: a truncating nonsense mutation and a missense mutation at a highly conserved amino acid. Cell culture studies suggest that PINK1 is mitochondrially located and may exert a protective effect on the cell that is abrogated by the mutations, resulting in increased susceptibility to cellular stress. These data provide a direct molecular link between mitochondria and the pathogenesis of PD.
McLaughlin Research Institute for Biomedical Sciences, 1520 23rd Street South, Great Falls, MT 59405, USA.
Eya1 is a critical gene for mammalian organogenesis. Mutations in human EYA1 cause branchio-oto-renal (BOR) syndrome, an autosomal dominant disorder characterized by varying combinations of branchial, otic and renal anomalies, whereas deletion of mouse Eya1 results in the absence of multiple organ formation. Eya1 and other Eya gene products share a highly conserved 271 amino acid Eya domain that is required for protein-protein interaction. Recently, several point mutations that result in single amino acid substitutions in the conserved Eya domain region of EYA1 have been identified in BOR patients; however, the molecular and developmental basis of organ defects that occurred in BOR syndrome is unclear. To understand how these point mutations cause disease, we have analyzed the functional importance of these Eya domain missense mutations with respect to protein complex formation and cellular localization. We have demonstrated that these point mutations do not alter protein localization. However, four mutations are crucial for protein-protein interactions in both yeast and mammalian cells. Our results provide insights into the molecular mechanisms of organ defects detected in human syndromes.
M Gigante, M G Matera, D Seripa, A M Izzo, R Venanzi, A Giannotti, M C Digilio, C Gravina, M Lazzari, G Monteleone, M Monteleone, B Dallapiccola, V M Fazio
Laboratorio Patologia Molecolare e Terapia Genica, I.R.C.C.S. Ospedale Casa Sollievo della Sofferenza, Opera di Padre Pio da Pietrelcina, San Giovanni Rotondo, Italy.
Osteochondromas represent the largest group of benign tumors of bone. Multiple osteochondromatosis or hereditary multiple exostoses (EXT) is an autosomal dominant inherited disorder characterized by the presence of multiple benign cartilage-capped exostoses. EXT is genetically heterogeneous with at least 3 chromosomal loci: EXT1 (8q24.1), EXT2 (11p11-p13), and EXT3 (19p). In <5% of EXT patients, the inactivation of both copies of EXT alleles (LOH) is associated with malignant transformation. We have analyzed the EXT1 and EXT2 genes in 9 unrelated EXT families and in a patient with a sporadic osteochondroma, all originating from Italy. Four families show an EXT1 mutation, consisting of a small deletion in 3 of them and a small insertion in the 4th. All these mutations lead to premature termination of translation and thus a truncated EXT1 protein. Three families presented EXT2 mutations consisting of nucleotide substitutions leading to alterations of the third intron splice-site, to an amino acid substitution and to a nonsense mutation. All these mutations cosegregate with the disease phenotype. The sporadic osteochondroma patient carried a novel missense mutation in exon 11 of EXT2 gene, leading to an amino acid substitution. Seven of these mutations have never been described before. EXT2 missense mutations were also confirmed by amino acids conservation between human and mouse and by analysis of a healthy control population. In conclusion, our study provide further evidence that loss of function of the EXT1 or EXT2 gene is the main cause of EXT supporting the putative tumor-suppressor function of these genes.