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provided by Elsevier - Publisher Connector ARTICLE in Contactin-1, a Neural Adhesion and , Cause a Familial Form of Lethal Congenital

Alison G. Compton,1,9 Douglas E. Albrecht,2,10 Jane T. Seto,1,3 Sandra T. Cooper,1,3 Biljana Ilkovski,1 Kristi J. Jones,1 Daniel Challis,4,5 David Mowat,4,6 Barbara Ranscht,7 Melanie Bahlo,8 Stanley C. Froehner,2 and Kathryn N. North1,3,*

We have previously reported a group of patients with congenital onset weakness associated with a deficiency of members of the syntro- phin-a-dystrobrevin subcomplex and have demonstrated that loss of and dystrobrevin from the of skeletal mus- cle can also be associated with denervation. Here, we have further studied four individuals from a consanguineous Egyptian family with a lethal congenital myopathy inherited in an autosomal-recessive fashion and characterized by a secondary loss of b2-syntrophin and a-dystrobrevin from the muscle sarcolemma, central nervous system involvement, and fetal akinesia. We performed homozygosity mapping and candidate analysis and identified a that segregates with disease within CNTN1, the gene encoding for the neural immunoglobulin family adhesion molecule, contactin-1. Contactin-1 transcripts were markedly decreased on gene-expres- sion arrays of muscle from affected family members compared to controls. We demonstrate that contactin-1 is expressed at the neuro- muscular junction (NMJ) in mice and man in addition to the previously documented expression in the central and peripheral nervous system. In patients with secondary dystroglycanopathies, we show that contactin-1 is abnormally localized to the sarcolemma instead of exclusively at the NMJ. The cntn1 null mouse presents with ataxia, progressive muscle weakness, and postnatal lethality, similar to the affected members in this family. We propose that loss of contactin-1 from the NMJ impairs communication or adhesion between nerve and muscle resulting in the severe myopathic phenotype. This disorder is part of the continuum in the clinical spectrum of congenital and congenital myasthenic syndromes.

Introduction phin (MIM 601017) and a-dystrobrevin localize to the sarcolemma of both fast and slow fibers; b1-syntrophin The -associated protein complex (DAPC) is (MIM 600026) is located predominantly at the sarcolemma a large, tightly associated oligomeric complex of of type 2 fibers, whereas b2-syntrophin (MIM 600027) is located within the membrane.1 The largely expressed at the sarcolemma of type 1 fibers.4 In DAPC is thought to connect the extracellular matrix to mice, however, b2-syntrophin and a-dystrobrevin-1 expres- the intracellular cytoskeleton and stabilize the plasma sion are largely restricted to the NMJ.5,6 membrane against stresses imposed during muscle con- Previously, we have identified 16 patients with congeni- traction or stretch (reviewed in Ervasti and Sonnemann2). tal onset muscle weakness and myopathic features on mus- Mutations in several members of the DAPC result in cle biopsy (out of 162 patients with congenital onset human muscle disease.3 Many members of the DAPC, myopathy or dystrophy of unknown cause) with isolated e.g., (MIM 128240), a- (MIM secondary deficiency of the syntrophin-a-dystrobrevin 128239), biglycan (MIM 301870), the , and subcomplex by immunohistochemistry.4 Primary muta- dystrobrevins, are also highly expressed at the neuromus- tions in members of the syntrophin-dystrobrevin complex cular junction (NMJ). The syntrophins and dystrobrevins were excluded in all patients. Although the functional con- form a cytoplasmic subcomplex within the DAPC, binding sequence of syntrophin and a-dystrobrevin loss is not directly to dystrophin (MIM 300377) and utrophin (MIM known in humans, several knockout mouse models that 128240). This subcomplex serves as a membrane scaffold, lack syntrophins and dystrobrevins have been generated targeting signaling proteins such as ion channels, kinases, to elucidate their roles in skeletal muscle and nerve. How- and nNOS (MIM 163731) to the sarcolemma. In normal ever, the loss of a1- and b2-syntrophin in mice does not human muscle, syntrophins and a-dystrobrevin (MIM affect muscle structure or function, and they do not 601239) also localize to the NMJ. In addition, a1-syntro- exhibit signs of a muscular dystrophy.7–9 Mice lacking

1Institute for Neuromuscular Research, The Children’s Hospital at Westmead, Sydney, NSW 2145, Australia; 2Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195, USA; 3Discipline of Pediatrics and Child Health, Faculty of Medicine, University of Sydney, NSW 2006, Australia; 4Department of Pediatrics, School of Women’s and Children’s Health, University of New South Wales, Sydney, NSW 2052, Australia; 5Division of Obstetrics and Maternal Fetal Medicine, Royal Hospital for Women, Randwick, NSW 2031, Australia; 6Department of Medical Genetics, Sydney Chil- dren’s Hospital, Sydney, NSW 2031, Australia; 7Burnham Institute for Medical Research, La Jolla, CA 92037, USA; 8Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia 9Present address: Mitochondrial and Metabolic Research Group, Murdoch Childrens Research Institute, Royal Children’s Hospital, Melbourne, VIC 3052, Australia 10Present address: The Jain Foundation, Inc., Redmond, WA 98052, USA *Correspondence: [email protected] DOI 10.1016/j.ajhg.2008.10.022. ª2008 by The American Society of Human Genetics. All rights reserved.

714 The American Journal of Human Genetics 83, 714–724, December 12, 2008 a-dystrobrevin exhibit a mild muscular dystrophy10,11 and of Mendelian and double-recombinant errors with MERLIN.18 cardiomyopathy.12 Interestingly, loss of either a-dystrobre- Error rates were low, indicating that the pedigree was correct. We vin or a1-syntrophin also result in abnormalities in NMJ carried out multipoint homozygosity mapping assuming a fully structure including reduced number of junctional fold penetrant recessive model with a rare allele frequency of 0.0001. openings to the synaptic cleft and reduced junctional The initial genome-wide scan LOD score based on the eight family members yielded a peak LOD of 2.9 at 12q. Analysis with the AChR levels,8–13 suggesting that the syntrophin-dystrobre- additional fine-mapping marker data and refinement of genetic vin subcomplex may have a role in synaptic maturation maps increased evidence for linkage to highly significant (LOD ¼ and remodeling. Adding additional weight to this hypoth- 3.99) and narrowed the linkage region to 14cM at 12p12.1-q12. esis is the finding that the syntrophin-dystrobrevin sub- Because the pedigree was too large (31 bits), the final multi- complex is differentially regulated during fetal develop- point mapping was carried out by dropping individuals from ment not only in mice but also in humans.14,15 Unlike the analysis until the pedigree was analyzable with exact multi- other members of the DAPC, the expression of members point methods. Haplotype results were combined to delineate of the syntrophin-dystrobrevin subcomplex is also altered the critical region. in response to innervation and denervation, implicating a participation in nerve-muscle communication15. On the basis of these observations, we hypothesized that DNA Isolation and Mutation Analysis Total DNA extraction from blood lymphocytes (n ¼ 8) or cheek the underlying cause of muscle weakness in our patient co- swab (n ¼ 1) was performed with proteinase K or sodium dodecyl hort may be neuropathic rather than myopathic. In this sulfate (SDS) digestion. Genomic DNA was isolated from 1 3 8 mm study, we used homozygosity mapping, gene-expression cryosection of frozen skeletal muscle (n ¼ 4) with the GenomiPhi array, and a candidate gene approach to identify the dis- DNA amplification kit (Amersham Biosciences). We synthesized ease-causing gene, CNTN1 (MIM 600016), in the affected cDNA by RT-PCR from mRNA extracted from frozen muscle family members from the large consanguineous Egyptian (patient V:4) and used PCR primers designed to amplify the entire family first described in Jones et al.4 The CNTN1 gene cDNA in overlapping segments (primer sequences for additional encodes for contactin-1, a neural adhesion molecule of screened available upon request). We analyzed PCR frag- the immunoglobulin (Ig) superfamily. We show contac- ments by direct sequencing using ABI Prism (ABI100 Version tin-1 expression at the NMJ in normal human and mouse 3.2). Sequences were compared with the published NCBI se- skeletal muscle, and its mislocalization to the sarcolemma quences. To confirm sequence changes found in cDNA, we ampli- fied the corresponding exons from genomic DNA using genome- in patients with secondary dystroglycanopathy. To our specific primers. We based codon numbering of CNTN1 on the knowledge, this is the first report implicating mutations reported sequence, using the first methionine as an in contactin-1 as a cause of human disease. We suggest initiation codon (NCBI accession numbers NM_001843.2 and that a spectrum exists including both severe lethal myopa- NM_175038). Control genomic DNA was screened by a PCR- thy associated with fetal akinesia and congenital myas- RFLP test utilizing a MnlI restriction site abolished by the CNTN1 thenic syndromes much like those seen in the synaptopa- 871 dupT mutation (primers REgCNTN-1: 50-GGGCCAAAATGT thies caused by mutation in proteins expressed at the NMJ, GACC-30, REgCNTN-2: 50-aagccctccatgtgattt-30). For all primers such as in AChR subunits and rapsyn.16,17 used for CNTN1 amplification, see Table S1 available online. Align- ments of contactin sequences from a variety of vertebrate species were performed with the alignment program MAFFT. Material and Methods

Patient Material In Silico Analysis Patient material was obtained from four affected infants and nine We used the University of California Santa Cruz (UCSC) Genome unaffected family members from a large consanguineous family of Browser (July 2003 freeze), the National Center for Biotechnology 4 Egyptian background that have been briefly reported previously. Information (NCBI) Genome Map Viewer, and the Ensembl Informed consent for tissue and DNA samples was obtained from Server to search for known genes, expressed parents and other contributing family members. Skeletal-muscle sequence tags, and putative genes in the 12p12.1-q12 region. biopsy samples were obtained from the four affected infants This region contained 174 known gene sequences; however, 91 se- for immunohistochemical and immunoblot analysis as well as quences were multiple entries of the same gene because of alterna- genomic DNA isolation and cDNA synthesis. Blood lymphocytes tive splicing and seven were annotation errors within the Browser (n ¼ 8) and buccal swab (n ¼ 1) was obtained for total DNA isola- (i.e., had no Unigene identifier) leaving 76 genes within our region. tion from unaffected family members. The study protocol was We performed bioinformatic analysis of public gene-expression approved by the ethics committees of The Children’s Hospital at databases to prioritize our candidate genes in this region to those Westmead, Sydney, Australia. expressed in muscle and nerve tissue. We used expression data from brain as a substitute for nerve because data is incomplete Linkage Analysis for in nerve and the spinal cord. Because public Genotyping was performed at the Australian Genome Research databases may contain high levels of inaccuracy, we used these Facility (AGRF), Melbourne, with the 5 cM ABI PRISM Linkage two independent data sets to allow crossvalidation of results, Gen- Mapping Set version 2.5 marker set. An additional 15 microsatel- eCards’ electronic Northern (eNorthern), and NCBI SAGEmap lite markers were genotyped for fine mapping in all individuals (Serial Analysis of Gene Expression). Of the 76 known genes in and an additional three individuals. The genotyping was cleaned our candidate region, 34 are expressed in muscle, and 44 are

The American Journal of Human Genetics 83, 714–724, December 12, 2008 715 expressed in the brain. Except for one gene (CFTR/MRP), all mus- confocal microscope. Immunoblot analysis was performed as de- cle genes are also expressed in the brain. scribed previously.22 We performed all immunohistochemical staining and immunoblot studies where possible using age- matched muscle biopsies with normal as controls. Expression-Array Analysis of Patients and Control Skeletal Muscle Total RNA was isolated from postmortem muscle biopsies taken from three patients (V:2, V:3, and V:4) and six normal controls Results of similar age-matched controls. RNA was extracted with Trizol re- agent (Invitrogen) in accordance with the manufacturer’s instruc- As part of our initial cohort,4 we ascertained four patients tions and then further purified with a QIAGEN RNeasy kit. The who belonged to a large consanguineous family of Egyp- concentration of total RNA was measured by absorbance at tian descent (Figure 1A). Individuals V:2, V:3, V:4, and 260 nm and RNA quality was assessed on an Agilent bioanalyzer V:7 had a similar clinical presentation. Reduced fetal move- 2100. RNA target labeling, hybridization to Affymetrix Human ments were detected on ultrasound during the second and Genome U133 plus 2.0 arrays, and chip scanning were performed by the Center for Array Technologies at the University of Washing- third trimester. All infants were born between 28 and 35 ton with standard Affymetrix protocols and an Affymetrix 3000 weeks’ gestation and pregnancy was complicated by multi- scanner. Data from the raw chip scans were uploaded to the Gen- ple problems including polyhydramnios and growth retar- esifter (Vizix Labs) server and normalized by mean intensity, and dation. Three of the children showed no spontaneous comparisons between the control and patient population were movements and died shortly after birth. One infant (V:7) performed with two sample t tests with a p value of 0.05. Genes survived to 1 month of age but required intubation and mapping to the region of 12 that contained the ventilation from birth. She remained ventilator dependent mutation were analyzed for significant changes. because of poor respiratory effort. However, there was no evidence of primary lung pathology requiring increased Immunohistochemistry and Immunoblotting ventilatory settings. Infant V:7 had low birth weight and Muscle biopsy samples were available from all four infants and was noted to be hypotonic at birth with absent deep ten- showed the muscle to be structurally normal with no signs of dys- don reflexes, no suck or swallow, an absent Moro reflex, trophy. Biopsies were snap frozen in liquid-nitrogen-cooled iso- and paucity or absence of spontaneous movement. She pentane and stored in liquid nitrogen until required. Indirect had dysmorphic features including scaphocephaly, an immunofluorescence was performed on 8-mm-thick cryosections oval face, hypertelorism, and a high arched palate. She with antibodies against all members of the DAPC especially those was unresponsive to external stimuli. Her hands were associated with congenital muscular dystrophy (dystrophin, [a-, small with simian creases, arachnodactyly with overlap- b-, d-, and g-] , laminin a2, b-spectrin, and a-dystro- glycan) according to standard procedures.4 All antibodies were ob- ping fingers, and marked camptodactyly. She had flexion tained from Novacastra Laboratories (Newcastle upon Tyne, UK) contractures in most joints but most notably the elbows unless otherwise stated. Antibodies raised against a1-syntrophin and hips. Echocardiogram and head ultrasound were nor- (SYN17), b1-syntrophin (SYN35), b2-syntrophin (SYN28)5,19 and mal. Muscle mass was generally reduced and all serum a-dystrobrevin-1 (DB670) and 2 (DB2)6 were provided by S.C levels were normal, including creatine kinase levels Froehner. A rabbit polyclonal antibody raised against (70 U/l; normal 0–230 U/l). She died 1 month after birth was generously provided by Dr. Louis Kunkel (Boston, MA). Mono- (equivalent of 36 weeks’ gestation developmentally) from clonal anti-laminin a2 (MAB1922) was obtained from Chemicon, respiratory failure precipitated by aspiration. An autopsy and clone 4H8-2 was obtained from Alexis. Antibodies to the cyto- confirmed that the gross morphology of all organs was nor- plasmic domain of the a7-integrin chain [A2(346) and B2(347)] mal including the central nervous system. Light micros- were kindly provided by S.J. Kaufman and used as previously copy of her skeletal muscle showed a minor variation in described.20,21 a-Bungarotoxin, Alexa Fluor 488 conjugate was obtained from Molecular Probes, and polyclonal contactin-1 anti- fiber size (7–15 microns) and a mild increase in collagen body (AF904) was obtained from R&D Systems. For a-dystroglycan fibrils between muscle fibers. Electron microscopy revealed (using clone VIA4-1 from Upstate Biotechnology), we treated the a moderate number of foci in which small numbers of con- muscle with ice-cold acetic acid:ethanol (1:1) for 1 min. For immu- tiguous were disrupted with disorganization of nodetection of a-dystrobrevin (DB670 and DB26), cryosections the Z-band (Figures 1B and 1B0) and a reduced number of were fixed with 3% paraformaldehyde at room temperature for mitochondria. The myofilaments were also somewhat dis- 10 min and then rinsed in PBS. Sections were blocked in 2% BSA ordered within these foci, reminiscent of minicores. The (bovine serum albumin) in PBS (phosphate-buffered saline) at nerve biopsy was histologically normal. Based on the clin- room temperature for 15 min. Sections were incubated in primary ical presentation and histological findings in muscle, the antibody (diluted in 2% BSA) at room temperature for 2 hr or 4C diagnosis in this family was concluded to be a severe con- overnight and then were washed three times in PBS. Anti-mouse genital myopathy. CY3-conjugated, anti-mouse Alexa-488-conjugated, anti-goat CY3-conjugated, and anti-rabbit CY3-conjugated secondary anti- The muscle biopsies from all four infants (V:2, V:3, V:4, bodies (Jackson Immunoresearch Laboratories) were applied, and and V:7) were stained with antibodies to dystrophin, a-, sections were incubated at room temperature for 1 hr. After an b-, g-, and d-, sarcospan, a-dystroglycan, lami- additional three washes in PBS, coverslips were applied over nin a2, b-dystroglycan, and collagen VI. All showed nor- Immumount. Images were captured with a Leica SP2 scanning mal expression and localization at the muscle membrane

716 The American Journal of Human Genetics 83, 714–724, December 12, 2008 Figure 1. Family Pedigree and Skeletal Muscle Pathology (A) Pedigree indicating family members studied with linkage anal- ysis (initial mapping performed on individuals marked with an asterisk) and genotype. Filled symbols indicate affected individ- uals and half-filled symbols indicate carrier status (assessed by genotyping). All empty symbols indicate family members for which samples were unavailable for testing. (B and B0) Electron micrograph of skeletal muscle from affected individual V:7. Fibers contained foci of sarcomeric disruption and z-line streaming. (B) shows a micrograph magnification at 45003, and (B0) shows a micrograph magnification at 10,0003. (C–H) Immunohistochemistry of muscle sections from patient V:7 (C, D, E, F, G, and H) and age-matched control (E0,F0, and G0) for members of the dystrophin-associated protein complex. Shown are (C) a-dystroglycan, (D) a1-syntrophin, (E and E0) b1-syntro- phin, (F and F0) a-dystrobrevin-2, (G and G0) b2-syntrophin, and (H) slow . All members of the dystrophin-associated protein complex were expressed normally except select members of the syntrophin-a-dystrobrevin subcomplex. All affected family mem- bers showed an absence of sarcolemmal membrane expression for b2-syntrophin (which is normally expressed at the sarcolemma of slow fibers (G0 marked with an asterisk in the age-matched control) and a-dystrobrevin. Slow myosin shows the presence of slow fibers in the patient biopsy. Scale bars represent 40 mm. Biopsies were taken from quadriceps at developmental age 35 weeks’ gestation.

(Figure 1C and data not shown). Integrin a7 staining was absent; however, sequencing of the ITGA7 (MIM 600536) gene identified no mutations within its coding region.23 Integrin a7 deficiency is now commonly recognized as a secondary phenomenon in inherited myopathies.23,24 We stained the muscle with antibodies to members of the syntrophin-a-dystrobrevin subcomplex and found consistent abnormalities in all four patients. In patients and age-matched controls, both a1-syntrophin and b1-syntrophin localized to the muscle membrane (Figures 1D and 1E). Developmentally, b1-syntrophin is expressed at the sarcolemmal membrane of all fiber types15 as is observed in Figures 1E and 1E0 (age-matched control). However, in all four patients, both b2-syntrophin and a-dystrobrevin were absent from the muscle membrane (Figures 1F and 1G). Immunohistochemistry with an anti- body raised against slow myosin heavy chain (Figure 1H) shows the presence of slow fibers (which would normally express b2-syntrophin) in the patient biopsy. Interestingly, a-dystrobrevin-1 and -2 were retained at the NMJ even though these molecules were lost from the sarcolemmal membrane (Figure 1F). We performed immunoblot analy- sis of b2-syntrophin on the skeletal muscle biopsy from patient V:4 compared to age-matched control. A diffuse band of ~59 kDa (the expected size for b2-syntrophin) was detected in normal control muscle but not in the patients’ muscle (data not shown). Antibodies raised against a-dystrobrevin do not work reliably on immuno- blotting to allow study. We previously sequenced the cod- ing regions of b2-syntrophin and a-dystrobrevin as well as a1-syntrophin in these patients and did not identify any mutations.4

The American Journal of Human Genetics 83, 714–724, December 12, 2008 717 Figure 2. Linkage and Mutation Analysis (A) Parametric multipoint linkage analysis results of the disease locus in this Egyptian family. An idiotype of chro- mosome 12 reports the position of the locus within the flanking recombination markers D12S1688 and D12S1663. Electro- pherograms show the mutation containing region in control, unaffected parent and affected infant. The duplication of a thy- mine between position 871 and 872 is indi- cated by an arrow. Both the control and the affected normal protein sequences are shown below the respective electrophero- gram. A schematic of the contactin-1 pro- tein shows the position of the amino acid mutation, S291fsX296, in relation to the full-length 1018 amino acid protein (gray ovals represent Ig-like domains, black boxes indicate FNIII domains, and the C-terminal arrow indicates the GPI-anchor). (B) The nucleotides and amino acids flank- ing the insertion (gray shaded) are highly conserved in different species (human contactin-1 nucleotides 848–894, amino acids 262–314). Orthologs in Homo sapiens, Pongo troglodytes, Pongo pygmaeus, Mus musculus, Rattus norvegicus, Bos taurus, Gal- lus gallus, Xenopus laevis, and Danio rerio are shown. Stars indicate conserved residues.

Human Genome Server); only some genes had identified functions, whereas others encode ‘‘hypotheti- cal’’ proteins with yet-to-be-deter- mined function. We constructed an in silico genomic map of the region, using public databases (GeneCards’ eNorthern and NCBI SAGEmap) and prioritized genes for mutation screen- ing on the basis of expression levels (i.e., present in skeletal muscle and We thus carried out multipoint homozygosity mapping brain) and putative function or known interactions. We on eight family members (including three of the four af- excluded a number of candidate genes by sequence analy- fected individuals, Figure 1A) and identified a single, fully sis (n ¼ 24; PEPP2, IAPP, LDHB, KCNJ8, ABCC9, KRAS2, informative region at 12p12.3-q13.11 of 25cM in which BHLHB3, SSPN, ITPR2, STK38L, OVCH1, IPO8, DDX11, only affected children were homozygous. The nonaffected MGC50559, LOC196394, FLJ10652, DNM1L, YARS2, family members (unaffected carriers) were heterozygous SYT10, KIF21A, FGD4, BICD1, MGC24039, and C12orf14). for this region. Further analysis with additional microsatel- Concurrently, we performed gene-expression-array as- lite markers confirmed coinheritance of the haplotype says on skeletal muscle RNA (matched for age and fiber with disease status in all family members (n ¼ 13) and fur- type proportion) from three patients (V:2, V:3, and V:4) ther narrowed the region of homozygosity to 14cM and six control samples to compare the expression levels (flanked by markers D12S1688 and D12S1663), with a para- of the 76 genes within the linkage region. Fourteen genes metric LOD score of 4 (Figure 2A). There were no other ob- within this region were identified as being differentially vious candidate loci. This region (12p12.1-q12) contains expressed in patients versus controls. We had previously 76 known genes (retrieved from the UCSC Genome excluded mutations in half of the genes identified, as being Browser, NCBI Entrez Genome Map Viewer, and Ensembl differentially expression in patients versus controls

718 The American Journal of Human Genetics 83, 714–724, December 12, 2008 Figure 3. Contactin-1 Localization at the NMJ in Both Human and Mouse Skel- etal Muscle Confocal immunofluorescence in normal human and mouse quadriceps muscle showing overlap between contactin-1 (CNTN) and a-bungarotoxin (Txn) used to highlight the acetylcholine receptors of the NMJ. Arrows are used to indicate an example of this staining.

Contactin-1 expression has not been extensively studied in tissues other than the peripheral and central nervous systems. However, very low levels of contactin-1 mRNA have pre- viously been reported in human skeletal muscle.26 The low levels of contactin-1 expression in skeletal (ABCC9, STK38L, DNM1L, DDX11, ITPR2, IAPP, and muscle is supported by our gene-expression-array control MGC24039), by sequence analysis. Of the remainder, the data, which in turn demonstrated a significant reduction most striking alteration in expression was seen for in the contactin-1 RNA transcript in muscle from affected CNTN1 with a 7.9- and 16.7-fold decrease in expression patients. Contactin-1 protein expression levels in normal in patients compared to controls depending on the probe human and mouse skeletal muscle were below detection set used (227202_at and 227209_at, respectively). In addi- levels by immunoblot analysis, although a correctly sized tion, analysis using Genesifter annotated contactin-1 as band could be detected in mouse cerebellum in which con- ‘‘present’’ for all six control samples but ‘‘absent’’ in all tactin-1 is abundantly expressed (see Berglund et al.27 and three patient samples. data not shown). Immunohistochemical studies revealed We sequenced the coding region of the contactin 1 gene that contactin-1 expression is restricted to the NMJ in (CNTN1) and identified a homozygous thymidine duplica- both mouse and human skeletal muscle (Figure 3,n¼ 3 tion, c.871dupT, within exon 8 in all four affected infants each). Because of sampling-site variability and lack of suffi- (Figure 2A). The thymidine duplication (c.871dupT) in cient patient muscle, additional examination of NMJ mor- exon 8 is predicted to result in a reading frameshift intro- phology could not be performed. However, immunohisto- ducing a premature stop codon (S291fsX296). The truncat- chemistry was performed on the quadriceps muscles from ing mutation occurs within the third Ig domain, present the cntn1 null27 and wild-type littermate mice for the syn- within both human contactin-1 transcripts25 and is likely trophins-dystrobrevin subcomplex. In contrast to loss of to be rapidly degraded by nonsense-mediated decay. The b2-syntrophin and a-dystrobrevin from the sarcolemma mutation was heterozygous in the parents of affected indi- in human muscle lacking contactin-1, no difference in viduals and cosegregated with disease in each family mem- staining was detected at the sarcolemmal membrane for ber. The mutation was not present on 121 healthy controls any members of this subcomplex in wild-type or cntn1 of mixed ethnicity (242 ) or 27 unrelated null mice (data not shown). Junctional staining was also myopathy patients (54 chromosomes). Multiple align- normal for these proteins. Specifically, b2-syntrophin ments of contactin-1 orthologs from vertebrates and inver- staining was restricted to the NMJ in both wild-type and tebrates demonstrated that both the nucleotide and amino cntn1 null mice, and no difference was detected in either acid sequences surrounding this duplication mutation the junctional or sarcolemmal staining of the different (nucleotides 871 and 872 in human CNTN1 sequence a-dystrobrevin isoforms (data not shown). NM_001843) are highly conserved during evolution The role of contactin-1 at the neuromuscular junction is (Figure 2B). We have also sequenced contactin-1 in the unknown. In Drosophila follicle cells, contactin forms other members of the patient cohort with secondary syn- a complex with neurexin IV and neuroglian, which are trophin-dystrobrevin deficiencies described in Jones necessary for septate-junction formation and localiza- et al.4 but did not identify any disease-causing mutations. tion.28 In the absence of a-dystroglycan, both neurexin This suggests that this clinical and immunohistochemical and contactin are promiscuously mislocalized, whereas phenotype is genetically heterogeneous, and we consider neuroglian expression is unaltered by loss of a-dystrogly- other proteins that interact with the syntrophins, dystro- can29. On this basis, we used immunochemistry to deter- brevins and now contactin-1 as potential disease candi- mine whether this relationship between contactin and dates for these patients. a-dystroglycan is maintained in human skeletal muscle.

The American Journal of Human Genetics 83, 714–724, December 12, 2008 719 Figure 4. Contactin-1 Is Mislocalized to the Sarcolemmal Membrane in the Absence of Glycosylated a-Dystroglycan Confocal immunofluorescence of skeletal muscle biopsies from secondary a-dystro- glycanopathy patients (mutations in FKRP [B and C] and POMTGnT1 [D]), (E) denerva- tion disorder (SMA [MIM 253300]), and (F) Duchenne muscular dystrophy (DMD) as compared to (A) control muscle with poly- clonal anti-contactin-1 antibodies. Con- tactin-1 expression is mislocalized to the sarcolemmal membrane in response to loss of glycosylated a-dystroglycan (indi- cated with arrows in [B], [C], and [D]) but not in response to generalized dystro- phic changes or denervation. In the con- trol (A), contactin-1 is retained at the NMJ (indicated with arrow). The control shown is not age matched for all biopsies because NMJs are not present in all human biopsies as a result of sampling.

In control skeletal muscle, contactin-1 expression was subunits and rapsyn.16,17 Both disorders can present with restricted to the NMJ (Figures 3 and 4A). However, in fetal akinesia sequence, characterized by paucity of move- patients with secondary dystroglycanopathies (due to ment in utero, and severe weakness and joint contractures mutations in the putative glycosyl transferases FKRP at birth. In this study, we report on a disease-causing muta- [MIM 606612], [MIM 253800], and POMTGnT tion in CNTN1—the gene encoding the glycosyl phospha- [MIM 606822]; n ¼ 6), contactin-1 was not restricted to tidylinositol (GPI)-anchored neural adhesion molecule the NMJ but was also found on the sarcolemmal mem- contactin-1—that results in a severe lethal congenital brane (Figures 4B–4D). Sarcolemmal expression of contac- myopathy with fetal akinesia and nonspecific myopathic tin-1 was not observed in either denervated (n ¼ 4) or dys- features on muscle biopsy. trophinopathy (MIM 310200, n ¼ 4) muscle (Figures 4E In skeletal muscle, contactin-1 is primarily localized to and 4F), suggesting that the loss of glycosylated mem- the NMJ. We propose that reduced expression of contac- brane-bound a-dystroglycan is associated with the contac- tin-1 at the NMJ causes a defect in neuromuscular trans- tin-1 mistargeting and is not secondary to the neuropathic mission, resulting in the severe myopathic phenotype. or dystrophic process. These data suggest that in humans, We have previously demonstrated that denervation results as in Drosophila, the glycosylation state of a-dystroglycan in secondary deficiency of members of the syntrophin-dys- influences the localization of contactin-1. trobrevin complex,15 which is normally localized to the NMJ. The loss of b2-syntrophin and a-dystrobrevin in affected family members is probably secondary to the Discussion defect in neuromuscular transmission or signaling. These data further emphasize the overlap in the clinical spectrum The congenital myopathies are a clinically and genetically of congenital myopathies with fetal akinesia and congeni- heterogeneous group of neuromuscular disorders that pres- tal myasthenic syndromes and suggest that it is not only ent with hypotonia and areflexia, poor muscle bulk, and the proteins necessary for neuromuscular signal transduc- generalized weakness and are usually defined by a patho- tion but also those related to NMJ adhesion that are impor- logical hallmark on muscle biopsy, e.g., nemaline bodies, tant for normal muscle development and growth. cores, and central nuclei. To date, the genes responsible Contactin-1 is a member of the immunoglobulin super- for inherited myopathies encode proteins with a variety family with a common structure of six C2 Ig-like domains, of structural and metabolic functions (reviewed in four fibronectin type III (FNIII) repeats, and a GPI anchor.25 North30) including components of the muscle contractile Ig-like and FNIII domains are common building blocks of machinery, proteins that mediate calcium release from many extracellular proteins involved in ligand recognition the , and proteins involved in and cell adhesion. They are also the main motifs of several cytoskeleton reorganization and endocytosis. There is clin- intracellular proteins associated with the contractile appa- ical overlap between severe congenital myopathies and ratus of muscles such as , obscurin, and myotilin. congenital myasthenic syndromes due to mutations in Contactin-1 expression has been extensively studied in components of the NMJ such as acetylcholine receptor the mouse central nervous system and is thought to play

720 The American Journal of Human Genetics 83, 714–724, December 12, 2008 a role in the establishment and stabilization of synaptic progressive paralysis of the hind limbs due to muscle atro- connections.31,32 In the mouse central and peripheral ner- phy during the second week of life and subsequent lethal- vous systems, contactin-1 is associated with several signal- ity in the first month of life. The med mouse also presented ing molecules present in the extracellular matrix of neu- with degeneration, much like the cntn1-null- rons and glia. Several lines of evidence suggest a critical mouse model. role for contactin-1 in the regulation of neurite growth, Unlike the majority of DAPC members, the syntrophins synapse formation, fasciculation, and myelin organization and dystrobrevins in skeletal muscle are regulated by neu- (reviewed in Falk et al.33). ral signaling; their expression levels and localization are Several different contactin-1 knockdown models have altered by loss of innervation (as seen in human denerva- been generated so that the in vivo functions of contac- tion disorders).15 Glycosylation of a-dystroglycan is regu- tin-1 could be determined. Knockdown of cntn1 expression lated in a similar manner.50 The potential interaction in Xenopus impairs neurogenesis and outgrowth of sensory between the syntrophin-dystrobrevin subcomplex and axons.34 Antibody perturbation induces misguidance of a-dystroglycan within the DAPC and contactin-1 may con- central axons of proprioceptive dorsal root ganglion neu- tribute to the central nervous system defects seen in both rons of chicks.35 Contactin-1 null mice are severely ataxic, patients with muscle disease and the knockout mouse exhibit muscle weakness and growth retardation, and die models (cntn1, med, and dmu). In several secondary a-dys- within the first 18 days of life. The mice show defects in troglycanopathies, loss or abnormal glycosylation of brain the micro-organization of the cerebellar circuitry,27 disrup- dystroglycan can also result in CNS defects as well as de- tion of paranodal junctions,36 and impaired synaptic plas- layed NMJ formation and a delay in muscle formation.51 ticity.37 In the affected infants in our study family, central Both the syntrophin (a1- and b2-) and dystrobrevin and peripheral nervous system involvement could not be (a- and b-) knockout mouse models are also reported to well documented because of their early death. The one exhibit subtle functional defects, such as performance in infant who survived for 1 month responded poorly to sensorimotor tests, that cannot be explained by NMJ external stimuli. Although thought to be a possible conse- abnormalities9,52. quence of birth asphyxia, this clinical phenotype may be The abnormalities in syntrophins and a-dystrobrevin in associated with contactin-1 mutations. Skeletal muscle patients with a mutation in CNTN1 suggests an association function and NMJ morphology and function have not between contactin-1 and the syntrophin-dystrobrevin sub- been studied in detail in our patient group or in the contac- complex. Another member of the DAPC, biglycan regu- tin-1 null mouse; however, leg splaying was evident in lates the membrane expression of the syntrophin-dystro- contactin-1 null mice, suggestive of muscle weakness and brevin complex in skeletal muscle. Biglycan null mice reminiscent of the ‘‘frog-leg’’ posture adopted by patients show abnormal syntrophin-dystrobrevin expression, simi- with congenital myopathy. lar to our patient cohort;53 we have excluded primary The mutation in CNTN1 described in this study intro- biglycan mutations in all of these patients.54 Biglycan duces a premature stop codon at position 296 within the interacts directly with a-dystroglycan and, in Drosophila, third Ig-like domain. Little is known about the contactin- expression of a-dystroglycan at the sarcolemmal mem- 1 binding partners in skeletal muscle; however, this brane precludes incorrect membrane localization of con- domain is crucial for tenascin binding in the nervous tactin.29 In this study, we show that contactin-1 is also mis- system38,39 and would be abolished with this mutation. localized to the sarcolemmal membrane in humans with Tenascin-C (MIM 187380) has been predominantly stud- secondary dystroglycanopathies. Dystroglycan is normally ied within the central nervous system; however, one study expressed in the brain, and loss of dystroglycan in the showed expression also at the neuromuscular and myoten- brain-specific dystroglycan knockout mouse results in dinous junctions.40 Tenascin-C-deficient mice exhibit structural and functional brain abnormalities.55 Similarly, long-term impairment of skeletal muscle reinnervation, in several secondary a-dystroglycanopathies, loss or abnor- suggesting that tenascin-C plays an important role in the mal glycosylation of brain dystroglycan also results in CNS formation and stabilization of the NMJ.41 Contactin-1 defects, as well as abnormal NMJ formation and delayed also interacts with specific sodium channel isoforms in terminal muscle formation.51 In combination, these data the central and peripheral nervous system,42–46 but suggest that contactin-1 may also contribute to disease whether this interaction occurs with the skeletal-muscle- pathogenesis in a wider spectrum of disease phenotypes specific isoforms has yet to be investigated. The syntro- including the dystroglycanopathies. Although the precise phins also directly associate with voltage-gated sodium function of contactin-1 in skeletal muscle is unknown, it channels in brain and skeletal muscle.47 In mice, muta- is possible that its absence results in disruption of signaling tions in the neuronal voltage-gated sodium channel, pathways through the DAPC and has a deleterious effect on

Nav1.6 (MIM 600702) cause an ataxic phenotype (i.e., muscle membrane function or integrity, accounting for the med mouse; neurological mutant with ‘‘motor endplate muscle weakness observed in patients with CNTN1 muta- disease’’48) or muscular dystrophy (i.e., a spontaneous tions. This hypothesis warrants further investigation. mutation resulting in muscle degeneration; dmu mouse49). In summary, we have identified a mutation within the Both mouse models are characterized by early-onset neural adhesion molecule, contactin-1 in patients with

The American Journal of Human Genetics 83, 714–724, December 12, 2008 721 lethal congenital myopathy, characterized by the second- Gallus gallus NP_001004381, Danio rerio NP_851300, Xenopus lae- ary loss of b2-syntrophin and a-dystrobrevin from the sar- vis BAA13100, Homo sapiens NP_001834, and NP_778203. The colemma and central nervous system involvement. To our cDNA sequences reported in this paper are as follows: Homo sapiens knowledge, this is the first time contactin-1 has been NM_001843 and NM_175038, Pan troglodytes XM_001167985, Pongo pygmaeus Mus musculus Rattus nor- linked to human disease. In patient muscle biopsies, we CR857808, NM_007727, vegicus NM_057118, Bos Taurus NM_174280, Gallus gallus show that contactin-1 transcript levels are significantly de- NM_001004381, Xenopus laevis D86505, and Danio rerio (1a) creased compared to controls and demonstrate that con- NM_180969. tactin-1 is localized to the NMJ. 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