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Genotype-phenotype correlations

ORIGINAL ARTICLE Genotypeephenotype correlation in CC2D2A-related reveals an association with ventriculomegaly and seizures Ruxandra Bachmann-Gagescu,1 Gisele E Ishak,2 Jennifer C Dempsey,1 Jonathan Adkins,1 Diana O’Day,1 Ian G Phelps,1 Meral Gunay-Aygun,3 Antonie D Kline,4 Krzysztof Szczaluba,5 Loreto Martorell,6 Abdulrahman Alswaid,7 Shatha Alrasheed,7 Shashidhar Pai,8 Louise Izatt,9 Anne Ronan,10 Melissa A Parisi,11 Heather Mefford,1 Ian Glass,1 Dan Doherty1,12

For numbered affiliations see ABSTRACT junction.1 2 Patients with JS manifest hypotonia, end of article. Background Joubert syndrome (JS) is a ataxia, developmental delay, abnormal eye move- characterised by a distinctive brain malformation (the ments and abnormal respiratory control.34In Correspondence to Dr Dan Doherty, Associate ‘molar tooth sign’), developmental delay, abnormal eye addition to the core neurological features, subsets Professor, Divisions of Genetic movements and abnormal breathing pattern. Retinal of patients with JS also display abnormalities in Medicine and Developmental dystrophy, cystic kidney disease, liver fibrosis and other organ systems, including retinal dystrophy, Medicine, University of polydactyly are variably present, resulting in significant chorioretinal coloboma, cystic kidney disease, liver Washington, Box 356320, phenotypic heterogeneity and overlap with other fi 5 RR-249, 1959 NE Pacific Street, brosis and polydactyly. Given this pleiotropic Seattle, WA 98195-6320, USA; . JS is also genetically heterogeneous, phenotypic presentation, substantial overlap with [email protected] resulting from mutations in 13 . These factors other disorders is observed, including Meckel, render clinical/molecular diagnosis and management SenioreLøken and BardeteBiedl syndromes, as well Received 5 October 2011 challenging. CC2D2A mutations are a relatively common as Leber congenital amaurosis and isolated neph- Revised 7 December 2011 fi Accepted 8 December 2011 cause of JS and also cause Meckel syndrome. The ronophthisis. These disorders have been classi ed as clinical consequences of CC2D2A mutations in patients ciliopathies, since their causative genes all encode with JS have been incompletely reported. involved in function of primary cilia, Methods Subjects with JS from 209 families were ubiquitous organelles essential in mechanical, evaluated to identify mutations in CC2D2A. Clinical and chemical and light transduction, as well as specific imaging features in subjects with CC2D2A mutations signalling pathways.6 were compared with those in subjects without CC2D2A JS, like most ciliopathies, is characterised by mutations and reports in the literature. significant genetic heterogeneity, since 13 causative Results 10 novel CC2D2A mutations in 20 subjects genes have been identified to date: AHI1, ARL13B, were identified; a summary is provided of all published CC2D2A, CEP290, INPP5E, KIF7, NPHP1, OFD1, CC2D2A mutations. Subjects with CC2D2A-related JS RPGRIP1L, TCTN1, TCTN2, TMEM67 (MKS3) and e were more likely to have ventriculomegaly (p<0.0001) TMEM216.7 20 These account individually for up and seizures (p¼0.024) than subjects without CC2D2A to 10% and collectively for w50% of patients with mutations. No mutation-specific genotypeephenotype JS, indicating that many genes are yet to be iden- correlations could be identified, but the findings confirm tified. In addition to the phenotypic overlap the observation that mutations that cause CC2D2A- between ciliopathies, significant genetic overlap is related JS are predicted to be less deleterious than observed, particularly between JS and Meckel mutations that cause CC2D2A-related Meckel syndrome. syndrome (MKS), which is characterised by ence- Missense variants in the coiled-coil and C2 domains, as phalocele, cystic/dysplastic kidney disease, ductal well as the C-terminal region, identify these regions as plate malformation of the liver, polydactyly and, important for the biological mechanisms underlying JS. typically, fetal or neonatal lethality.21 Of the 13 Conclusions CC2D2A testing should be prioritised in genes responsible for JS, six also cause MKS patients with JS and ventriculomegaly and/or seizures. (CC2D2A,21 CEP290,22 RPGRIP1L,23 TCTN2,24 Patients with CC2D2A-related JS should be monitored TMEM6712 and TMEM21614), leading to the for hydrocephalus and seizures. concept that some forms of JS and MKS represent mild and severe presentations of the same biological disorder. Consistent with this concept, patients with MKS more often have truncating mutations INTRODUCTION in CC2D2A, and patients with JS more often have Joubert syndrome (JS) is a recessive disorder char- at least one missense mutation.21 Of note, no other acterised by cerebellar vermis hypoplasia, thick- genotypeephenotype correlations have been ened, mis-oriented superior cerebellar peduncles reported in patients with CC2D2A mutations. and an abnormally deep interpeduncular fossa CC2D2A mutations represent a major cause of resulting in the pathognomonic ‘molar tooth sign’ both JS and MKS since they are responsible for (MTS) on axial imaging of the mid-hind brain w10% of each disorder.4 721CC2D2A encodes

126 J Med Genet 2012;49:126e137. doi:10.1136/jmedgenet-2011-100552 e Genet Med J Table 1 Summary of phenotypic information in subjects with CC2D2A mutations, single heterozygous variants, and variants of unclear functional significance identified in this cohort CC2D2A CC2D2A P2 score, Ctrl allele % Age Identification no mutations, cDNA mutations, Div/Var EU/AA (years) MTS EC DD/ ID Sz A/T Nyst, OMA RD Kidney Liver PD Other

2012; CC2D2A mutations and phenotypic summary UW36-37 c.3364C>T homM,P p.P1122S 1.0/0.99 0 8 + e + e + eeeee 49 UW41-37 c.2848C>T homM,P p.R950X hom 04+e + ee ++eeeCortic vis imp :126 UW46-1 N c.3289 delGP p.V1097Ffs*1 ND 19 + e + ee + ee e e e c.4289T>CM p.V1430A 1.0/0.86 0 3.doi:10.1136/jmedgenet-2011-100552 137. UW46-2 N c.3289 delGP p.V1097Ffs*1 ND 22 + e + ee + ee e eScoliosis c.4289T>CM p.V1430A 1.0/0.86 0 UW47-37 c.3055C>TM p.R1019X 0 19 + e ++++ ee e e Downloaded from c.3288G>CP p.Q1096H 0.64/0.12 0 UW48-37 c.3364C>T homM,P p.P1122S 1.0/0.99 0 7* + e + e + + + + (ESRF/TXPT) eeScoliosis UW49-330 c.3289 delGM p.V1097Ffs*1 ND 24 + e ++++ e + + (TXPT) e Coloboma c.4582C>TP p.R1528C 1.0/0.97 0 COACH UW50-37 c.4582C>T homM,P p.R1528C hom 1.0/0.97 0 12 + e + ee e ee e eSibling w/ EC UW50-67 c.4582C>T homM,P p.R1528C hom 1.0/0.97 0 8 + + + ee e ee e e jmg.bmj.com UW67-330 c.3145C>T p.R1049X 0 7+e + e ++ e + + fibrosis e c.3347C>T p.T1116M 1.0/0.97 0.03/0 UW75-3N c.1676T>CM p.L559P 1.0/0.99 0 18 + e + e ++ ee e e c.3892_3 del(GT)P p.V1298Ffs*16 ND N P UW76-3 c.3134T>C p.V1045A 0.98/0.6 0 3+e + e + eeeee onJanuary18,2012-Publishedby c.3850C>TM p.R1284C 1.0/1.0 0 UW78-3 N c.3055C>TM p.R1019X 0 1+e + ee + e ND ND e c.4667A>TP p.D1556V 0.98/0.74 0.03/0.03 UW79-3 N c.(1263_4InsGGCATGT p.S423Gfs*19 ND 26 + e + e ++ ee e e TTTGGC; c.1268G>A)P p.V1151A 1.0/0.39 0 c.3452T>CM 0 UW79-4 N c.(1263_4InsGGCATGT p.S423Gfs*19 ND 24 + e ++++ ee e e TTTGGC; 1268G>A)P p.V1151A 1.0/0.39 0 c.3452T>CM 0 UW80-3 c.3289 delG p.V1097Ffs*1 ND 1+e +++ND e + (cysts) ND e c.3347C>T p.T1116M 1.0/0.97 0.03/0 UW81-3 N c.4179+1delG p.E1393Efs*1 ND 11 + e + ee + ee e e c.4667A>T p.D1556V 0.98/0.74 0.03/0.03 N P correlations Genotype-phenotype UW82-3 c.3976-3C>A p.IVS30(-3) 0 8+e ++++ ee e e group.bmj.com c.4844_4847delCTCTM p.S1615Lfs*15 ND UW102-3 N c.3772-1G>TP p.IVS29(-1) 0 4+e + e + eeeeND Cortic vis imp c.4582C>TM p.R1528C 1.0/0.97 0 UW103-3 N c.3289delG p.V1097Ffs*1 ND 3+e + ee e ee e eSibling w/ MKS c.3851G>A p.R1284H 1.0/1.0 0 n¼20 subjects from 17 families 20/20 1/20 20/20 5/20 12/20 13/19 2/20 4/19 2/18 0/19

Single heterozygous changes and variants of unclear significance UW06-3 N c.351T>G p.S117R 0.84/0.36 0.2/0.07 14 + e + +F + + + + (ESRF, TXPT) eeVPI UW54-3 c.1519A>G p.K507E 0.99/0.70 0.8/0.08 10 + e + e ++ e + (ESRF, TXPT) + (TXPT) e Coloboma TMEM67 UW88-3 N c.3989G>A p.R1330Q 1.0/0.94 0 10 + e + ee + ee e e UW88-4 N c.3989G>A p.R1330Q 1.0/0.94 0 13 + e + ee e ee e e UW106-3 c.4667A>T p.D1556V 0.98/0.74 0.03/0.03 4 + e + ee e ee e e

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a 1620 amino acid protein containing coiled-coiled domains and a C2 domain. Coiled-coil domains are involved in proteineprotein interactions and are very common among ciliary proteins, while C2 domains are phospholipid-binding ND TCTN2 Dextro-cardia domains and have been implicated in calcium-dependent vesicle fusion and other membrane interactions.25 e e The present study focuses on genotype phenotype analysis of 20 subjects with JS caused by mutations in both copies of CC2D2A. Most subjects carried at least one missense mutation, and these mutations clustered in the C2 domain and C-terminal ND + Coloboma region. Given the genetic heterogeneity of JS, correlations between the causative and the clinical presentation can guide diagnostic testing strategies and monitoring for compli- variants are listed in the lower part of the table. Control cations in specific patients. Our data suggest that CC2D2A testing should be prioritised in patients with ventriculomegaly CC2D2A eee and/or seizures and patients with CC2D2A mutations should be monitored for these complications. In the future, these data will fi eeee e e e Twins e ee e e ee ee e e be essential for interpreting variants identi ed through genome- wide approaches such as whole-exome or whole-genome sequencing.26 + + +NDNDND METHODS Subjects In cooperation with the Joubert Syndrome and Related Disor- ND + + ND ND ND Sibling with MTS ++ + ND ND + ND + +NDND ders Foundation and clinical collaborators throughout the world, the University of Washington Joubert Center has enrolled >300 ee ee e ee e e e families with JS under approved protocols at the University of Washington and Seattle Children’s Hospital. All participants or their legal representatives provided written informed consent. + + + + ++++ + + + + +NN + + Inclusion criteria were: (1) MTS on brain imaging (or cerebellar vermis hypoplasia on CT scan when an MRI was not available); e e e e e e e e e (2) clinical findings of JS (intellectual impairment, hypotonia, mutations of unclear functional significance and single heterozygous ataxia).

CC2D2A Clinical and imaging data Clinical information was collected using a structured intake 1* + 1+ Age (years) MTS EC DD/ ID Sz A/T Nyst, OMA RD Kidney Liver PD Other < 13 + < 6+ form and review of medical records. Seizure status was based on documentation of seizures in medical records and/or parental report of seizures. At the time of enrolment, we reviewed brain MRI and/or CT scans to confirm the MTS and to evaluate for ventriculomegaly, agenesis of the corpus callosum, poly- 01ND Ctrl allele % EU/AA 0.01/2.4 0/0.5 3/ND 0/0.03 3/ND 0.03/0.03 microgyria, heterotopias and occipital encephalocele. When MRI or CT images were not available, we abstracted information from the MRI or CT report. 0.99/0.700.99/0.70 0.8/0.08 0.8/0.08 2 2 + + P2 score, Div/Var 0.05/0.02 0 0.004/0.005 0.003/0.004 0.98/0.74 Genetic mapping and mutation identification In consanguineous families, we first excluded known JS loci using homozygosity mapping as previously described.7 Known JS genes within heterozygous intervals in a subject were considered excluded, while genes present in homozygous inter- vals were sequenced. The common NPHP1 deletion was evalu- CC2D2A mutations, protein p.K507E p.K507E p.L701V p.V660I p.L684I p.E229del p.IVS11(+1) p.E229del p.D1556V ated by microsatellite genotyping as previously described.27 AHI1, ARL13B, CC2D2A, CEP290, OFD1, RPGRIP1L, TCTN2, TMEM67/MKS3 and TMEM216 were sequenced in the subjects

A without a known genetic aetiology at the time each gene was , mutation not previously reported. >

N 7 G G T A TG p.T1116M 1.0/0.97 0.03/0 2 + A T p.R1019X discovered as previously described ; therefore not all genes have G p.S117R 0.84/0.36 0.2/0.07 2 + > > > > > > > > > been sequenced in all families. Clinical genetic testing results were also included in the analysis. Segregation of mutations c.3055C c.3347C c.2101C c.351T c.1978G c.2050T c.685_687delGAA c.1017+1G CC2D2A mutations, cDNA c.4667A within a family was evaluated when parental and/or sibling DNA was available. PolyPhen-228 was used to determine the , maternal alleles; N ND N M Continued N N N likelihood that mutations were deleterious. In subjects harbouring a single heterozygous CC2D2A variant predicted to be deleterious, we performed array comparative UW107-3UW107-4UW108-3 c.1519A c.1519A UW110-3 UW109-3 UW104-3 UW77-3 UW111-3 UW105-3 c.685_687delGAA Deceased. , paternal; allele frequency is based on exome data (see Methods: Controls). UW50-3 and UW50-6 are first cousins; all other subjects sharing the same UW number are siblings. Nonsense or frameshift mutations are in bold. MutationsP predicted as benign by Polyphen-2 are italicised. Subjects with * A/T, apnoea/tachypnoea; cortic vis imp,end cortical visual stage impairment; renal Ctrl failure; alleleindicated); freq hom, PD, % homozygous; EU/AA, polydactyly; control F, RD, allele febrile frequency retinal seizures; as dystrophy; percentage MKS, Sz, in Meckel European seizures; Americans/African syndrome; TXPT, Americans; MTS, DD/ID, transplant; molar developmental VPI, delay/intellectual tooth velopalatal disability; sign; del, insufficiency. NN, deletion; EC, neonatal encephalocele; seizures; ESRF, ND, not documented; Nyst, nystagmus; OMA, oculomotor apraxia; P2 score, Polyphen-2 scores (both ‘Hdiv and Hvar’ are

Table 1 Identification no genomic hybridisation to identify deletions or duplications

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Genotype-phenotype correlations

Figure 1 CC2D2A protein schematic and variant summary. Schematic of the CC2D2A protein showing the coiled-coil domains (CC) and the C2 domain (C2). (A, B) Missense variants and in-frame deletions reported in other cohorts (A) and in our cohort (B) are indicated above the protein. (C, D) Truncating mutations (nonsense, frameshift) and splice site mutations detected in our cohort (C) and in other cohorts (D) are indicated below the protein. Novel variants from the current study are indicated (N). The number of times a specific variant was identified is indicated before the variant (n3). Variants reported in patients with Joubert syndrome are in bold. Missense variants predicted to be damaging by Polyphen-2 are black, while common variants and missense variants predicted to be benign are depicted in grey. Variants identified only as single heterozygous changes are indicated (het). within the CC2D2A gene. Genomic DNA was labelled and fied by clinical testing, bringing the number of subjects with hybridised as previously described29 to a custom array with CC2D2A-related JS to 20. high-density probe coverage in the CC2D2A gene (average probe spacing within the gene of 41 bp). Causative CC2D2A mutations Detailed mutation analysis of all 17 CC2D2A families revealed Controls 12 different missense mutations (five novel), eight nonsense or The frequency of missense variants in subjects without severe frameshift mutations (three novel), and two novel splice-site congenital disorders was examined using data available through mutations (table 1). Missense mutations were clustered in the the NHLBI Exome Sequencing Project, Seattle, WA (URL: C2 domain (amino acids 1020e1205) and in the C-terminal http://snp.gs.washington.edu/EVS/) (accessed 29 November region. All of these mutations were present in <1% of controls 2011). For the p.E229del variant, we sequenced exon 9 in 96 drawn from the publicly available Exome Variant Server (URL: European American control samples. http://snp.gs.washington.edu/EVS), and familial segregation of the mutation was consistent with recessive inheritance when RESULTS parental samples were available (table 1). In 10/17 families, one Prevalence of CC2D2A mutations in JS missense mutation was inherited with a truncating mutation We identified causative CC2D2A mutations in 16 of 166 families (nonsense or frameshift), while in 4/17 families, two missense from our JS cohort (19 individuals; table 1). To calculate the mutations were found. In one family, a splice-site mutation was prevalence of causative CC2D2A mutations in our cohort, we inherited with a frameshift mutation, and in another family used as a denominator the number of families sequenced for a different splice-site mutation was associated with a missense CC2D2A (n¼166) + the number of families excluded at this mutation. Only one subject harboured a homozygous nonsense by single-nucleotide polymorphism mapping (n¼16) + the mutation. Most mutations are unique or recur infrequently number of families not sequenced for CC2D2A because muta- between different families (figure 1), and we observed very little tions in another gene had been identified as causative (n¼27). overlap between mutations in our cohort compared with Therefore the prevalence of CC2D2A-related JS in our cohort previously published patients (figure 1 and table 2). In summary, is 7.7% (16/209). In addition to these 16 families, we later CC2D2A mutations in JS recur rarely and generally include at enrolled one family (UW103) with CC2D2A mutations identi- least one missense mutation in the C2 domain or the C-terminal

J Med Genet 2012;49:126e137. doi:10.1136/jmedgenet-2011-100552 129 130 eoyepeoyecorrelations Genotype-phenotype

Table 2 Summary of all published CC2D2A mutations, single heterozygous variants, and variants of unclear significance with phenotypic information CC2D2A CC2D2A Ctrl allele Identification mutations, cDNA mutations, protein P2 Div/Var % EU/AA Dx MTS EC DD/ID OMA Nyst RD Kidney Liver PD Reference Review of all reported CC2D2A mutations/variants with phenotypic summary Noor et al31 c.2338+1G>C p.V728Efs*13 ND JS + e ++ + NDNDe Noor et al31 F871 c.4652T>C hom p.L1551P 0.86/0.27 0 JS CVH e ++ e + eeGorden et al7 Downloaded from F434-21 c.517C>T p.R173X 0.01/0 JS CVH e ++ eeeeOtto et al32 c.1676T>C p.L559P 1.0/0.99 0 JBS-006 c.2161C>T hom p.P721S 1.0/0.99 0 JS + e ++ ND eeeMougou-Zerelli et al21 JBS-008 c.3341C>T p.T1114M 1.0/0.99 0 JS + e ++ ND eeeMougou-Zerelli et al21 c.4667A>T p.D1556V 0.98/0.74 0.03/0.03 A2421-21 c.3544T>C p.W1182R 1.0/0.99 0 MKS NA + NA NA NA + + ND Otto et al32 jmg.bmj.com c.3774_5insT p.E1259fs*1 ND Tallila et al33 c.1762C>T p.V587Efs*29 0 MKS NA + NA NA NA + + + Tallila et al33 MKS-84D c.3399_3975del hom p.A1134Nfs*9 ND MKS NA OM NA NA NA + + e Mougou-Zerelli et al21 MKS-54 c.517C>T hom p.R173X 0.01/0 MKS NA + NA NA NA + + + Mougou-Zerelli et al21 21 MKS-160 c.3399e3C>A hom IVS27(-3) 0 MKS NA + NA NA NA + ND + Mougou-Zerelli et al onJanuary18,2012-Publishedby MKS-414 c.3584delT hom p.F1195Sfs*11 ND MKS NA + NA NA NA + ND + Mougou-Zerelli et al21 MKS-977 c.3084delG hom p.K1029Rfs*3 ND MKS NA + NA NA NA + ND e Mougou-Zerelli et al21 MKS-011 c.3145C>G hom p.R1049X 0 MKS NA OM NA NA NA + + + Mougou-Zerelli et al21 MKS-143 c.3522_3523insTG p.H1175Cfs*13 ND MKS NA OM NA NA NA + ND + Mougou-Zerelli et al21 c.4496+2T>A IVS36(+2) 0 MKS-982 c.1538T>A p.W513X 0 MKS NA Anencep NA NA NA + + + Mougou-Zerelli et al21 c.4179+1delG IVS33(+1) ND MKS-365 c.2673C>T p.R925X 0 MKS NA OM NA NA NA + + + Mougou-Zerelli et al21 c.2486+1G>C IVS20(+1) 0 e Genet Med J

Single heterozygous changes and variants of unclear significance A3208-21 c.1519A>G het p.K507E 0.99/0.7 0 JS ND ND ND ND ND + ND ND Otto et al32 A1421-21 c.3056G>A het p.R1019Q 1.0/1.0 0 JS ND ND ND ND ND + ND ND Otto et al32 2012; MKS-987 c.1339delG het p.A447Rfs*11 ND MKS NA + NA NA NA + + + Mougou-Zerelli et al21 group.bmj.com 21 49 MKS-692 c.834delG het p.L279Cfs*40 ND MKS NA DW NA NA NA + + + Mougou-Zerelli et al

:126 F16-21 c.1519A>G het p.K507E 0.99/0.79 1.0/0.07 SLNS ND ND ND ND + (ND) + ND ND Otto et al32 32 e A559-22 c.2161C>T het p.P721S 1.0/0.99 0 NPHP ND ND ND ND ND + ND ND Otto et al 3.doi:10.1136/jmedgenet-2011-100552 137. A944-21 c.2161A>T het p.P721S 1.0/0.99 0 NPHP ND ND ND ND ND + ND ND Otto et al32 A1347-21 c.2161C>T het p.P721S 1.0/0.99 0 NPHP ND ND ND ND ND + ND ND Otto et al32 A2426-21 c.685_7delGAA p.E229del 3/ND MKS NA + NA NA NA + e ND Otto et al32 c.3893T>A p.V1298D 1.0/0.98 0 Nonsense or frameshift mutations are in bold. Anencep, anencephaly; Ctrl allele freq % EU/AA, control allele frequency as percentage in European Americans/African Americans; CVH, cerebellar vermis hypoplasia; DD/ID, developmental delay/ intellectual disability; DW, DandyeWalker; Dx, diagnosis; EC, encephalocele; het, heterozygous; hom, homozygous; JS, Joubert syndrome; MKS, Meckel syndrome; MTS, molar tooth sign; NA, not applicable; ND, not documented; NPHP, ; Nyst, nystagmus; OM, occipital meningocele; OMA, oculomotor apraxia; PD, polydactyly; RD, retinal dystrophy; SLNS, SenioreLøken syndrome. Downloaded from jmg.bmj.com on January 18, 2012 - Published by group.bmj.com

Genotype-phenotype correlations region of the protein, combined with a second missense or oculomotor apraxia were reported in 13/19 (68%). Retinal a truncating mutation. dystrophy was found in 2/20 subjects (10%), kidney disease in 4/ 19 (21%) and liver disease in 2/18 (11%), with one subject requiring Heterozygous CC2D2A variants a kidney transplant and another requiring a liver transplant. The We identified nine families with heterozygous CC2D2A variants: subject with severe liver disease requiring transplantation also had in seven families, only a single heterozygous CC2D2A variant kidney disease and chorioretinal coloboma and was previously was identified (one nonsense and six missense variants, of reported to have a diagnosis of COACH syndrome.30 No subjects which five were predicted to be deleterious by Polyphen-2). with CC2D2A mutations had polydactyly. Although these Variants p.D1556V, p.T1116M and p.R1019X were also identi- frequencies of organ involvement are somewhat lower than what fied in compound heterozygosity with a second deleterious is generally reported in the literature for JS,4534the number of CC2D2A variant in other subjects (table 1), while variants subjects is too small to draw definitive conclusions. p.R1330Q, p.K507E, p.S117R and p.L701V were found only in the heterozygous state. None of these were listed in dbSNP, and Enrichment for abnormal neuroimaging findings, in particular their frequency in control samples ranged from 0% to 0.8%. ventriculomegaly, in CC2D2A-related JS Deletions (or duplications) of the second CC2D2A allele were In addition to the MTS, a variety of abnormal neuroimaging not identified by high-density custom array comparative features are rarely reported in patients with JS.35 We reviewed genomic hybridisation. Between seven and eight other known JS full brain MRI studies from 13 of our 20 subjects with CC2D2A- genes were sequenced in these families without identification of related JS, and identified a variety of additional neuroimaging the genetic cause. Two families carried a predicted damaging abnormalities (table 3 and figure 2). Based on the imaging review CC2D2A variant in the heterozygous state along with causal and information from medical records, 13/17 (76%) subjects mutations in both alleles of another JS gene: family UW111 with CC2D2A mutations had ventriculomegaly (figure 2A), carried variant p.S117R in CC2D2A (Polyphen-2 score 0.507) compared with 21/105 (20%) subjects without CC2D2A muta- along with two causative mutations in TCTN2 (and one tions (p<0.0001, Fisher exact test). Three subjects had hydro- predicted damaging mutation in ARL13B). Family UW54 carried cephalus requiring ventriculoperitoneal shunting, and one variant p.K507E in CC2D2A (Polyphen-2 score 0.859) and two subject without ventriculomegaly (UW47-3) had a small but causative mutations in TMEM67/MKS3. clear increase in ventricle size between 3 and 11 years of age (based on imaging at these ages). CC2D2A variants of unclear significance Additional infratentorial abnormalities included asymmetrical In one family (UW77), we identified two missense variants that cerebral peduncles (n¼2), occipital cephalocele (n¼1), posterior were both predicted to be benign by Polyphen-2: neither L684I fossa fluid collections (two retrocerebellar (figure 2D), and one nor V660I (rs16892134) are common in European American originating in the 4th ventricle and herniating through the control but occur in 0.5% and 2.4% of African tentorium (figure 2C)), heterotopias (two interpeduncular, one American controls, respectively. Five additional JS genes were inferior to the pons (figure 2B), one cerebral peduncle), foramen sequenced in this family with negative results. Family UW79 magnum cephalocele (n¼2), and abnormal dorsal prominence at harboured a missense variant (c.1268G>A) downstream of a 13 the cervicomedullary junction (n¼2). Additional supratentorial bp insertion on the same allele, making it impossible to evaluate abnormalities included agenesis and/or dysgenesis of the corpus the effect of the missense variant. callosum (one subject with complete agenesis, two with posterior In 17 families, we identified a 3 bp deletion in CC2D2A agenesis (figure 2C), four with global thinning), polymicrogyria (c.685_7 delGAA) resulting in loss of a glutamic acid at position (one with seizures, one without seizures), cortical heterotopias 229 (p.E229del). This variant, which is listed in dbSNP version (two subependymal (figure 2A), one transmantle), increased T2/ 134 (rs112367037), was previously reported as causative for FLAIR signal in the white matter (n¼3), and malformed hippo- MKS, in association with a missense mutation (V1298D)32; campi (n¼1). Three subjects also had striking dolichocephaly. In however, we detected the p.E229del variant in 3% of control chromosomes, indicating that it is a polymorphism. Moreover, CC2D2A two of the families carrying this variant also had deleterious Table 3 Neuroimaging features in subjects with -related JS mutations in both alleles of another JS gene, explaining their Poretti Poretti CC2D2A 35 35 phenotype. Nonetheless, it is possible that this variant has Feature 2011 JS 2011 OFD some functional effect given that two other families with the Supratentorial findings p.E229del variant (UW104 and UW105) also harboured a second Ventriculomegaly 13/17 4/71 2/4 CC2D2A variant strongly predicted to be damaging (IVS11(+1) Agenesis of the corpus callosum 1/13 1/71 0/4 G>A and D1556V), and four more families harboured single Dysgenesis of the corpus callosum 6/13 5/71 0/4 heterozygous mutations in other JS genes. In five other families, Polymicrogyria 3/12* 1/71 3/4 we detected another common variant (E376A, rs16892095), but Periventricular nodular heterotopias 2/13 2/71 1/4 this variant was present in 2.3% of combined European and Transmantle heterotopias 1/13 0/71 0/4 African American controls. White matter increased T2/FLAIR signal 3/12 ND ND Infratentorial findings Highly variable phenotypic presentation Asymmetrical cerebral peduncles 2/13 10/71 2/4 Occipital cephalocele 1/13 8/71 1/4 of CC2D2A-related JS CC2D2A Retrocerebellar fluid collection 2/13 14/71 3/4 Phenotypic features of JS in the 20 individuals with - Brainstem and midbrain heterotopias 3/13y 3/71 0/4 related JS were drawn from medical records, direct examination Foramen magnum cephalocele 2/13 5/71 1/4 and/or family intake forms (table 1). The MTS and developmental Prominence at cervicomedullary junction 2/13 2/71 1/4 delays/intellectual disability were present in all individuals. Alter- *Imaging was not sufficient to identify or exclude polymicrogyria in one imaging study. nating apnoea/tachypnoea was reported in 12/20 subjects (60%), yOne interpeduncular, one cerebral peduncle, one pontomedullary. and abnormal eye movements including nystagmus and/or JS, Joubert syndrome; OFD, oral-facial-digital syndrome.

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Figure 2 Selected atypical neuroimaging features in subjects with CC2D2A-related Joubert syndrome. (A) Ventriculomegaly and multiple subependymal heterotopias (black arrowheads) in UW103-3. (B) Interpeduncular and infrapontine heterotopias (black arrows), dysplastic tectum (asterisk) and abnormal prominence of the cervicomedullary junction (bracket) in UW82-3. (C) Agenesis of the posterior corpus callosum (bracket indicates remaining corpus callosum) and severe inferior cerebellar ectopia (white arrowhead) in UW49-3. Note the 4th ventricular cyst herniating superiorly through the tentorium (area of low signal around the asterisk) and the inferior pseudomeningocele due to prior posterior fossa decompression surgery (area of low signal around the white arrowhead). (D) Retrocerebellar fluid collection (asterisk) and foramen magnum cephalocele (white arrow) in UW79-4. The “A” is on the original MRI image and refers to Anterior. MRI sequences are as follows: (A) axial T2-weighted, (B and D) sagittal T2-weighted and (C) sagittal T1-weighted. addition, fetal siblings of UW50-3 and UW103-3 were reported to a rare finding in patients with JS, were reported in 5/17 families have encephaloceles. In summary, ventriculomegaly and other with CC2D2A mutations (27%) (figure 3A). Seizure status in all neuroimaging findings rarely observed in patients with JS were subjects was based on information available at the time the substantially enriched in subjects with CC2D2A-related JS. subject was enrolled (parental report and/or medical records). To reduce potential bias due to selective inclusion of subjects with Enrichment for seizures in CC2D2A-related JS CC2D2A mutations, we repeated the analysis using only the In addition to the striking enrichment of ventriculomegaly in families enrolled at the time we sequenced the entire cohort for subjects with CC2D2A-related JS, we noted that seizures, also CC2D2A and for whom CC2D2A status had been clarified (by

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mutations) had a single seizure immediately after liver trans- plant, thought to be secondary to high ciclosporin levels. Finally, the diagnosis of seizures was considered questionable in two patients, where no formal diagnosis had been made and where the parental description appeared ambiguous. On the basis of this additional information, a seizure disorder was present in 4/16 families with CC2D2A mutations and 12/193 families without CC2D2A mutations (p¼0.024, Fisher exact test), while six subjects (one with CC2D2A mutations) were excluded because they only had neonatal or provoked seizures. Of the 16 subjects with a bona fide seizure disorder and known CC2D2A mutation status, four had definitive CC2D2A mutations, one had compound heterozygous CC2D2A variants of unknown significance (UW77-3), one each had causal muta- tions in TMEM216, NPHP1 and AHI1, and eight did not have a known cause (figure 3A).

Neurological and neurodevelopmental outcome in CC2D2A-related JS Since subjects with JS due to CC2D2A mutations have a higher prevalence of seizures, ventriculomegaly and other neuroimaging findings, we further investigated their neurological and neuro- developmental outcomes. For this purpose, we sent a basic developmental survey to all 17 families with CC2D2A muta- tions, leading to 13 responses (survey available upon request). We found a wide range of developmental outcomes, similar to previous descriptions of outcomes in patients with JS (table 4). Independent walking was achieved in 8/17 subjects (age range e Figure 3 Seizures in Joubert syndrome (JS). (A) Pie chart indicating 2 9 years). A surprisingly high number of parents reported the relative contributions of different genes in subjects with seizures. involuntary movements (5/20 subjects) and tics (3/20 subjects). Absolute numbers are indicated for each gene. ‘Unknown aetiology’ Verbal communication was achieved in 7/15 subjects, but two of indicates that the underlying genetic cause has not been identified. (B) these seven subjects also used sign language. Sign language was Bar graph showing the different types of seizures in subjects with JS. the main mode of communication for 4/15 subjects, and an Black bars indicate CC2D2A subjects, and white bars indicate the rest of augmentative communication device or a picture communica- the subjects with seizures. ‘Unknown’ indicates that the seizure type tion system was used in 3/14. One subject was non-verbal, but could not be determined. no additional information about other modes of communication was available. All subjects for whom we have educational sequencing (n¼166), mapping (n¼16) or if another causative information were enrolled in special education, with the highest gene was identified (n¼27) for a total of 209 subjects with functioning individual attending post-secondary school. Behav- known CC2D2A status). In this analysis, seizures were present ioural problems reported by the families included aggression/ in 5/16 families with CC2D2A mutations while they were violence (n¼4), temper outbursts (n¼1), anxiety (n¼1) and reported in 17/193 families without CC2D2A mutations autism spectrum disorder (n¼2). Additional neurological prob- (p¼0.0163, Fisher’s exact test). lems reported included recurrent facial palsy, cortical blindness To gain additional information on seizure type, age of onset and hemifacial spasms (table 4). In summary, the increased and severity, we sent a survey to all subjects with reported prevalence of ventriculomegaly and seizures in CC2D2A-related seizures (survey available upon request). Sixteen of 22 surveys JS does not appear to be associated with a markedly increased were returned, which, in combination with the previously frequency/severity of other neurological features or an obviously available records, enabled classification of the seizures for 19 of poorer developmental outcome compared with published e 22 subjects (figure 3B). In the remaining three subjects, we reports.4 36 38 were unable to obtain any additional information beyond the initial report of seizures (classified as ‘unknown’ seizure type in figure 3B). We observed a variety of seizure types among the 22 DISCUSSION patients with seizures, regardless of the genotype. In the Mutations in CC2D2A account for 7.7% of JS in our large cohort CC2D2A subgroup, we observed complex partial seizures in and for 10% of MKS,21 highlighting the importance of this gene two subjects, primary generalised seizures in one subject, and in human ciliopathies. As a consequence, genotypeephenotype unknown seizure type in one subject (table 4). In non-CC2D2A correlations for CC2D2A mutations are of direct use in patient subjects, absence seizures and simple partial seizures were also care, especially for JS, since genotypeephenotype correlations reported. After analysis of survey responses, several subjects did can guide genetic testing, optimise monitoring for medical not meet criteria for a seizure disorder: three subjects had complications and, ideally, provide better prognostic informa- seizures exclusively in the neonatal period or in infancy and tion for newly diagnosed patients. In the future, genotypee were currently seizure-free off medication; two other subjects phenotype information will be critical for the interpretation of had clearly provoked seizures: one (without CC2D2A muta- variants identified by genome-wide sequencing technologies that tions) had febrile seizures and the second (with CC2D2A will soon make their way into clinical use.

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Table 4 Neurological features and neurodevelopmental outcome of CC2D2A- related Joubert syndrome Downloaded from Age Identification no (years) MRI findings VM/HC Ataxia Walking Speech Education Seizures Behaviour Other neuro UW36-3 8 MTS, malformed hippocampi VM Yes Wheelchair, ND High school cert, Sp Ed e Aggressive e household ambulator UW41-3 9 MTS VM Yes Walking with aid Non-verbal Preschool, Sp Ed e Aut behaviour Stereotyped mvts UW46-1 19 MTS VM Yes Indep age 3 years Verbal Reads in KG, Sp Ed ee Involuntary mvts, tics UW46-2 22 MTS VM Yes Indep age 6 years Signs College Sp Ed ee e jmg.bmj.com UW47-3 19 MTS e Yes Indep age 10 years Signs Lifeskills program Complex partial Anxiety e UW48-3 7* MTS, perivent WM abnl VM ND ND ND ND ee e UW49-3 24 MTS, partial ACC HC, VP shunt Yes Wheelchair, walks Verbal Mainstreamed Sp Ed yes (provoked, Violent outbursts e

a few steps single post-txpt) onJanuary18,2012-Publishedby UW50-3 12 MTS, FMC e Yes Indep age 9 years Partially verbal Preschool Sp Ed ee Involuntary mvts, tics UW50-6 8 MTS, ACC, small EC VM Yes Not walking Non-verbal Preschool Sp Ed ee Involuntary mvts, tics UW67-3 7 MTS, PMG, TM het, MB het VM ND Walker Non-verbal, ACD ND ee e UW75-3 18 MTS ND Yes Indep age 4 years Verbal Mainstreamed Sp ed ee Hemifacial spasms (unsteady) UW76-3 3 MTS ND ND Not walking age 3 year Signs NA ee Recurrent facial palsy UW78-3 1 MTS e Yes ND ND ND ee e UW79-3 26 MTS ND Yes Indep age 5 years Verbal and signs Sp Ed e Aggressive Involuntary mvts e Genet Med J UW79-4 24 MTS, CMP, FMC VM Yes Indep age 7 years Verbal and signs Sp Ed Primary generalised Aut behaviour, violence Involuntary mvts UW80-3 1 MTS HC, VP shunt ND ND ND ND yes (type ND) ND e UW81-3 11 MTS e Yes Indep age 2 years Verbal Primary school Sp Ed e Temper outbursts e UW82-3 8 MTS, Nod het, BS het, VM Yes Walker Non-verbal, ACD Preschool Sp Ed Complex partial ee 2012; MB het, CMP group.bmj.com UW102-3 4 MTS, MB het HC, VP shunt ND Not walking Signs ND ee Cortical visual impairment 49

:126 UW103-3 3 MTS, PMG, Nod het VM Yes Not walking age 3 years Non-verbal, NA ee e picture comm e

3.doi:10.1136/jmedgenet-2011-100552 137. *Deceased.

ACC, agenesis of the corpus callosum; ACD, augmentative communication device; Aut behaviour, autistic behaviour; BS het, brain stem heterotopia; cert, certificate; CMP, cervicomedullary prominence; EC, encephalocele; FMC, foramen magnum cephalocele; Indep, independent; KG, kindergarten; MB het, midbrain heterotopia; MTS, molar tooth sign; mvts, movements; NA, not applicable; ND, not documented; Nod het, nodular heterotopias; perivent, periventricular; picture comm, picture communication system; PMG, polymicrogyria; Sp Ed, special education; TM het, transmantle heterotopia; txpt, transplant; VM/HC, ventriculomegaly/hydrocephalus; VP, ventriculoperitoneal; W, Walker; WM, white matter. Downloaded from jmg.bmj.com on January 18, 2012 - Published by group.bmj.com

Genotype-phenotype correlations

Genotypeephenotype correlations truncating mutations in our JS subjects are located in the C- Our work identifies two neurological features uncommon in JS, terminal region of the protein, suggesting that at least some namely ventriculomegaly and seizures, as significantly enriched partially functional protein might be produced. This contrasts in subjects with CC2D2A-related JS. In particular, ventriculo- with the finding that patients with MKS have two truncating megaly was much more common in subjects with CC2D2A- mutations, and that the majority of these are within or before related JS compared with our other subjects and previously the C2 domain, indicating that functional protein is unlikely to published patients.35 At least some of the ventriculomegaly is be produced.21 Therefore patients with CC2D2A-related JS are due to hydrocephalus (in three subjects), but we also have likely to have some residual CC2D2A protein function, whereas evidence for very slow volume loss on serial MRIs in one subject. patients with MKS are more likely to have severe loss-of-function Given the small sample size, the associations between CC2D2A or complete absence of CC2D2A. mutations and ventriculomegaly/seizures will need to be vali- dated in other JS cohorts; however, our findings indicate that Impact of genetic modifiers on phenotype genetic testing should be preferentially directed towards Reliable genotypeephenotype correlations are rare in the clini- CC2D2A sequencing in patients with ventriculomegaly/hydro- cally and genetically heterogeneous ciliopathies. Nevertheless, cephalus and/or seizures. Conversely, the threshold for seizure causative mutations in some genes appear to be more highly work-up and neurological consultation should be low when associated with specific features, as highlighted by the associa- seizures are suspected in patients with CC2D2A-related JS. tion of TMEM67 mutations with liver involvement41 and of Despite the more prevalent neuroimaging abnormalities and CC2D2A mutations with ventriculomegaly and seizures (present seizures in CC2D2A-related JS, these subjects did not have study). Moreover, the severity of the causative mutations markedly more severe neurodevelopmental outcomes, nor did correlates at least in some cases with the severity of the they have more common involvement of the retina, kidneys and phenotypic presentation (CC2D2A mutations leading to MKS liver. The one possible exception to this conclusion is the vs JS). Beyond these associations, major phenotypic variability is recurrent reports of involuntary movements and tics, which are observed. This could be explained by a prominent hypothesis in inconsistently reported in patients with JS. the field that proposes that the phenotype in a given patient results from the sum of the effects of variants in ciliary (and Limitations other) genes, also termed ‘mutational load’.42 Support for this This study has several limitations that are typical for research hypothesis comes from BardeteBiedl syndrome, which in some e on the natural history of rare disorders. Although we report instances exhibits oligogenic inheritance.43 45 For JS and its the largest number of subjects with CC2D2A-related JS to allelic disorders (ie, nephronophthisis, Leber congenital amau- date, the numbers remain small. In addition, known and rosis, SenioreLøken syndrome and MKS), specific variants in unknown ascertainment biases are likely. For instance, families RPGRIP1L and AHI1 have been proposed to act as genetic of children with more severe disease may be more likely to enrol modifiers of retinal disease risk.46 47 Similarly, MKS1 variants in research studies. Furthermore, we are not able to perform have been proposed to be associated with seizures in patients standardised assessments on subjects who live at a distance and with BardeteBiedl syndrome.48 Although we lack the statistical therefore may misclassify subjects with respect to clinical power to determine their significance, we observed several features. The progressive retinal, kidney and liver complications intriguing combinations of heterozygous variants in multiple JS may also be underascertained in younger patients. To improve genes within individual subjects. Taking all these data together, on the information in this report, future studies would it is likely that the effects of mutations in a causal gene drive have to use more standard assessments on larger numbers of much of the phenotype in a given patient, while variants in subjects. modifier genes explain a substantial portion of the phenotypic variability between patients with mutations in the same causal Implications for CC2D2A function gene. Integrating comprehensive sequence variant data with Analysis of the CC2D2A mutations in our JS cohort suggests reliable phenotypic information to develop predictive models for a number of points relevant for CC2D2A protein function. C2 phenotype, as well as identifying the highest-yield molecular domains are phospholipid-binding domains that are often targets for therapeutic interventions, remain challenges for involved in vesicle trafficking and fusion. Recent evidence future research. implicates Cc2d2a in Rab8-dependent vesicle trafficking in 39 Author affiliations zebrafish photoreceptors, similar to what has been described 1 40 Division of Genetic Medicine, Department of Pediatrics, University of Washington, for other JS proteins such as AHI1. Clustering of mutations in Seattle, Washington, USA the C2 domain is consistent with a role for CC2D2A in vesicle 2Department of Radiology, University of Washington, Seattle Children’s Hospital, trafficking, and a role for aberrant trafficking in the molecular Seattle, Washington, USA 3 mechanism underlying CC2D2A-related JS. Multiple mutations Medical Genetics Branch, National Research Institute National Institutes of Health, Bethesda, Maryland, USA are also present in the C-terminal portion of the CC2D2A 4Harvey Institute for Human Genetics, Greater Baltimore Medical Center, Baltimore, protein, which does not contain defined functional domains, but Maryland, USA shares similarity with centrosomal protein CEP76.7 Indeed, 5Department of Medical Genetics, Institute of Mother and Child, Warsaw, Poland 6 a BLAST search with the C-terminal CC2D2A sequence (amino Molecular Genetics Section, Hospital Sant Joan de Deu, Barcelona, Spain 7Department of Pediatrics, King Abdulaziz Medical City, Riyadh, Saudi Arabia acids 1205e1620) retrieves CEP76 and vice versa. This finding 8 fi Department of Pediatrics, Medical University of South Carolina, Charleston, South suggests the presence of an as yet unidenti ed functional Carolina, USA domain. 9Clinical Genetics Department, Guy’s and St Thomas’ NHS Foundation Trust, London, In addition, we confirm the previous finding that patients UK 10 with JS harbour at least one missense mutation in CC2D2A, Hunter Genetics Unit, Waratah, New South Wales, Australia 11Eunice Kennedy Shriver National Institute of Child Health and Human Development, since only one of our 20 subjects had a homozygous nonsense National Institutes of Health, Bethesda, Maryland, USA mutation, while all others had either two missense mutations or 12Division of Developmental Medicine, Department of Pediatrics, University of one missense and one truncating mutation. Moreover, several Washington, Seattle, Washington, USA

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Acknowledgements We would like to thank all participating families with Joubert 14. Valente EM, Logan CV, Mougou-Zerelli S, Lee JH, Silhavy JL, Brancati F, syndrome and the Joubert Syndrome and Related Disorders Foundation. Iannicelli M, Travaglini L, Romani S, Illi B, Adams M, Szymanska K, Mazzotta A, Lee JE, Tolentino JC, Swistun D, Salpietro CD, Fede C, Gabriel S, Russ C, Cibulskis K, Funding This work was supported by National Institute of Health grants Sougnez C, Hildebrandt F, Otto EA, Held S, Diplas BH, Davis EE, Mikula M, Strom CM, 5KL2RR025015 and R01NS064077 to DD, and K12HD043376 to RB-G, by Basil Ben-Zeev B, Lev D, Sagie TL, Michelson M, Yaron Y, Krause A, Boltshauser E, O’Connor Start Scholar Research Grant No 5-FY09-13 from the March of Dimes Elkhartoufi N, Roume J, Shalev S, Munnich A, Saunier S, Inglehearn C, Saad A, Foundation to DD, and by an Arc of Washington Trust Fund grant to DD. We would Alkindy A, Thomas S, Vekemans M, Dallapiccola B, Katsanis N, Johnson CA, also like to thank the NHLBI GO Exome Sequencing Project and its ongoing studies Attie-Bitach T, Gleeson JG. Mutations in TMEM216 perturb ciliogenesis and cause which produced and provided exome variant calls for comparison: the Lung GO Joubert, Meckel and related syndromes. Nat Genet 2010;42:619e25. Sequencing Project (HL-102923), the WHI Sequencing Project (HL-102924), the Broad 15. Sang L, Miller Julie J, Corbit Kevin C, Giles Rachel H, Brauer Matthew J, Otto Edgar GO Sequencing Project (HL-102925), the Seattle GO Sequencing Project (HL-102926) A, Baye Lisa M, Wen X, Scales Suzie J, Kwong M, Huntzicker Erik G, Sfakianos and the Heart GO Sequencing Project (HL-103010). Mindan K, Sandoval W, Bazan JF, Kulkarni P, Garcia-Gonzalo Francesc R, Seol Allen D, O’Toole John F, Held S, Reutter Heiko M, Lane William S, Rafiq Competing interests None. Muhammad A, Noor A, Ansar M, Devi Akella Radha R, Sheffield Val C, Slusarski Diane C, Vincent John B, Doherty Daniel A, Hildebrandt F, Reiter Jeremy F, Jackson Patient consent Obtained. Peter K. Mapping the NPHP-JBTS-MKS protein network reveals ciliopathy disease Ethics approval Ethics approval was provided by University of Washington IRB and genes and pathways. Cell 2011;145:513e28. Seattle Children’s Hospital IRB. 16. Dafinger C, Liebau MC, Elsayed SM, Hellenbroich Y, Boltshauser E, Korenke GC, Fabretti F, Janecke AR, Ebermann I, Nu¨rnberg G, Nu¨rnberg P, Zentgraf H, Koerber F, Contributors RBG performed the study and wrote the manuscript; DD designed the Addicks K, Elsobky E, Benzing T, Schermer B, Bolz HJ. Mutations in KIF7 link Joubert study, reviewed the neuroimaging and wrote the manuscript; JD collected and syndrome with Sonic Hedgehog signaling and microtubule dynamics. J Clin Invest analysed clinical data; GEI reviewed the neuroimaging; HM performed comparative 2011;121:2662e7. genomic hybridisation; JA, DO’D and IGP performed sequencing; IG, MGA, ADK, KS, 17. Garcia-Gonzalo FR, Corbit KC, Sirerol-Piquer MS, Ramaswami G, Otto EA, Noriega LM, AA, SA, SP, LI, AR and MAP participated in subject recruitment and edited the TR, Seol AD, Robinson JF, Bennett CL, Josifova DJ, Garcia-Verdugo JM, Katsanis N, manuscript. Hildebrandt F, Reiter JF. A transition zone complex regulates mammalian ciliogenesis and ciliary membrane composition. Nat Genet 2011;43:776e84. Provenance and peer review Not commissioned; externally peer reviewed. 18. Bielas SL, Silhavy JL, Brancati F, Kisseleva MV, Al-Gazali L, Sztriha L, Bayoumi RA, Zaki MS, Abdel-Aleem A, Rosti RO, Kayserili H, Swistun D, Scott LC, Bertini E, Boltshauser E, Fazzi E, Travaglini L, Field SJ, Gayral S, Jacoby M, Schurmans S, REFERENCES Dallapiccola B, Majerus PW, Valente EM, Gleeson JG. 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J Med Genet 2012;49:126e137. doi:10.1136/jmedgenet-2011-100552 137 Downloaded from jmg.bmj.com on January 18, 2012 - Published by group.bmj.com

Genotype−phenotype correlation in CC2D2A -related Joubert syndrome reveals an association with ventriculomegaly and seizures

Ruxandra Bachmann-Gagescu, Gisele E Ishak, Jennifer C Dempsey, et al.

J Med Genet 2012 49: 126-137 doi: 10.1136/jmedgenet-2011-100552

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