HUMAN MUTATION Mutation in Brief 31: E1319-E1331 (2010) Online MUTATION IN BRIEF HUMAN MUTATION OFFICIAL JOURNAL Novel TMEM67 Mutations and Genotype-phenotype Correlates in Meckelin-related Ciliopathies www.hgvs.org Miriam Iannicelli,1# Francesco Brancati,1,2# Soumaya Mougou-Zerelli,3 Annalisa Mazzotta,1 Sophie Thomas,3 Nadia Elkhartoufi,4 Lorena Travaglini,1 Céline Gomes,3 Gian Luigi Ardissino,5 Enrico Bertini,6 Eugen Boltshauser,7 Pierangela Castorina,5 Stefano D’Arrigo,8 Rita Fischetto,9 Brigitte Leroy,10 Philippe Loget,11 Maryse Bonnière,12 Lena Starck,13 Julia Tantau,14 Barbara Gentilin,5 Silvia Majore,15 Dominika Swistun,16 Elizabeth Flori,17 Faustina Lalatta,5 Chiara Pantaleoni,8 Johannes.Penzien,18 Paola Grammatico,15 the International JSRD Study Group, Bruno Dallapiccola,6 Joseph G. Gleeson,16 Tania Attie-Bitach,3,4* and Enza Maria Valente,1,19* 1Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo and CSS-Mendel Institute, Rome, Italy; 2CeSI, Aging Research Centre, and Dept. of Biomedical Sciences, G. d’Annunzio University Foundation, Chieti, Italy; 3INSERM U-781, Hôpital Necker-Enfants Malades and Université René Descartes, Paris V, France; 4Dept of Genetics, Hôpital Necker-Enfants Malades, Assistance Publique Hôpitaux de Paris (APHP), Paris, France; 5Foundation IRCCS Ospedale Maggiore Policlinico Mangiagalli e Regina Elena, Milan, Italy; 6 IRCCS Ospedale Pediatrico Bambino Gesù, Rome, Italy; 7Dept. of Neurology, Children's University Hospital, Zurich, Switzerland; 8Division of Neurologia dello Sviluppo, Carlo Besta Neurologic Institute Foundation, Milan, Italy; 9UO Metabolic Disease-Medical Genetics, PO Giovanni XXIII-AOU Policlinico Consorziale, Bari, Italy;10Service d’Anatomie et Cytologie Pathologiques, CHI de Poissy, Saint Germain-en-Laye; 11Cabinet d’Anatomie et Cytologie Pathologiques Richier, Rennes, France;12Anatomopathologie, Lille, France; 13Sachs´Children´s Hospital, Stockholm,Sweden; 14Service d’Anatomopathologie, Hôpital Saint-Vincent de Paul, AP-HP, Paris; 15 Dept. of Experimental Medicine, Sapienza University,S.Camillo-Forlanini Hospital, Rome, Italy; 16Neurogenetics Laboratory, Howard Hughes Medical Institute, Dept. of Neurosciences, University of California, San Diego, La Jolla (CA), USA; 17Service de cytogénétique, Fédération de génétique, Hôpitaux Universitaires de Strasbourg, Strasbourg, France; 18Children's Hospital, Augsburg, Germany; 19Dept. of Medical and Surgical Pediatric Sciences, University of Messina, Messina, Italy. # These authors contributed equally to this work. *Correspondence to: Enza Maria Valente, Neurogenetics Unit, CSS-Mendel Institute, viale Regina Margherita 261 I-00198 Rome, Italy; Tel.: +39 06 4416 0537; Fax: +39 06 4416 0548; E-mail: [email protected]; or to: Tania Attie- Bitach, E-mail: [email protected] Contract grant sponsor: Italian Ministry of Health, MIUR, the March of Dimes, Burroughs Wellcome Fund NINDS, NIH. Contract grant number: Ricerca Corrente 2009 (to BD); Ricerca Finalizzata 2006 ex art. 56 (to EMV); Telethon grant n. GGP08145 (to EB and EMV). Communicated by Ravi Savarirayan Received 7 November 2009; accepted revised manuscript 16 February 2010. © 2010 WILEY-LISS, INC. DOI: 10.1002/humu.21239 E1320 Iannicelli et al. ABSTRACT: Human ciliopathies are hereditary conditions caused by defects of proteins expressed at the primary cilium. Among ciliopathies, Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS) and nephronophthisis (NPH) present clinical and genetic overlap, being allelic at several loci. One of the most interesting gene is TMEM67, encoding the transmembrane protein meckelin. We performed mutation analysis of TMEM67 in 341 probands, including 265 JSRD representative of all clinical subgroups and 76 MKS fetuses. We identified 33 distinct mutations, of which 20 were novel, in 8/10 (80%) JS with liver involvement (COACH phenotype) and 12/76 (16%) MKS fetuses. No mutations were found in other JSRD subtypes, confirming the strong association between TMEM67 mutations and liver involvement. Literature review of all published TMEM67 mutated cases was performed to delineate genotype-phenotype correlates. In particular, comparison of the types of mutations and their distribution along the gene in lethal versus non lethal phenotypes showed in MKS patients a significant enrichment of missense mutations falling in TMEM67 exons 8 to 15, especially when in combination with a truncating mutation. These exons encode for a region of unknown function in the extracellular domain of meckelin. ©2010 Wiley-Liss, Inc. KEY WORDS: TMEM67, MKS3, Joubert syndrome, Meckel syndrome, congenital hepatic fibrosis, COACH syndrome. INTRODUCTION Human ciliopathies represent an expanding group of autosomal or X-linked disorders caused by defects of proteins expressed at the primary cilium or its apparatus (Hildebrandt and Zhou, 2007; Sharma et al., 2008). Primary cilia are microtubule-based structures found in most tissues, that function as cellular sensors and control axonal migration and cell polarity during development (Gerdes et al., 2009; Marshall, 2008). The wide expression pattern of ciliary proteins well explains the multiorgan involvement seen in most ciliary disorders, mainly implicating the central nervous system (CNS), retina, kidneys and liver (Lancaster and Gleeson, 2009). There is striking clinical and genetic overlap among some ciliopathies, namely Joubert syndrome and related disorders (JSRD; MIM# 213300), Meckel syndrome (MKS; MIM# 249000), and Nephronophthisis (NPH; MIM# 256100). These conditions are known to be allelic at several gene loci, albeit genotype-phenotype correlations are only partly understood (Lancaster and Gleeson, 2009). Among these genes, great interest has recently arisen on TMEM67 (MIM# 609884) that encodes meckelin (Dawe et al., 2007; Smith et al., 2006). This is a 995 aminoacid protein, with an extracellular N-terminus containing a signal peptide and a cysteine rich domain, a transmembrane portion and an intracellular C-terminus including a coiled-coil domain (Khaddour et al., 2007; Smith et al., 2006). TMEM67 was first identified as causative of MKS (Smith et al., 2006), a lethal disorder displaying CNS malformations (typically occipital encephalocele), multicystic kidneys, ductal plate dysplasia with congenital hepatic fibrosis (CHF) and postaxial polydactyly (Salonen, 1984; Salonen and Paavola, 1998). Large mutation screenings identified TMEM67 mutations in 23 of 195 (12%) MKS fetuses (Baala et al., 2007; Consugar et al., 2007; Khaddour et al., 2007; Smith et al., 2006; Tallila et al., 2009), including some fetuses with Meckel-like phenotypes. These lacked at least one MKS diagnostic criterion, and their brain pathology often resembled the “molar tooth sign” (MTS) (Baala et al., 2007). The MTS defines a specific constellation of cerebellar and brainstem abnormalities that is peculiar of JSRD, another heterogeneous group of ciliopathies with CNS, retinal, renal and hepatic manifestations (Valente et al., 2008). Within the JSRD spectrum, there is strong correlation between TMEM67 mutations and the subgroup of JS plus liver involvement (COACH syndrome; MIM# 216360), with an overall mutation frequency of 73% (Baala et al., 2007; Brancati et al., 2009; Doherty et al., 2009). Two additional TMEM67-related ciliopathies were recently delineated. Otto and collaborators reported mutations in five of 62 (8%) probands with NPH and CHF (Otto et al., 2009). Finally, TMEM67 was found mutated in two families with a peculiar association of polycystic kidney (mimicking autosomal recessive polycystic kidney disease - ARPKD), NPH, CHF and midbrain-hindbrain abnormalities within the MTS spectrum, defined as ARPKD-like syndrome (Gunay-Aygun et al., 2009). This extreme clinical heterogeneity associated with mutations in one and the same gene is intriguing, and calls for the delineation of specific clinical-genetic correlates. The allelic spectrum of TMEM67 is broad and includes missense, truncating and splice site mutations, as well as rare multiexon deletions. A preliminary correlation has Novel TMEM67 Mutations in Joubert and Meckel Syndromes E1321 been observed between truncating mutations and the occurrence of MKS, while missense mutations are more frequently detected in association to less severe phenotypes such as COACH and NPH. However, a systematic analysis of the phenotypic burden of TMEM67 mutations has not been performed yet. Here, we present the molecular screening of TMEM67 in two cohorts of patients, including JSRD cases representative of all clinical subgroups, and MKS fetuses. We describe and characterize 20 novel mutations, perform a detailed review of all published mutations and discuss genotype–phenotype correlates. MATERIALS AND METHODS Patients A total of 341 probands were analyzed. The first group included 265 probands with clinically and neuroradiologically confirmed diagnosis of JSRD. Detailed clinical data were obtained by referring clinicians of the International JSRD Study Group through a standardized questionnaire, allowing to assign patients to the following subgroups: pure JS (n=101), JS plus retinopathy (n=51), JS plus renal disease (n=11), cerebello-oculo- renal syndrome (n=80), JS plus liver disease (n=10), oro-facio-digital syndrome type VI (n=12). The second group included 76 fetuses diagnosed with MKS according on established criteria (Salonen, 1984). Pregnancies were terminated after genetic counseling, in accordance with local laws. Subjects were recruited worldwide and collected under