Publications by authors named "Wolfgang Höhne"

42 Publications

SSBP1 mutations in dominant optic atrophy with variable retinal degeneration.

Ann Neurol 2019 09 31;86(3):368-383. Epub 2019 Jul 31.

School of Optometry and Vision Sciences, Cardiff University, Cardiff, United Kingdom.

Objective: Autosomal dominant optic atrophy (ADOA) starts in early childhood with loss of visual acuity and color vision deficits. OPA1 mutations are responsible for the majority of cases, but in a portion of patients with a clinical diagnosis of ADOA, the cause remains unknown. This study aimed to identify novel ADOA-associated genes and explore their causality.

Methods: Linkage analysis and sequencing were performed in multigeneration families and unrelated patients to identify disease-causing variants. Functional consequences were investigated in silico and confirmed experimentally using the zebrafish model.

Results: We defined a new ADOA locus on 7q33-q35 and identified 3 different missense variants in SSBP1 (NM_001256510.1; c.113G>A [p.(Arg38Gln)], c.320G>A [p.(Arg107Gln)] and c.422G>A [p.(Ser141Asn)]) in affected individuals from 2 families and 2 singletons with ADOA and variable retinal degeneration. The mutated arginine residues are part of a basic patch that is essential for single-strand DNA binding. The loss of a positive charge at these positions is very likely to lower the affinity of SSBP1 for single-strand DNA. Antisense-mediated knockdown of endogenous ssbp1 messenger RNA (mRNA) in zebrafish resulted in compromised differentiation of retinal ganglion cells. A similar effect was achieved when mutated mRNAs were administered. These findings point toward an essential role of ssbp1 in retinal development and the dominant-negative nature of the identified human variants, which is consistent with the segregation pattern observed in 2 multigeneration families studied.

Interpretation: SSBP1 is an essential protein for mitochondrial DNA replication and maintenance. Our data have established pathogenic variants in SSBP1 as a cause of ADOA and variable retinal degeneration. ANN NEUROL 2019;86:368-383.
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http://dx.doi.org/10.1002/ana.25550DOI Listing
September 2019

Mutations of KIF14 cause primary microcephaly by impairing cytokinesis.

Ann Neurol 2017 Oct 14;82(4):562-577. Epub 2017 Oct 14.

Cologne Center for Genomics, University of Cologne, Cologne, Germany.

Objective: Autosomal recessive primary microcephaly (MCPH) is a rare condition characterized by a reduced cerebral cortex accompanied with intellectual disability. Mutations in 17 genes have been shown to cause this phenotype. Recently, mutations in CIT, encoding CRIK (citron rho-interacting kinase)-a component of the central spindle matrix-were added. We aimed at identifying novel MCPH-associated genes and exploring their functional role in pathogenesis.

Methods: Linkage analysis and whole exome sequencing were performed in consanguineous and nonconsanguineous MCPH families to identify disease-causing variants. Functional consequences were investigated by RNA studies and on the cellular level using immunofluorescence and microscopy.

Results: We identified homozygous mutations in KIF14 (NM_014875.2;c.263T>A;pLeu88*, c.2480_2482delTTG; p.Val827del, and c.4071G>A;p.Gln1357=) as the likely cause in 3 MCPH families. Furthermore, in a patient presenting with a severe form of primary microcephaly and short stature, we identified compound heterozygous missense mutations in KIF14 (NM_014875.2;c.2545C>G;p.His849Asp and c.3662G>T;p.Gly1221Val). Three of the 5 identified mutations impaired splicing, and 2 resulted in a truncated protein. Intriguingly, Kif14 knockout mice also showed primary microcephaly. Human kinesin-like protein KIF14, a microtubule motor protein, localizes at the midbody to finalize cytokinesis by interacting with CRIK. We found impaired localization of both KIF14 and CRIK at the midbody in patient-derived fibroblasts. Furthermore, we observed a large number of binucleated and apoptotic cells-signs of failed cytokinesis that we also observed in experimentally KIF14-depleted cells.

Interpretation: Our data corroborate the role of an impaired cytokinesis in the etiology of primary and syndromic microcephaly, as has been proposed by recent findings on CIT mutations. Ann Neurol 2017;82:562-577.
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http://dx.doi.org/10.1002/ana.25044DOI Listing
October 2017

Effects of Different Variants in the ENPP1 Gene on the Functional Properties of Ectonucleotide Pyrophosphatase/Phosphodiesterase Family Member 1.

Hum Mutat 2016 11 23;37(11):1190-1201. Epub 2016 Aug 23.

Department of General Pediatrics, Muenster University Children's Hospital, Albert-Schweitzer-Campus 1, Muenster, 48149, Germany.

Ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (E-NPP1), encoded by ENPP1, is a plasma membrane protein that generates inorganic pyrophosphate (PP ), a physiologic inhibitor of hydroxyapatite formation. In humans, variants in ENPP1 are associated with generalized arterial calcification of infancy, an autosomal-recessive condition causing premature onset of arterial calcification and intimal proliferation resulting in stenoses. ENPP1 variants also cause pseudoxanthoma elasticum characterized by ectopic calcification of soft connective tissues. To determine the functional impact of ENPP1 missense variants, we analyzed 13 putative pathogenic variants in vitro regarding their functional properties, that is, activity, localization, and PP generation. Transfection of eight of the 13 variants led to complete loss of NPP activity, whereas four mutants (c.1412A > G, p.Tyr471Cys; c.1510A > C, p.Ser504Arg; c.1976A > G, p.Tyr659Cys; c.2330A > G, p.His777Arg) showed residual activity compared with wild-type E-NPP1. One putative pathologic variant (c.2462 G > A, p.Arg821His) showed normal activity. The five mutants with normal or residual E-NPP1 enzyme activity were still able to generate PP and localized in the plasma membrane. In this study, we identified a functional ENPP1 polymorphism, which was expected to be pathogenic till now. Furthermore, we identified four mutants (p.Tyr471Cys, p.Ser504Arg, p.Tyr659Cys, p.His777Arg) with residual E-NPP1 function, which would be potential therapeutical targets for conformational-stabilizing agents.
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http://dx.doi.org/10.1002/humu.23057DOI Listing
November 2016

Bi-allelic Mutations in KLHL7 Cause a Crisponi/CISS1-like Phenotype Associated with Early-Onset Retinitis Pigmentosa.

Am J Hum Genet 2016 Jul;99(1):236-45

Department of General Pediatrics, Münster University Children's Hospital, 48149 Münster, Germany.

Crisponi syndrome (CS)/cold-induced sweating syndrome type 1 (CISS1) is a very rare autosomal-recessive disorder characterized by a complex phenotype with high neonatal lethality, associated with the following main clinical features: hyperthermia and feeding difficulties in the neonatal period, scoliosis, and paradoxical sweating induced by cold since early childhood. CS/CISS1 can be caused by mutations in cytokine receptor-like factor 1 (CRLF1). However, the physiopathological role of CRLF1 is still poorly understood. A subset of CS/CISS1 cases remain yet genetically unexplained after CRLF1 sequencing. In five of them, exome sequencing and targeted Sanger sequencing identified four homozygous disease-causing mutations in kelch-like family member 7 (KLHL7), affecting the Kelch domains of the protein. KLHL7 encodes a BTB-Kelch-related protein involved in the ubiquitination of target proteins for proteasome-mediated degradation. Mono-allelic substitutions in other domains of KLHL7 have been reported in three families affected by a late-onset form of autosomal-dominant retinitis pigmentosa. Retinitis pigmentosa was also present in two surviving children reported here carrying bi-allelic KLHL7 mutations. KLHL7 mutations are thus associated with a more severe phenotype in recessive than in dominant cases. Although these data further support the pathogenic role of KLHL7 mutations in a CS/CISS1-like phenotype, they do not explain all their clinical manifestations and highlight the high phenotypic heterogeneity associated with mutations in KLHL7.
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http://dx.doi.org/10.1016/j.ajhg.2016.05.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5005468PMC
July 2016

Skeletal dysplasia in a consanguineous clan from the island of Nias/Indonesia is caused by a novel mutation in B3GAT3.

Hum Genet 2015 Jul 19;134(7):691-704. Epub 2015 Apr 19.

Cologne Center for Genomics (CCG), University of Cologne, Cologne, Germany.

We describe a large family with disproportionate short stature and bone dysplasia from Nias in which we observed differences in severity when comparing the phenotypes of affected individuals from two remote branches. We conducted a linkage scan in the more severely affected family branch and determined a critical interval of 4.7 cM on chromosome 11. Sequencing of the primary candidate gene TBX10 did not reveal a disease-causing variant. When performing whole exome sequencing we noticed a homozygous missense variant in B3GAT3, c.419C>T [p.(Pro140Leu)]. B3GAT3 encodes β-1,3-glucuronyltransferase-I (GlcAT-I). GlcAT-I catalyzes an initial step of proteoglycan synthesis and the mutation p. (Pro140Leu) lies within the donor substrate-binding subdomain of the catalytic domain. In contrast to the previously published mutation in B3GAT3, c.830G>A [p.(Arg277Gln)], no heart phenotype could be detected in our family. Functional studies revealed a markedly reduced GlcAT-I activity in lymphoblastoid cells from patients when compared to matched controls. Moreover, relative numbers of glycosaminoglycan (GAG) side chains were decreased in patient cells. We found that Pro140Leu-mutant GlcAT-I cannot efficiently transfer GlcA to the linker region trisaccharide. This failure results in a partial deficiency of both chondroitin sulfate and heparan sulfate chains. Since the phenotype of the Nias patients differs from the Larsen-like syndrome described for patients with mutation p.(Arg277Gln), we suggest mutation B3GAT3:p.(Pro140Leu) to cause a different type of GAG linkeropathy showing no involvement of the heart.
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http://dx.doi.org/10.1007/s00439-015-1549-2DOI Listing
July 2015

A specific IFIH1 gain-of-function mutation causes Singleton-Merten syndrome.

Am J Hum Genet 2015 Feb 22;96(2):275-82. Epub 2015 Jan 22.

Department of Pediatrics, Academic Medical Center, University of Amsterdam, Amsterdam 1105 AZ, the Netherlands.

Singleton-Merten syndrome (SMS) is an infrequently described autosomal-dominant disorder characterized by early and extreme aortic and valvular calcification, dental anomalies (early-onset periodontitis and root resorption), osteopenia, and acro-osteolysis. To determine the molecular etiology of this disease, we performed whole-exome sequencing and targeted Sanger sequencing. We identified a common missense mutation, c.2465G>A (p.Arg822Gln), in interferon induced with helicase C domain 1 (IFIH1, encoding melanoma differentiation-associated protein 5 [MDA5]) in four SMS subjects from two families and a simplex case. IFIH1 has been linked to a number of autoimmune disorders, including Aicardi-Goutières syndrome. Immunohistochemistry demonstrated the localization of MDA5 in all affected target tissues. In vitro functional analysis revealed that the IFIH1 c.2465G>A mutation enhanced MDA5 function in interferon beta induction. Interferon signature genes were upregulated in SMS individuals' blood and dental cells. Our data identify a gain-of-function IFIH1 mutation as causing SMS and leading to early arterial calcification and dental inflammation and resorption.
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http://dx.doi.org/10.1016/j.ajhg.2014.12.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4320263PMC
February 2015

Mutations in PLK4, encoding a master regulator of centriole biogenesis, cause microcephaly, growth failure and retinopathy.

Nat Genet 2014 Dec 26;46(12):1283-1292. Epub 2014 Oct 26.

Medical Research Council (MRC) Human Genetics Unit, Institute of Genetics and Molecular Medicine (IGMM), University of Edinburgh, Edinburgh, UK.

Centrioles are essential for ciliogenesis. However, mutations in centriole biogenesis genes have been reported in primary microcephaly and Seckel syndrome, disorders without the hallmark clinical features of ciliopathies. Here we identify mutations in the genes encoding PLK4 kinase, a master regulator of centriole duplication, and its substrate TUBGCP6 in individuals with microcephalic primordial dwarfism and additional congenital anomalies, including retinopathy, thereby extending the human phenotypic spectrum associated with centriole dysfunction. Furthermore, we establish that different levels of impaired PLK4 activity result in growth and cilia phenotypes, providing a mechanism by which microcephaly disorders can occur with or without ciliopathic features.
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http://dx.doi.org/10.1038/ng.3122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4676084PMC
December 2014

A missense mutation in the PISA domain of HsSAS-6 causes autosomal recessive primary microcephaly in a large consanguineous Pakistani family.

Hum Mol Genet 2014 Nov 20;23(22):5940-9. Epub 2014 Jun 20.

Institute of Human Genetics and

Asymmetric cell division is essential for normal human brain development. Mutations in several genes encoding centrosomal proteins that participate in accurate cell division have been reported to cause autosomal recessive primary microcephaly (MCPH). By homozygosity mapping including three affected individuals from a consanguineous MCPH family from Pakistan, we delineated a critical region of 18.53 Mb on Chromosome 1p21.3-1p13.1. This region contains the gene encoding HsSAS-6, a centrosomal protein primordial for seeding the formation of new centrioles during the cell cycle. Both next-generation and Sanger sequencing revealed a homozygous c.185T>C missense mutation in the HsSAS-6 gene, resulting in a p.Ile62Thr substitution within a highly conserved region of the PISA domain of HsSAS-6. This variant is neither present in any single-nucleotide polymorphism or exome sequencing databases nor in a Pakistani control cohort. Experiments in tissue culture cells revealed that the Ile62Thr mutant of HsSAS-6 is substantially less efficient than the wild-type protein in sustaining centriole formation. Together, our findings demonstrate a dramatic impact of the mutation p.Ile62Thr on HsSAS-6 function and add this component to the list of genes mutated in primary microcephaly.
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http://dx.doi.org/10.1093/hmg/ddu318DOI Listing
November 2014

CDK6 associates with the centrosome during mitosis and is mutated in a large Pakistani family with primary microcephaly.

Hum Mol Genet 2013 Dec 4;22(25):5199-214. Epub 2013 Aug 4.

Cologne Center for Genomics (CCG).

Autosomal recessive primary microcephaly (MCPH) is characterized by reduced head circumference, reduction in the size of the cerebral cortex with otherwise grossly normal brain structure and variable intellectual disability. MCPH is caused by mutations of 11 different genes which code for proteins implicated in cell division and cell cycle regulation. We studied a consanguineous eight-generation family from Pakistan with ten microcephalic children using homozygosity mapping and found a new MCPH locus at HSA 7q21.11-q21.3. Sanger sequencing of the most relevant candidate genes in this region revealed a homozygous single nucleotide substitution c.589G>A in CDK6, which encodes cyclin-dependent kinase 6. The mutation changes a highly conserved alanine at position 197 into threonine (p.Ala197Thr). Post hoc whole-exome sequencing corroborated this mutation's identification as the causal variant. CDK6 is an important protein for the control of the cell cycle and differentiation of various cell types. We show here for the first time that CDK6 associates with the centrosome during mitosis; however, this was not observed in patient fibroblasts. Moreover, the mutant primary fibroblasts exhibited supernumerary centrosomes, disorganized microtubules and mitotic spindles, an increased centrosome nucleus distance, reduced cell proliferation and impaired cell motility and polarity. Upon ectopic expression of the mutant protein and knockdown of CDK6 through shRNA, we noted similar effects. We propose that the identified CDK6 mutation leads to reduced cell proliferation and impairs the correct functioning of the centrosome in microtubule organization and its positioning near the nucleus which are key determinants during neurogenesis.
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http://dx.doi.org/10.1093/hmg/ddt374DOI Listing
December 2013

Intellectual disability and hemizygous GPD2 mutation.

Am J Med Genet A 2013 May 29;161A(5):1044-50. Epub 2013 Mar 29.

Department of Clinical Genetics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

We report on a 25-year-old female with intellectual disability, mildly unusual face, and a pervasive developmental disorder, in whom routine aCGH showed a 298 kb de novo deletion at chromosome 2q24.1(156869529-157167986 × 1). The region contained two genes (NR4A2; GPD2). Molecular studies in the proposita showed an additional variant in GPD2 (c.614C > T, p.Pro205Leu), which was predicted to be pathogenic. The variant was also present in the healthy mother and sister. Functional analysis showed absent GPD2 activity in the proposita and 50% activity in mother and sister. We conclude that we have been able to find circumstantial evidence for the causative effect of the hemizygous GPD2 mutation but full proof remained lacking. Total costs for the work-up in these patients were high (€21,975 [$27,029]). Similar results will increasingly be found when Next Generation Techniques will be applied widely in patients with intellectual disability, and proving pathogenicity by functional studies or in animal models will be expensive. We advocate the use of freely accessible international databases combining phenotype and genotype data using standard nomenclatures to facilitate proving pathogenicity of research data and to decrease costs of health care.
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http://dx.doi.org/10.1002/ajmg.a.35873DOI Listing
May 2013

Mutations in ABCD4 cause a new inborn error of vitamin B12 metabolism.

Nat Genet 2012 Oct 26;44(10):1152-5. Epub 2012 Aug 26.

Division of Metabolism, Children's Research Center (CRC), University Children's Hospital, Zürich, Switzerland.

Inherited disorders of vitamin B12 (cobalamin) have provided important clues to how this vitamin, which is essential for hematological and neurological function, is transported and metabolized. We describe a new disease that results in failure to release vitamin B12 from lysosomes, which mimics the cblF defect caused by LMBRD1 mutations. Using microcell-mediated chromosome transfer and exome sequencing, we identified causal mutations in ABCD4, a gene that codes for an ABC transporter, which was previously thought to have peroxisomal localization and function. Our results show that ABCD4 colocalizes with the lysosomal proteins LAMP1 and LMBD1, the latter of which is deficient in the cblF defect. Furthermore, we show that mutations altering the putative ATPase domain of ABCD4 affect its function, suggesting that the ATPase activity of ABCD4 may be involved in intracellular processing of vitamin B12.
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http://dx.doi.org/10.1038/ng.2386DOI Listing
October 2012

Generalized arterial calcification of infancy and pseudoxanthoma elasticum can be caused by mutations in either ENPP1 or ABCC6.

Am J Hum Genet 2012 Jan 29;90(1):25-39. Epub 2011 Dec 29.

Department of General Pediatrics, Münster University Children's Hospital, Germany.

Spontaneous pathologic arterial calcifications in childhood can occur in generalized arterial calcification of infancy (GACI) or in pseudoxanthoma elasticum (PXE). GACI is associated with biallelic mutations in ENPP1 in the majority of cases, whereas mutations in ABCC6 are known to cause PXE. However, the genetic basis in subsets of both disease phenotypes remains elusive. We hypothesized that GACI and PXE are in a closely related spectrum of disease. We used a standardized questionnaire to retrospectively evaluate the phenotype of 92 probands with a clinical history of GACI. We obtained the ENPP1 genotype by conventional sequencing. In those patients with less than two disease-causing ENPP1 mutations, we sequenced ABCC6. We observed that three GACI patients who carried biallelic ENPP1 mutations developed typical signs of PXE between 5 and 8 years of age; these signs included angioid streaks and pseudoxanthomatous skin lesions. In 28 patients, no disease-causing ENPP1 mutation was found. In 14 of these patients, we detected pathogenic ABCC6 mutations (biallelic mutations in eight patients, monoallelic mutations in six patients). Thus, ABCC6 mutations account for a significant subset of GACI patients, and ENPP1 mutations can also be associated with PXE lesions in school-aged children. Based on the considerable overlap of genotype and phenotype of GACI and PXE, both entities appear to reflect two ends of a clinical spectrum of ectopic calcification and other organ pathologies, rather than two distinct disorders. ABCC6 and ENPP1 mutations might lead to alterations of the same physiological pathways in tissues beyond the artery.
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http://dx.doi.org/10.1016/j.ajhg.2011.11.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257960PMC
January 2012

Defining the immunoreactive epitope for the monoclonal anti-human glutathione peroxidase-4 antibody anti-hGPx4 Mab63-1.

Immunol Lett 2010 Oct 5;133(2):85-93. Epub 2010 Aug 5.

Institute of Biochemistry, University Medicine Berlin - Charité, Monbijoustr. 2, D-10117 Berlin, Federal Republic of Germany.

Glutathione peroxidases (GPx) form a heterogeneous enzyme family and GPx4-isoforms have been implicated in anti-oxidative defense, brain development, neuroinjury and sperm maturation. In humans seven GPx isoforms (GPx1-GPx7) can be separated. To selectively quantify the expression of GPx4-isoforms we have raised a monoclonal antibody (anti-hGPx4 Mab63-1) against the pure recombinant Sec46Cys mutant of human cytosolic GPx4 and used it for immunoblotting, immunoprecipitation and immunohistochemistry. The antibody recognizes human GPx4, its mouse ortholog but neither reacted with rat GPx4 nor other human GPx-isoforms. Sequence alignment of human and rat GPx4 proteins indicated three different amino acids (S18, F35, K99 in humans, A18, C35, R99 in rats) and a S18A exchange in the human enzyme completely abolished immunoreactivity. To further characterize the immunological epitope we synthesized a set of 12-mer peptides flanking S18* of human GPx4 and found that the sequence SMHEFS*AKDIDG exhibited strongest immunoreactivity. Substitution analysis and peptide length variation narrowed down the essential epitope to FS*AKDI and indicated that most mutations in this region strongly impaired immunoreactivity. In silico blast searches of public protein databases failed to identify proteins with potential immunoreactivity suggesting that the antibody exhibits a high specificity for human and mouse GPx4 and may not cross-react with unrelated proteins.
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http://dx.doi.org/10.1016/j.imlet.2010.07.006DOI Listing
October 2010

Crystallization and preliminary X-ray crystallographic analysis of the [NiFe]-hydrogenase maturation factor HypF1 from Ralstonia eutropha H16.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2010 Apr 31;66(Pt 4):452-5. Epub 2010 Mar 31.

Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Chausseestrasse 117, D-10115 Berlin, Germany.

The hydrogenase maturation factor HypF1 is a truncated but functional version of the HypF protein. HypF is known to be involved in the supply of the CN(-) ligands of the active site of [NiFe]-hydrogenases, utilizing carbamoyl phosphate as a substrate. The first crystallization and preliminary X-ray studies of HypF1 from Ralstonia eutropha H16 are reported here. Crystals of HypF1 (394 amino acids, 40.7 kDa) were obtained by the sitting-drop vapour-diffusion technique using sodium formate as a precipitant. The crystals belonged to space group I222, with unit-cell parameters a = 79.7, b = 91.6, c = 107.2 A. Complete X-ray diffraction data sets were collected at 100 K from native crystals and from a platinum derivative to a maximum resolution of 1.65 A.
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http://dx.doi.org/10.1107/S1744309110006196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2852342PMC
April 2010

Three novel mutations in the ANK membrane protein cause craniometaphyseal dysplasia with variable conductive hearing loss.

Am J Med Genet A 2010 Apr;152A(4):870-4

Institute of Medical Genetics, Charité Universitätsmedizin, Berlin, Germany.

Craniometaphyseal dysplasia (CMD) is a rare, sclerosing skeletal disorder caused by mutations in ANKH, which encodes a putative pyrophosphate transporting membrane protein. Six distinct ANKH mutations have been described to date. We report here on three novel mutations in simplex patients with CMD. The c.1015T>C (p.Cys339Arg) mutation found in Patient A was associated with congenital facial palsy, early-onset conductive hearing loss, and a generalized undermodeling of the long bones. The c.1172T>C (p.Leu391Pro) mutation in Patient B was associated with facial palsy, progressive conductive hearing loss, and generalized undermodeling of tubular bones. A milder phenotype without cranial nerve affection was observed in Patient C, associated with a c.1001T>G (p.Leu334Arg) mutation. All affected residues lie in evolutionarily conserved sequence blocks. These additional cases and the associated mutations contribute to an improved appreciation of the variability of this rare skeletal dysplasia. (c) 2010 Wiley-Liss, Inc.
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http://dx.doi.org/10.1002/ajmg.a.33301DOI Listing
April 2010

Insights into lysosomal cobalamin trafficking: lessons learned from cblF disease.

J Mol Med (Berl) 2010 May 20;88(5):459-66. Epub 2010 Feb 20.

Department of General Pediatrics, Münster University Children's Hospital, Münster, Germany.

Vitamin B(12) (cobalamin) is essential in animals and humans for metabolism of methylmalonic acid, for the remethylation of homocysteine to methionine and, consequently, for all S-adenosylmethionine-dependent methylation reactions, including DNA synthesis. In man, cobalamin deficiency leads to anemia and neurologic and cognitive impairment. In the cblF inborn error of vitamin B(12) metabolism, free vitamin accumulates in lysosomes and cannot be converted to cofactors for mitochondrial methylmalonyl-CoA mutase and cytosolic methionine synthase. Recent work has shown that this defect is caused by mutations in the lysosomal membrane protein LMBD1, which shows significant homology to lipocalin membrane receptors, thereby indicating that LMBD1 is a lysosomal membrane exporter for cobalamin.
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http://dx.doi.org/10.1007/s00109-010-0601-xDOI Listing
May 2010

Identification of a putative lysosomal cobalamin exporter altered in the cblF defect of vitamin B12 metabolism.

Nat Genet 2009 Feb 11;41(2):234-9. Epub 2009 Jan 11.

Department of General Pediatrics, Münster University Children's Hospital, Albert-Schweitzer-Strasse 33, D-48149 Münster, Germany.

Vitamin B(12) (cobalamin) is essential in animals for metabolism of branched chain amino acids and odd chain fatty acids, and for remethylation of homocysteine to methionine. In the cblF inborn error of vitamin B(12) metabolism, free vitamin accumulates in lysosomes, thus hindering its conversion to cofactors. Using homozygosity mapping in 12 unrelated cblF individuals and microcell-mediated chromosome transfer, we identified a candidate gene on chromosome 6q13, LMBRD1, encoding LMBD1, a lysosomal membrane protein with homology to lipocalin membrane receptor LIMR. We identified five different frameshift mutations in LMBRD1 resulting in loss of LMBD1 function, with 18 of the 24 disease chromosomes carrying the same mutation embedded in a common 1.34-Mb haplotype. Transfection of fibroblasts of individuals with cblF with wild-type LMBD1 rescued cobalamin coenzyme synthesis and function. This work identifies LMBRD1 as the gene underlying the cblF defect of cobalamin metabolism and suggests that LMBD1 is a lysosomal membrane exporter for cobalamin.
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http://dx.doi.org/10.1038/ng.294DOI Listing
February 2009

Mutations and polymorphisms in the human argininosuccinate synthetase (ASS1) gene.

Hum Mutat 2009 Mar;30(3):300-7

Universitätsklinikum Münster, Klinik und Poliklinik für Kinder- und Jugendmedizin, Münster, Germany.

Citrullinemia type I is an autosomal recessive disorder that is caused by a deficiency of the urea cycle enzyme argininosuccinate synthetase (ASS1). Deficiency of ASS1 shows various clinical manifestations encompassing severely affected patients with fatal neonatal hyperammonemia as well as asymptomatic individuals with only a biochemical phenotype. This is a comprehensive report of all 87 mutations found to date in the ASS1 gene on chromosome 9q34.1. A large proportion of the mutations (n=27) are described here for the first time. Mutations are distributed throughout exons 3 to 15, most of them being identified in exons 5, 12, 13, and 14. The mutation G390R in exon 15 is the single most common mutation in patients with the classical phenotype. Certain mutations clearly link to specific clinical courses but the clinical phenotype cannot be anticipated in all patients. This update presents a survey of the correlation between mutations in the ASS1 gene and the respective clinical courses as described so far. It also sheds light on the geographic incidence of the mutations. Enzymatic studies have been done in bacterial and human cell systems. However, the prognostic value of genetic aberrations with respect to their effect on protein function and clinical manifestation remains uncertain.
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http://dx.doi.org/10.1002/humu.20847DOI Listing
March 2009

tRNA splicing endonuclease mutations cause pontocerebellar hypoplasia.

Nat Genet 2008 Sep;40(9):1113-8

Cologne Center for Genomics and Institute for Genetics, University of Cologne, Cologne, Germany.

Pontocerebellar hypoplasias (PCH) represent a group of neurodegenerative autosomal recessive disorders with prenatal onset, atrophy or hypoplasia of the cerebellum, hypoplasia of the ventral pons, microcephaly, variable neocortical atrophy and severe mental and motor impairments. In two subtypes, PCH2 and PCH4, we identified mutations in three of the four different subunits of the tRNA-splicing endonuclease complex. Our findings point to RNA processing as a new basic cellular impairment in neurological disorders.
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http://dx.doi.org/10.1038/ng.204DOI Listing
September 2008

Phage display screening for peptidic chitinase inhibitors.

J Mol Recognit 2008 Nov-Dec;21(6):401-9

Institute of Biochemistry, Charité- Universitätsmedizin Berlin, Monbijoustrasse 2, D-10117 Berlin, Germany.

A phage display library with disulfide-cyclized peptides was screened for peptides binding to chitinases from Serratia marcescens. One of those peptides was found to efficiently inhibit chitinase A and two others were inhibitors of chitinase B. Complete substitutional analysis of all three peptides using cellulose-bound peptide spot synthesis revealed key interaction positions and allowed optimization of the chitinase B inhibitory peptides towards higher affinity, with inhibitory constants in the lower nanomolar range. Inhibition by all peptides proved to be competitive and highly specific for the chitinase used to select them, as shown with a series of chitinases from different organisms.
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http://dx.doi.org/10.1002/jmr.911DOI Listing
January 2009

The structure of the anti-c-myc antibody 9E10 Fab fragment/epitope peptide complex reveals a novel binding mode dominated by the heavy chain hypervariable loops.

Proteins 2008 Nov;73(3):552-65

Institut für Biochemie, Charité, Universitätsmedizin Berlin, Monbijoustr. 2, D-10117 Berlin, Germany.

The X-ray structure of the Fab fragment from the anti-c-myc antibody 9E10 was determined both as complex with its epitope peptide and for the free Fab. In the complex, two Fab molecules adopt an unusual head to head orientation with the epitope peptide arranged between them. In contrast, the free Fab forms a dimer with different orientation. In the Fab/peptide complex the peptide is bound to one of the two Fabs at the "back" of its extended CDR H3, in a cleft with CDR H1, thus forming a short, three-stranded antiparallel beta-sheet. The N- and C-terminal parts of the peptide are also in contact with the neighboring Fab fragment. Comparison between the CDR H3s of the two Fab molecules in complex with the peptide and those from the free Fab reveals high flexibility of this loop. This structural feature is in line with thermodynamic data from isothermic titration calorimetry.
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http://dx.doi.org/10.1002/prot.22080DOI Listing
November 2008

Investigation of citrullinemia type I variants by in vitro expression studies.

Hum Mutat 2008 Oct;29(10):1222-7

Universitätsklinikum Münster, Klinik und Poliklinik für Kinder- und Jugendmedizin, Münster, Germany.

Mild citrullinemia is an allelic variant of classical citrullinemia type I also caused by deficiency of the urea cycle enzyme argininosuccinate synthetase (ASS). Affected patients comprise a biochemical but no clinical phenotype. However, there is no reliable parameter allowing conclusions regarding the course of the disorder or its type of manifestation. The aim of this study was to test the importance of varying levels of ASS residual activities for the severity at diagnosis. Bacterial in vitro expression studies allowed the enzymatic analysis of purified wild-type and the mutant ASS proteins p.Ala118Thr (c.352G>A), p.Trp179Arg (c.535T>C), p.Val263Met (c.787G>A), p.Arg265Cys (c.793C>T), p.Met302Val (c.904A>G), p.Gly324Ser (c.970G>A), p.Gly362Val (c.1085G>T), and p.Gly390Arg (c.1168G>A). In the chosen system, classical mutations do not show any significant enzymatic activity, whereas mutations associated with a mild course yield significant ASS activity levels. The mutation p.Ala118Thr (c.352G>A) impresses by a high residual activity (62%) but a severe reduction of affinity toward the substrates citrulline and aspartate. This mutation was identified in a hitherto healthy female adult with no history of known citrullinemia who had died during the postpartum period from hyperammonemic coma. The results of this study suggest that even a high level of residual ASS activity is not a reliable prognostic marker for an uneventful clinical course. Determination of ASS residual activities, therefore, cannot help in anticipating the risk of metabolic derangement. This study should guide clinicians as well as patients with mild citrullinemia toward a lifelong awareness of the disorder.
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http://dx.doi.org/10.1002/humu.20784DOI Listing
October 2008

HLA-DR alpha 2 mediates negative signalling via binding to Tirc7 leading to anti-inflammatory and apoptotic effects in lymphocytes in vitro and in vivo.

PLoS One 2008 Feb 13;3(2):e1576. Epub 2008 Feb 13.

CellAct Pharma GmbH, Berlin, Germany.

Classically, HLA-DR expressed on antigen presenting cells (APC) initiates lymphocyte activation via presentation of peptides to TCR bearing CD4+ T-Cells. Here we demonstrate that HLA-DR alpha 2 domain (sHLA-DRalpha2) also induces negative signals by engaging TIRC7 on lymphocytes. This interaction inhibits proliferation and induces apoptosis in CD4+ and CD8+ T-cells via activation of the intrinsic pathway. Proliferation inhibition is associated with SHP-1 recruitment by TIRC7, decreased phosphorylation of STAT4, TCR-zeta chain & ZAP70, and inhibition of IFN-gamma and FasL expression. HLA-DRalpha2 and TIRC7 co-localize at the APC-T cell interaction site. Triggering HLA-DR - TIRC7 pathway demonstrates that sHLA-DRalpha2 treatment inhibits proinflammatory-inflammatory cytokine expression in APC & T cells after lipopolysaccaride (LPS) stimulation in vitro and induces apoptosis in vivo. These results suggest a novel antiproliferative role for HLA-DR mediated via TIRC7, revise the notion of an exclusive stimulatory interaction of HLA-DR with CD4+ T cells and highlights a novel physiologically relevant regulatory pathway.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001576PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2217592PMC
February 2008

Noncompaction of the ventricular myocardium is associated with a de novo mutation in the beta-myosin heavy chain gene.

PLoS One 2007 Dec 26;2(12):e1362. Epub 2007 Dec 26.

Cologne Center for Genomics and Institute for Genetics, University of Cologne, Cologne, Germany.

Noncompaction of the ventricular myocardium (NVM) is the morphological hallmark of a rare familial or sporadic unclassified heart disease of heterogeneous origin. NVM results presumably from a congenital developmental error and has been traced back to single point mutations in various genes. The objective of this study was to determine the underlying genetic defect in a large German family suffering from NVM. Twenty four family members were clinically assessed using advanced imaging techniques. For molecular characterization, a genome-wide linkage analysis was undertaken and the disease locus was mapped to chromosome 14ptel-14q12. Subsequently, two genes of the disease interval, MYH6 and MYH7 (encoding the alpha- and beta-myosin heavy chain, respectively) were sequenced, leading to the identification of a previously unknown de novo missense mutation, c.842G>C, in the gene MYH7. The mutation affects a highly conserved amino acid in the myosin subfragment-1 (R281T). In silico simulations suggest that the mutation R281T prevents the formation of a salt bridge between residues R281 and D325, thereby destabilizing the myosin head. The mutation was exclusively present in morphologically affected family members. A few members of the family displayed NVM in combination with other heart defects, such as dislocation of the tricuspid valve (Ebstein's anomaly, EA) and atrial septal defect (ASD). A high degree of clinical variability was observed, ranging from the absence of symptoms in childhood to cardiac death in the third decade of life. The data presented in this report provide first evidence that a mutation in a sarcomeric protein can cause noncompaction of the ventricular myocardium.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001362PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2137931PMC
December 2007

Engineering a circularly permuted GFP scaffold for peptide presentation.

J Mol Recognit 2007 Sep-Oct;20(5):367-78

Institut für Biochemie, Charité-Universitätsmedizin Berlin, Monbijoustrasse 20, D-10117 Berlin, Germany.

The use of peptides as in vivo and in vitro ligand binding agents is hampered by the high flexibility, low stability and lack of intrinsic detection signal of peptide aptamers. Recent attempts to overcome these limitations included the integration of the binding peptide into a stable protein scaffold. In this paper, we present the optimization and testing of a circularly permuted variant of the green fluorescent protein (GFP). We examined the ability of the optimized scaffold to accept peptide insertions at three different regions. The three regions chosen are localized in close spatial proximity to each other and support different conformations of the inserted peptides. In all the three regions peptides with a biased, but still comprehensive, amino acid repertoire could be presented without disturbing the function of the optimized GFP-scaffold.
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http://dx.doi.org/10.1002/jmr.844DOI Listing
June 2008

Structure of an anti-cholera toxin antibody Fab in complex with an epitope-derived D-peptide: a case of polyspecific recognition.

J Mol Recognit 2007 Jul-Aug;20(4):263-74

Charité-Universitätsmedizin Berlin, Institute of Biochemistry, Monbijoustr. 2, D-10117 Berlin, Germany.

The structure of a complex of the anti-cholera toxin antibody TE33 Fab (fragment antibody) with the D-peptide vpGsqhyds was solved to 1.78 A resolution. The D-peptide was derived from the linear L-peptide epitope VPGSQHIDS by a stepwise transformation. Despite the very similar amino acid sequence-the only difference is a tyrosine residue in position 7-there are marked differences in the individual positions with respect to their contribution to the peptide overall affinity as ascertained by a complete substitutional analysis. This is reflected by the X-ray structure of the TE33 Fab/D-peptide complex where there is an inverted orientation of the D-peptide as compared with the known structure of a corresponding complex containing the epitope L-peptide, with the side chains establishing different contacts within the binding site of TE33. The D- and L-peptide affinities are comparable and the surface areas buried by complex formation are almost the same. Thus the antibody TE33 provides a typical example for polyspecific binding behavior of IgG family antibodies.
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http://dx.doi.org/10.1002/jmr.838DOI Listing
November 2007

Structural basis for catalytic activity and enzyme polymerization of phospholipid hydroperoxide glutathione peroxidase-4 (GPx4).

Biochemistry 2007 Aug 13;46(31):9041-9. Epub 2007 Jul 13.

Institute of Biochemistry, University Medicine Berlin-Charité, Monbijoustr. 2, D-10117 Berlin, Germany.

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a moonlighting selenoprotein, which has been implicated in anti-oxidative defense, sperm development, and cerebral embryogenesis. Among GPx-isoforms, GPx4 is unique because of its capability to reduce complex lipid hydroperoxides and its tendency toward polymerization, but the structural basis for these properties remained unclear. To address this, we solved the crystal structure of the catalytically active U46C mutant of human GPx4 to 1.55 A resolution. X-ray data indicated a monomeric protein consisting of four alpha-helices and seven beta-strands. GPx4 lacks a surface exposed loop domain, which appears to limit the accessibility of the active site of other GPx-isoforms, and these data may explain the broad substrate specificity of GPx4. The catalytic triad (C46, Q81, and W136) is localized at a flat impression of the protein surface extending into a surface exposed patch of basic amino acids (K48, K135, and R152) that also contains polar T139. Multiple mutations of the catalytic triad indicated its functional importance. Like the wild-type enzyme, the U46C mutant exhibits a strong tendency toward protein polymerization, which was prevented by reductants. Site-directed mutagenesis suggested involvement of the catalytic C46 and surface exposed C10 and C66 in polymer formation. In GPx4 crystals, these residues contact adjacent protein monomers.
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http://dx.doi.org/10.1021/bi700840dDOI Listing
August 2007

Crisponi syndrome is caused by mutations in the CRLF1 gene and is allelic to cold-induced sweating syndrome type 1.

Am J Hum Genet 2007 May 30;80(5):971-81. Epub 2007 Mar 30.

Istituto di Neurogenetica e Neurofarmacologia-Consiglio Nazionale delle Ricerche, Cittadella Universitaria di Monserrato, Monserrato, Italy.

Crisponi syndrome is a severe autosomal recessive condition that is phenotypically characterized by abnormal, paroxysmal muscular contractions resembling neonatal tetanus, large face, broad nose, anteverted nares, camptodactyly, hyperthermia, and sudden death in most cases. We performed homozygosity mapping in five Sardinian and three Turkish families with Crisponi syndrome, using high-density single-nucleotide polymorphism arrays, and identified a critical region on chromosome 19p12-13.1. The most prominent candidate gene was CRLF1, recently found to be involved in the pathogenesis of cold-induced sweating syndrome type 1 (CISS1). CISS1 belongs to a group of conditions with overlapping phenotypes, also including cold-induced sweating syndrome type 2 and Stuve-Wiedemann syndrome. All these syndromes are caused by mutations of genes of the ciliary neurotrophic factor (CNTF)-receptor pathway. Here, we describe the identification of four different CRLF1 mutations in eight different Crisponi-affected families, including a missense mutation, a single-nucleotide insertion, and a nonsense and an insertion/deletion (indel) mutation, all segregating with the disease trait in the families. Comparison of the mutation spectra of Crisponi syndrome and CISS1 suggests that neither the type nor the location of the CRLF1 mutations points to a phenotype/genotype correlation that would account for the most severe phenotype in Crisponi syndrome. Other, still-unknown molecular factors may be responsible for the variable phenotypic expression of the CRLF1 mutations. We suggest that the syndromes can comprise a family of "CNTF-receptor-related disorders," of which Crisponi syndrome would be the newest member and allelic to CISS1.
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http://dx.doi.org/10.1086/516843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1852730PMC
May 2007

Insights into functional aspects of centrins from the structure of N-terminally extended mouse centrin 1.

Vision Res 2006 Dec 6;46(27):4568-74. Epub 2006 Oct 6.

Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Ziegelstr. 5-9, D-10098 Berlin, Germany.

Centrins are members of the family of Ca(2+)-binding EF-hand proteins. In photoreceptor cells, centrin isoform 1 is specifically localized in the non-motile cilium. This connecting cilium links the light-sensitive outer segment with the biosynthetic active inner segment of the photoreceptor cell. All intracellular exchanges between these compartments have to occur through this cilium. Three-dimensional structures of centrins from diverse organisms are known, showing that the EF-hand motifs of the N-terminal domains adopt closed conformations, while the C-terminal EF-hand motifs have open conformations. The crystal structure of an N-terminally extended mouse centrin 1 (MmCen1-L) resembles the overall structure of troponin C in its two Ca(2+) bound form. Within the N-terminal extension in MmCen1-L, residues W24 and R25 bind to the C-terminal domain of centrin 1 in a target-protein-like geometry. Here, we discuss this binding mode in connection with putative interaction sites of the target-protein transducin and the self-assembly of centrins.
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http://dx.doi.org/10.1016/j.visres.2006.07.034DOI Listing
December 2006