Disrupted auto-regulation of the spliceosomal gene SNRPB causes cerebro-costo-mandibular syndrome

Abstract

Elucidating the function of highly conserved regulatory sequences is a significant challenge in genomics today. Certain intragenic highly conserved elements have been associated with regulating levels of core components of the spliceosome and alternative splicing of downstream genes. Here we identify mutations in one such element, a regulatory alternative exon of SNRPB as the cause of cerebro-costo-mandibular syndrome. This exon contains a premature termination codon that triggers nonsense-mediated mRNA decay when included in the transcript. These mutations cause increased inclusion of the alternative exon and decreased overall expression of SNRPB. We provide evidence for the functional importance of this conserved intragenic element in the regulation of alternative splicing and development, and suggest that the evolution of such a regulatory mechanism has contributed to the complexity of mammalian development.

ARTICLE

Received 7 Feb 2014 | Accepted 23 Jun 2014 | Published 22 Jul 2014

DOI: 10.1038/ncomms5483 OPEN

Disrupted auto-regulation of the spliceosomal gene SNRPB causes cerebrocostomandibular syndrome

Danielle C. Lynch1, Timothe Revil2, Jeremy Schwartzentruber3, Elizabeth J. Bhoj4, A. Micheil Innes1,5, Ryan E. Lamont1,5, Edmond G. Lemire6, Bernard N. Chodirker7,8, Juliet P. Taylor9, Elaine H. Zackai4,D. Ross McLeod1,5, Edwin P. Kirk10,11, Julie Hoover-Fong12, Leah Fleming13, Ravi Savarirayan14, Care4Rare Canadaz, Jacek Majewski2,3, Loydie A. Jerome-Majewska2,15, Jillian S. Parboosingh1,5,* & Francois P. Bernier1,5,*

Elucidating the function of highly conserved regulatory sequences is a signicant challenge in genomics today. Certain intragenic highly conserved elements have been associated with regulating levels of core components of the spliceosome and alternative splicing of downstream genes. Here we identify mutations in one such element, a regulatory alternative exon of SNRPB as the cause of cerebrocostomandibular syndrome. This exon contains a premature termination codon that triggers nonsense-mediated mRNA decay when included in the transcript. These mutations cause increased inclusion of the alternative exon and decreased overall expression of SNRPB. We provide evidence for the functional importance of this conserved intragenic element in the regulation of alternative splicing and development, and suggest that the evolution of such a regulatory mechanism has contributed to the complexity of mammalian development.

1 Department of Medical Genetics, University of Calgary, Calgary, Alberta, Canada T2N 4N1. 2 Department of Human Genetics, McGill University, Montral, Quebec, Canada H3A 1B1. 3 McGill University and Gnome Qubec Innovation Centre, Montral, Quebec, Canada H3A 0G1. 4 Division of Genetics, The Childrens Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA. 5 Alberta Childrens Hospital Research Institute for Child and Maternal Health, Calgary, Alberta, Canada T3B 6A8. 6 Division of Medical Genetics, Department of Pediatrics, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 0W8. 7 Department of Pediatrics and Child Health, University of Manitoba, Winnipeg, Manitoba, Canada R3A 1S1. 8 Department of Biochemistry and Medical Genetics, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R3A 1S1. 9 Genetic Health Service, Auckland 1142,New Zealand. 10 Sydney Childrens Hospital, Randwick, New South Wales 2031, Australia. 11 School of Womens and Childrens Health, University of New South Wales, Randwick, New South Wales 2031, Australia. 12 Greenberg Center for Skeletal Dysplasias, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, Maryland 21287, USA. 13 National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. 14 Department of Pediatrics, McGill University Health Centre, Montreal, Quebec H3Z 2Z3, Canada. 15 Department of Pediatrics, McGill University, Montreal Childrens Hospital, Montreal, Quebec, Canada H3H 1P3. z Steering committee members and their afliations appear at the end of the paper. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to F.P.B. (email: mailto:[email protected]

Web End [email protected] ).

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Although only 1.5% of the human genome consists of regions that are translated into proteins, a higher proportion (57%) has been shown to be under

evolutionary constraint1,2. These non-coding conserved elements (NCEs) have been subclassied by somewhat arbitrary length and conservation criteria, and include ultraconserved3 and highly conserved4 elements. The nding that many NCEs exhibit a higher level of conservation5 and constraint6 than protein-coding sequences initially perplexed the genomics community. The elucidation that some NCEs have functional roles as long range enhancers of anking genes, splicing regulators, functional co-activators79 and their frequent association with developmental genes with the potential to regulate spatiotemporal expression7,10, imply a largely regulatory role. The evolution of these complex regulatory networks may therefore have underpinned the emergence of our organismal complexity6,11.

Evidence continues to emerge of a critical relationship between NCEs and alternative splicing (AS), the mechanism by which over 95% of human multi-exon genes create additional protein diversity12,13. Intragenic NCEs are preferentially associated with genes involved in pre-mRNA splicing3, and are also often involved in the regulation of the expression of this class of genes by coupling AS with nonsense-mediated decay (NMD)1416. Many genes involved in pre-mRNA splicing have ultra and highly conserved NCEs containing premature termination codons (PTCs), which can be alternatively spliced into the mature mRNAs to induce NMD in order to auto-regulate their expression1416. These ASNMD-mediated mechanisms are presumed to be crucial to the homeostatic maintenance of the core spliceosome components and the regulation of AS in a spatiotemporally specic manner by gene auto- and cross-regulation17,18.

Here we present mutations in a highly conserved, alternative PTC-containing exon of the small nuclear ribonucleoprotein polypeptides B and B1 (SNRPB) gene (Fig. 1) as the cause of cerebrocostomandibular syndrome (CCMS), a human multiple malformation disorder characterized by posterior rib gaps and Pierre Robin sequence (micrognathia, glossoptosis and cleft palate). This nding provides biological evidence of a direct link between conserved genomic elements, regulation of AS and human development, and therefore novel insight in the regulatory and developmental role of NCEs.

ResultsA combination of whole-exome sequencing and Sanger sequencing was used to identify causative mutations in a cohort of 10

unrelated families with CCMS, a rare genetic disorder characterized by micrognathia and posterior rib gaps19 (Supplementary Fig. 1). All patients have typical features of CCMS except one patient (Family D) who had a more severe disease (Supplementary Methods, Supplementary Fig. 2 and Supplementary Table 1). Nine of the 10 patients had heterozygous regulatory mutations in SNRPB. Overall, six distinct, novel mutations in SNRPB were identied. Five mutations are within the alternative PTC-containing exon (chr20:g.2447838_2447961) of SNRPB. These mutations cluster at the 50 and 30 ends of this exon within areas of high conservation (Fig. 1c). A single patient had a 50 untranslated region (UTR) mutation, which is predicted to introduce an outof-frame translation initiation site (TIS) leading to a stop codon after 25 amino acids (Fig. 1b). In the SNRPB-positive families, mutation analysis conrms that CCMS is an autosomal dominant disorder. We observed a high rate of de novo mutations and two instances of non-penetrance. One individual with classic CCMS was negative for sequence or copy-number variants in the coding and UTRs of SNRPB.

SNRPB encodes the protein isoforms SmB and SmB0, which are core components of the U1, U2, U4/U6 and U5 small ribonuclear protein (snRNP)20 subunits of the major spliceosome. The highly conserved alternative exon within the second of six introns in SNRPB contains a PTC and has been shown to auto-regulate SNRPB levels through NMD18. The alternate exon, which has a sub-optimal 50 splice site, is less frequently included when U1 snRNP levels are low as a result of SmB/B0 depletion18. Conversely, it is more frequently included with SmB/B0 overexpression16. We hypothesized that the mutations identied within this exon would alter the homeostatic balance between the coding full-length mRNA and alternative exon-containing transcripts targeted for degradation. Thus, we determined the effect of two of the alternative exon mutations using a splicing reporter minigene assay21. In the presence of the wild-type exon, 23% of all transcripts include this alternative exon, while introduction of either the chr20:g.2447951C4G or chr20:g.2447847G4T mutation shifts the proportion to 78% and 80%, respectively (Fig. 2a,b). Inclusion of the alternative PTC-containing exon was also assessed by quantitative reverse transcription PCR (qRTPCR) in patient broblasts with the chr20:g.2447951C4G, chr20:g.2449752C4G, and chr20:g.2447847G4T mutations. Expression of the PTC-containing transcript increased, whereas overall expression of SNRPB decreased compared with control cells (Fig. 2c,d).

1 kb

NM_003091

NM_198216

PTC-containing transcript phylop

Mammalin Conservation

WT SNRPB

g.2451408 G>T (c.1-72G>T)

50 bp

50 bp

Alternative

PTC-containingexonphylopMammalianConservation g.2447951 C>G n =1g.2447951 C>A n =1g.2447952 C>G n = 4

g.2447846 G > A n =1g.2447847 G > T n =1

Figure 1 | SNRPB mutations in CCMS. (a) The transcript isoforms encoding SmB (NM_003091), SmB0 (NM_198216), and the alternative PTC-containing transcript. (b) One patient (D II-1) had a mutation in the 50 UTR predicted to introduce an upstream out-of-frame TIS, leading to a PTC after 25 amino acids (SOM text). The green boxes represent translation initiation codons, and the red box and asterisk represents a translation termination codon. (c) Five mutations within the alternative PTC-containing exon were identied in CCMS patients. These cluster at four nucleotides at the 50 and 30 ends of the exon within blocks of high conservation. WT, wild type.

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*

Relative expressionof PTC-containg transcript

Relative expressionof all SNRPBtranscripts

3

2

1

2

1

0 Ctrl pt 1 pt 2 pt 3

100 bpLadder NT WT Mut 1 Mut 2

** **

345 bp

221 bp

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Figure 2 | SNRPB mutations in the alternative (alt) PTC-containing exon cause increased exon inclusion. (a,b) Cloning of the alternative exon with either the chr20:g.2447951C4G (mut1) or chr20:g.2447847G4T (mut2) mutation into a splicing minigene reporter and transfection into HEK297 cells shows 78% and 80% exon inclusion, respectively, compared with 23% for the wild-type (WT) sequence. Results shown are from one representative experimental replicate of three. A 100-bp DNA ladder was used as a size marker in a. NT, no template control. (c) Patient broblasts with the chr20:g.2447951C4G (pt 1), chr20:g.2447952C4G (pt 2) and chr20:g.2447847G4T (pt 3) mutations show increased expression of the PTC-containing transcript by qRTPCR. (d) The same three patients show decreased total SNRPB expression by qRTPCR. In c,d, the grey columns represent the normalized average expression from three anonymous controls (Ctrl). The experiment was performed three times. For bd, statistical signicance was determined with a Students t-test. Bars indicate s.d. * indicates 0.005oPo0.05, ** indicates 0.0005oPo0.005 with *** indicating Po0.001 and ****Po0.0001.

ESE

ESS

4

0

4

g.2,447,952 C>G n = 4

g.2,447,951 C > G n =1g.2,447,951 C > A n =1

g.2,447,847 G > T n =1g.2,447,846 G > A n =1

Figure 3 | CCMS mutations overlap with highly conserved ESSs. The bar height shows mammalian PhyloP conservation scores within andanking the alternative PTC-containing exon. The colours represent the strength of exonic splicing enhancers (ESEs) and silencers (ESSs) identied by deletion mutagenesis of miniSmB (17).

DiscussionCollectively, these results implicate the deregulation of SNRPB expression as the main disease mechanism for CCMS. Mutations in the alternative PTC-containing exon cluster at two sites, which overlap with known exonic splicing silencers (ESSs)22. In an experiment by Saltzman et al.18, deletion of both of these regions resulted in increased inclusion of the alternative exon in HeLa cells. Our results support the functional signicance of these ESSs, which are perfectly conserved across placental mammals (Fig. 3 and Supplementary Fig. 3), and suggest that the identied mutations weaken their silencing function. This would lead to the observed increase in the inclusion of this exon in CCMS, which is presumably the cause of the decreased overall SNRPB expression seen in patient cells (Fig. 4).

The mutations identied in the alternative exon appear to cause a reduction in the amount of SmB/B0 that is consistent with a hypomorphic, but not a null, allele. qRTPCR experiments in three patients show a narrow range of total SNRPB expression(0.530.66 relative to controls). In the minigene experiment, exclusion of the alternative exon was not eliminated in mutant transcripts, but occurred 2022% of the time. We also have evidence that null alleles might result in a more severe pheno-type as one patient without an alternative exon mutation has a 50 UTR mutation predicted to result in a null allele causing

haploinsufciency (Supplementary Discussion). This patients phenotype was more severe than the remainder of the cohort, with only ve pairs of poorly ossied ribs, a poorly ossied spine, cystic hygroma and multiple pterygia (Supplementary Table 1). Since none of the other patients carry truncating mutations in the gene (which would be much more likely to occur by chance than point mutations at two specic loci), and truncating mutations in or deletions encompassing SNRPB have not been reported23,24, we suggest that SNRPB haploinsufciency may cause a more severe and likely lethal phenotype that is distinct from classic CCMS.

CCMS joins a growing list of developmental disorders caused by mutations in core spliceosomal genes. Of particular interest are those with an overlapping craniofacial phenotype, such as Nager syndrome and the EFTUD2-related disorders2527. Interestingly, all of the above are caused by dominant mutations that are predicted to reduce expression of a component of the major spliceosome. It is known that the abundance of the spliceosomal machinery inuences AS28,29. In the case of SNRPB, RNAseq experiments have shown that specic AS exons are more sensitive to changes in SmB/B0 levels18. Among genes containing such exons, nucleic acid binding and RNA processing genes are over-represented. The SNRPB mutations presented here are therefore predicted to cross-regulate AS and expression of downstream

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2

3

Unaffected allele

CCMS alleles

1

2

3

4

5

6

7

SmB/SmB protein levels decrease

2

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Nonsense-mediated decay

SmB/SmB protein levels increase

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? ?

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Figure 4 | A model of disrupted SNRPB regulation in CCMS. Unknown repressor proteins (red circles) bind the ESS regulatory sequences (red squares) in the alternatively spliced exon (in blue) of SNRPB. Their binding leads to exclusion of this alternative exon, and thus an increase of SmB/SmB0 protein levels.

Higher levels of these proteins then favour inclusion of the alternative exon, by an unknown mechanism, leading to NMD and a reduction of SmB/SmB0 protein levels. In alleles mutated in CCMS patients, the binding of repressor proteins is thought to be abolished or reduced due to the mutations present in the regulatory sequences. This leads to continued inclusion of the alternative exon, and reduced SmB/SmB0 protein levels due to NMD.

genes. However, it is perplexing that a spliceosomal deciency could cause such a strikingly specic phenotype. It may be that this deciency affects a small number of transcripts that are particularly sensitive to spliceosomal protein levels. The identity of such transcripts remains speculative, however, animal models suggest that craniofacial abnormalities commonly found in CCMS are likely due to abnormal cell proliferation30, whereas the rib abnormalities have long been postulated to be a consequence of abnormal cartilage formation31. Given the common craniofacial phenotypes associated with the abovementioned disorders, it is possible that a common gene or network of genes is perturbed in all of these. Another possible explanation for the specicity of the CCMS phenotype is that the spliceosomal deciency is exacerbated in a critical tissue or developmental stage owing to increased demand for spliceosomal activity. Studies of the two spliceosome-associated disorders spinal muscular atrophy and retinitis pigmentosa have shown that the retina and spinal cord, tissues that appear to be sensitive to a spliceosomal deciency, show increased demand for spliceosomal proteins32,33.

Our study highlights the importance of accurate AS in development, alludes to the broad network of splicing regulation, and demonstrates the regulatory and developmental importance of a highly conserved regulatory element. The alternative exon of SNRPB has high conservation at the nucleotide level throughout placental mammals (average GERP score 4.08), although to a lesser extent than the ultra-conserved elements (Supplementary Fig. 3). In general, shorter human conserved elements are conserved among mammals, but not with other species3. It has been suggested that evolution of these elements is ongoing in vertebrates, and that specic specializations may reect cladespecic adaptive regulatory changes3. It is then possible that auto-regulation of SNRPB has evolved in mammals with the function of guiding specic cellular and developmental processes. Broadly, we therefore speculate that NCEs may have a signicant

role in regulating the phenotypic variation on which natural selection acts to drive the evolution of complex and highly integrated traits.

Methods

Patients. A cohort of 10 CCMS families was assembled through the Finding Of Rare disease GEnes (FORGE) Canada Consortium (now called Care4Rare). All patients provided informed consent, and the study was approved by and complies with the ethical regulations of the institutional review board at the University of Calgary. An experienced clinical geneticist was responsible for each diagnosis of CCMS. Exclusion criteria included absence of micrognathia and posterior rib gaps. Other variable features include scoliosis, short stature, conductive hearing loss and congenital heart defects. Although intellectual disability is reported to be a common feature of CCMS, this was not prevalent in our cohort (Supplementary Fig. 2 and Supplementary Table 1). Family A had a sibling recurrence with unaffected parents, families E and F had parentchild transmission, and the seven remaining cases were sporadic (Supplementary Fig. 1).

Exome sequencing. DNA was extracted from whole blood. Exome sequencing was performed for six unrelated cases and seven family members at the McGill University and Gnome Qubec Innovation Centre. The SureSelect 50 Mb Human All Exon kit (Agilent) was used for exon capture; v3 was used for families A, B and C, and v5 was used for families D, E and F. Captured regions were sequenced on a HiSeq 2000 sequencer (Illumina) with 100 bp paired-end reads. Reads were aligned to the hg19/GRCh37 human reference sequence using the Burrows-Wheeler Aligner34, and indel realignment was done with GATK35. Duplicate reads were then marked using Picard (http://picard.sourceforge.net/

Web End =http://picard.sourceforge.net/) and excluded from downstream analyses. Coverage of consensus coding sequence (CCDS) bases was assessed using the GATK, which showed that samples had on average 494% of

CCDS bases covered by at least 10 reads, and 490% of CCDS bases covered by at least 20 reads. Single-nucleotide variants and short insertions and deletions were called with SAMtools mpileup36 with the extended base alignment quality adjustment (-E). Only variants that were supported by Z20% of reads were returned. These were annotated using both Annovar37 and custom scripts to identify whether they affected protein-coding sequence, and whether they had previously been seen in the 1,000 genomes data set (April 2012), the National Institutes of Health Heart, Lung, Blood Institute, Grand Opportunity Exome Sequencing Project (NHLBI GO) exomes, or in B700 exomes previously sequenced at our center.

To identify de novo variants in the probands of the three families for which trios were sequenced, we ltered out all proband variants seen in a parent or in the

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1,000 genomes or NHLBI exome data sets, and manually reviewed remaining candidates. For family D a de novo 50 UTR variant was seen that introduced a potential out-of-frame TIS in SNRPB. We used TIS miner (http://dnafsminer.bic.nus.edu.sg/Tis.html

Web End =http:// http://dnafsminer.bic.nus.edu.sg/Tis.html

Web End =dnafsminer.bic.nus.edu.sg/Tis.html ) to predict the effect of this variant.

Sanger sequencing. For all individuals in the cohort, Sanger sequencing of the alternative exon including the anking intronic regions was performed. For patient D II-1, the 50 UTR was sequenced to conrm the presence of the variant identied by exome sequencing. For patient G II-3, the coding regions, including the anking intronic sequences, and the UTRs of SNRPB were sequenced. Primers were designed with Oligo 6 (Molecular Biology Insights). Sequences can be found in Supplementary Table 2. An amount of 2.5 ml of 50 ng ml 1 DNA was used in a 25-ml PCR using the HotStar Taq amplication system. Thermocycler conditions were as follows: 96 C for 5:00, 35 cycles of 96 C for 0:30, 58 C for 0:30 and 72 C for 0:30, and a nal elongation step at 72 C for 7:00. An amount of 5 ml was analysed on a 1% agarose gel. A quantity of 1.2 ml of 1/20 dilution of the PCR product was puried in a reaction with 1 ml ExoSAP-IT (Affymetrix) and 3 ml H2O The product of this reaction was added to a sequencing reaction with 2.2 ml H2O, 1.875 ml of 5

sequencing buffer, 0.5 ml primer and 0.25 ml BigDye Terminator v1.1 (Life Technologies). Unincorporated nucleotides were removed from the sequencing reaction by passage through a Sephadex column. The products were then analysed on a 3130xL Genetic Analyzer (Applied Biosystems).

Copy-number variant analysis by qPCR. qPCR of all exons of SNRPB was used to search for copy-number variants in patient G II-3. One microliter of 5 ng ml 1

DNA was used in a 20-ml reaction with 1 ml of 10 mM primer mix, 10 ml of SYBR Green (Life Technologies) and 8 ml of H2O. Primer sequences can be found in Supplementary Table 3. Reactions were run on a 7900HT Fast Real-Time PCR System (Applied Biosystems). Cycling conditions were as follows: 95 C for 10 min, 40 cycles of 95 C for 15 s and 60 C for 1 min, and a dissociation step with 95 C for 15 s, 60 C for 15 s, and 95 C for 15 s. Relative expression was calculated using the DDCt method36, with ALB used as a reference gene.

Cloning. GeneArt fragments with mutations were ordered from Invitrogen. Primer sequences can be found in Supplementary Table 4 and GeneArt fragment sequences can be found in Supplementary Table 5. These, along with the miniSmB plasmid (a gift of Dr Benjamin Blencowe), described in ref. 18, were digested with XhoI and NotI and gel puried. Ligation was done using the Quick Ligation kit (NEB), transformed and selected on ampicillin plates. Minipreps were prepared from selected clones and sequenced at the McGill University and Genome Quebec Innovation Centre.

Transfections. HEK293 cells were plated in 24-well plates at 4050% conuency. The following day, cells were transfected with 0.25 mg of DNA and 0.75 ml of Fugene 6 (Promega) in 2 ml of DMEM 10% FBS. Eighteen hours later, cells were

rinsed with Dulbeccos PBS and lysed with 1 ml of TRIzol (Invitrogen). RNA extraction was done using the manufacturers protocol and the resulting puried RNA was resuspended in 20 ml of diethylpyrocarbonate (DEPC)-treated H2O.

Transfection was performed three times.

RTPCR and analysis. Half of the RNA (10 ml) was treated with 2 units of DNase I (NEB) by incubation at 25 C for 15 min, then 1 ml of EDTA 25 mM was added, followed by incubation at 65 C for 10 min. One-eighth of this reaction (2.5 ml) was used in a 10 ml reverse transcriptase reaction using SuperScript III (Invitrogen), using the manufacturers protocol, with random primers and a reaction temperature of 50 C. RNase H treatment was performed by adding 1 unit of the enzyme and incubating at 37 C for 20 min. A quantity of 0.75 ml of cDNA was usedin a 30 ml PCR, of which 10 ml was analysed on a 2% agarose gel and 20 ml was analysed on a BioAnalyzer (Agilent). Primer sequences are available in the Supplementary Material.

Patient broblast culture and RNA extraction. Skin biopsies were collected from patient II-1 from family C, patient II-2 from family E and patient II-1 from familyF. Three anonymous control broblast lines were obtained from The Centre for Applied Genomics. Fibroblasts were cultured at 37 C in Amniomax media (Invitrogen) (15% Amiomax supplement, 0.5% glutamine and 0.005% fungizome). At conuency, cells were treated with 3 ml of Hanks balanced salt solution (Invitrogen), then 1 ml of trypsin-EDTA (Invitrogen) and centrifuged at 1,100 r.p.m. for 10 min. Cells were then either resuspended in fresh media for growth of the next passage or used for RNA extraction. Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturers protocol, with a DNase I digestion performed at the wash step (10 ml DNase I in 70 ml buffer RDD (Pre-

AnalytiX). RNA was extracted from three subsequent cell passages.

qRTPCR primer design and efciency testing. Primers were designed using Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/primer3/

Web End =http://bioinfo.ut.ee/primer3-0.4.0/primer3/) to overlap exonexon junctions to prevent amplication of genomic DNA. Sequences can be found in

Supplementary Table 6. For each primer pair, efciency was determined by tracing a standard curve of the Ct values of four serial dilutions of cDNA. Primers with efciency between 90 and 110% were selected. The SNRPBaltexon_qRTPCR_F and R primers amplify the transcript including the alternative PTC-containing exon; SNRPBtotal_qRTPCR_F and R primers amplify all transcripts.

qRTPCR analysis of SNRPB expression. Two microlitres of RNA were used in a 20 ml reverse transcriptase reaction using SuperScript III (Invitrogen), according to the manufacturers protocol, with oligo d(T) primers. The resulting cDNA was diluted by one-fth and used in a qRTPCR. All qRTPCRs had a 20 ml volume, with 1 ml cDNA, 1 ml of 10 mM primer mix, 10 ml of SYBR Green (Life Technologies) and 8 ml of H2O. Reactions were run on a 7900HT Fast Real-Time PCR System (Applied Biosystems). Cycling conditions were as follows: 95 C for 10 min, 40 cycles of 95 C for 15 s and 60 C for 1 min, and a dissociation step with 95 C for 15 s, 60 C for 15 s and 95 C for 15 s. Relative expression was calculated using the DDCt method36, with EIF1B used as a reference gene. The experiment was performed three times. Statistical signicance of observed differences was calculated with a Students t-test.

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Acknowledgements

Supporting information can be found in the online Supplementary Material. We thank the patients and their families who participated in this study. Cell culture for the qRTPCR experiments was performed by Nadine Gamache. The miniSmB construct was a gift from Dr Benjamin Blencowe. Drs Roy Gravel, Kym Boycott and Benedikt Hallgrimsson provided critical review of the manuscript. This work was performed under the Care4Rare Canada Consortium funded by Genome Canada, the Canadian Institutes of Health Research, the Ontario Genomics Institute, Ontario Research Fund, Genome

Quebec and Childrens Hospital of Eastern Ontario Research Foundation. We acknowledge the contribution of the high-throughput sequencing platform of the McGill University and Gnome Qubec Innovation Centre, Montral, Canada. We would like to thank Taila Hartley (Clinical Coordinator) and Chandree Beaulieu (Project Manager) at the Childrens Hospital of Eastern Ontario Research Institute for their contribution to the infrastructure of Care4Rare. D.C.L. is supported by a graduate studentship from Alberta InnovatesHealth Solutions. T.R. is supported in part by a fellowship from the RI MUHCFoundation of Stars at the Montreal Childrens Hospital. L.A.J.-M. is a member of the Research Institute of the McGill University Health Centre, which is supported in part by the Fonds de recherche du QubecSant.

Author contributions

D.C.L. performed variant ltering, validation by Sanger sequencing, qRTPCR in patient broblasts, and was the primary manuscript author. T.R. performed the minigene experiment, contributed gs 3 and 4, and edited the manuscript. J.S. performed bioinformatic analysis and edited the manuscript. E.J.B., E.H.Z., E.G.L., B.N.C., J.P.T., D.R.M., E.P.K., J.H.-F., L.F., R.S. and F.P.B. were responsible for the diagnosis of CCMS in this cohort, critical discussion regarding the CCMS phenotype and edited the manuscript. The Care4Rare Canada consortium performed whole-exome sequencing in patients. A.M.I., R.E.L., J.M., L.A.J.-M., J.S.P. and F.P.B. were involved in study design, data interpretation and editing of the manuscript. F.P.B. conceived the study and edited the manuscipt.

Additional information

Accession codes: Sequence data for CCMS patients have been deposited in the PhenomeCentral repository (https://phenomecentral.org

Web End =https://phenomecentral.org) under the following accession codes: P0000462 60_11-03316 (for Family A II-3), P0000456 60_10-8546 (for Family B II-2), P0000459 60_11-00920 (for Family C II-2), P0000463 60_12-2740 (for FamilyD II-1), P0000435 60_12-4200 (for Family E II-2) and P0000454 60_12-5745(for Family F II-1).

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

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Competing nancial interests: The authors declare no competing nancial interests.

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How to cite this article: Lynch, D. C. et al. Disrupted auto-regulation of the spliceosomal gene SNRPB causes cerebrocostomandibular syndrome. Nat. Commun. 5:4483doi: 10.1038/ncomms5483 (2014).

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Kym Boycott16, Alex MacKenzie16, Jacek Majewski2,3, Michael Brudno17,18, Dennis Bulman16 & David Dyment16

16Childrens Hospital of Eastern Ontario Research Institute, University of Ottawa, 401 Smyth Road, Ottawa, Ontario, Canada K1H 8L1. 17Department of Computer Science, University of Toronto, Ontario, Canada. 18Donnelly Centre and Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada.

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