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Haploinsufficiency of SF3B4, a Component of the Pre-mRNA Spliceosomal Complex, Causes Nager Syndrome

2012; Elsevier BV; Volume: 90; Issue: 5 Linguagem: Inglês

10.1016/j.ajhg.2012.04.004

1537-6605

François P. Bernier, Oana Caluseriu, Sarah Ng, Jeremy Schwartzentruber, Kati J. Buckingham, A. Micheil Innes, Ethylin Wang Jabs, Jeffrey W. Innis, Jane L. Schuette, Jerome L. Gorski, Peter H. Byers, Grégor Andelfinger, Victoria Mok Siu, Julie Lauzon, Bridget A. Fernandez, Margaret J. McMillin, Richard H. Scott, Hilary Racher, Jacek Majewski, Deborah A. Nickerson, Jay Shendure, Michael J. Bamshad, Jillian S. Parboosingh,

Developmental Biology and Gene Regulation

Nager syndrome, first described more than 60 years ago, is the archetype of a class of disorders called the acrofacial dysostoses, which are characterized by craniofacial and limb malformations. Despite intensive efforts, no gene for Nager syndrome has yet been identified. In an international collaboration, FORGE Canada and the National Institutes of Health Centers for Mendelian Genomics used exome sequencing as a discovery tool and found that mutations in SF3B4, a component of the U2 pre-mRNA spliceosomal complex, cause Nager syndrome. After Sanger sequencing of SF3B4 in a validation cohort, 20 of 35 (57%) families affected by Nager syndrome had 1 of 18 different mutations, nearly all of which were frameshifts. These results suggest that most cases of Nager syndrome are caused by haploinsufficiency of SF3B4. Our findings add Nager syndrome to a growing list of disorders caused by mutations in genes that encode major components of the spliceosome and also highlight the synergistic potential of international collaboration when exome sequencing is applied in the search for genes responsible for rare Mendelian phenotypes. Nager syndrome, first described more than 60 years ago, is the archetype of a class of disorders called the acrofacial dysostoses, which are characterized by craniofacial and limb malformations. Despite intensive efforts, no gene for Nager syndrome has yet been identified. In an international collaboration, FORGE Canada and the National Institutes of Health Centers for Mendelian Genomics used exome sequencing as a discovery tool and found that mutations in SF3B4, a component of the U2 pre-mRNA spliceosomal complex, cause Nager syndrome. After Sanger sequencing of SF3B4 in a validation cohort, 20 of 35 (57%) families affected by Nager syndrome had 1 of 18 different mutations, nearly all of which were frameshifts. These results suggest that most cases of Nager syndrome are caused by haploinsufficiency of SF3B4. Our findings add Nager syndrome to a growing list of disorders caused by mutations in genes that encode major components of the spliceosome and also highlight the synergistic potential of international collaboration when exome sequencing is applied in the search for genes responsible for rare Mendelian phenotypes. Nager syndrome (MIM 154400), first described by Nager and De Reynier in 1948,1Nager F. Reynier D. Das Gehorogan bei den angeborenen Kopfmissnildungen.Pract. Otorhinolaryngol. (Basel). 1948; 10: 1-128Google Scholar is the prototype for a group of disorders collectively referred to as the acrofacial dysostoses (AFDs), which are characterized by malformations of the craniofacial skeleton and the limbs (Figure 1).2Halal F. Herrmann J. Pallister P.D. Opitz J.M. Desgranges M.F. Grenier G. Differential diagnosis of Nager acrofacial dysostosis syndrome: Report of four patients with Nager syndrome and discussion of other related syndromes.Am. J. Med. Genet. 1983; 14: 209-224Crossref PubMed Scopus (57) Google Scholar, 3Opitz J.M. Nager “syndrome” versus “anomaly” and its nosology with the postaxial acrofacial dysostosis syndrome of Genée and Wiedemann.Am. J. Med. Genet. 1987; 27: 959-963Crossref PubMed Scopus (21) Google Scholar The major facial features of Nager syndrome include downslanted palpebral fissures, midface retrusion, and micrognathia, the latter of which often requires the placement of a tracheostomy in early childhood. Limb defects typically involve the anterior (i.e., radial) elements of the upper limbs and manifest as small or absent thumbs, triphalangeal thumbs, radial hypoplasia or aplasia, and radioulnar synostosis. Phocomelia of the upper limbs and, occasionally, lower-limb defects have also been reported.4Le Merrer M. Cikuli M. Ribier J. Briard M.L. Acrofacial dysostoses.Am. J. Med. Genet. 1989; 33: 318-322Crossref PubMed Scopus (17) Google Scholar The presence of anterior upper-limb defects as opposed to posterior upper-limb defects and the typical lack of lower-limb involvement distinguishes Nager syndrome from Miller syndrome (MIM 263750), another rare AFD.5Miller M. Fineman R. Smith D.W. Postaxial acrofacial dysostosis syndrome.J. Pediatr. 1979; 95: 970-975Abstract Full Text PDF PubMed Scopus (72) Google Scholar However, distinguishing Nager syndrome from other AFDs, including Miller syndrome, can be challenging, and there are numerous reports of individuals and families that have been difficult for researchers to categorize. Nager syndrome is rare, and, to date, fewer than 100 cases have been reported.6Schlieve T. Almusa M. Miloro M. Kolokythas A. Temporomandibular joint replacement for ankylosis correction in Nager syndrome: Case report and review of the literature.J. Oral Maxillofac. Surg. 2012; 70: 616-625Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Most cases are sporadic, but both autosomal-dominant7Aylsworth A.S. Lin A.E. Friedman P.A. Nager acrofacial dysostosis: Male-to-male transmission in 2 families.Am. J. Med. Genet. 1991; 41: 83-88Crossref PubMed Scopus (30) Google Scholar, 8Hall B.D. Nager acrofacial dysostosis: Autosomal dominant inheritance in mild to moderately affected mother and lethally affected phocomelic son.Am. J. Med. Genet. 1989; 33: 394-397Crossref PubMed Scopus (22) Google Scholar, 9McDonald M.T. Gorski J.L. Nager acrofacial dysostosis.J. Med. Genet. 1993; 30: 779-782Crossref PubMed Scopus (51) Google Scholar and autosomal-recessive10Chemke J. Mogilner B.M. Ben-Itzhak I. Zurkowski L. Ophir D. Autosomal recessive inheritance of Nager acrofacial dysostosis.J. Med. Genet. 1988; 25: 230-232Crossref PubMed Scopus (44) Google Scholar, 11Kennedy S.J. Teebi A.S. Newly recognized autosomal recessive acrofacial dysostosis syndrome resembling Nager syndrome.Am. J. Med. Genet. A. 2004; 129A: 73-76Crossref PubMed Scopus (13) Google Scholar segregation have been reported. This has led to the widespread speculation that Nager syndrome is genetically heterogeneous. The rarity of this syndrome, which typically occurs as a simplex case, and the different modes of inheritance have made identification of the causal gene(s) intractable to conventional gene-discovery approaches. Given the phenotypic overlap between Nager and Miller syndromes and given our recent finding that Miller syndrome is caused by mutations in DHODH (MIM 126064),12Ng S.B. Buckingham K.J. Lee C. Bigham A.W. Tabor H.K. Dent K.M. Huff C.D. Shannon P.T. Jabs E.W. Nickerson D.A. et al.Exome sequencing identifies the cause of a mendelian disorder.Nat. Genet. 2010; 42: 30-35Crossref PubMed Scopus (1484) Google Scholar we and others suspected that the disorders might be allelic or caused by genes in the pyrimidine biosynthetic pathway. However, screening of families affected by Nager syndrome (n > 12) did not reveal any mutations in these genes (Bamshad et al., unpublished data). To identify a gene for Nager syndrome, investigators at the University of Calgary as part of the FORGE (Finding of Rare Disease Genes) Canada Consortium and investigators at the University of Washington (UW) as one of the National Institutes of Health Centers for Mendelian Genomics independently performed exome sequencing.13Bamshad M.J. Ng S.B. Bigham A.W. Tabor H.K. Emond M.J. Nickerson D.A. Shendure J. Exome sequencing as a tool for Mendelian disease gene discovery.Nat. Rev. Genet. 2011; 12: 745-755Crossref PubMed Scopus (1261) Google Scholar Between both institutions, a total of 35 independent families (a total of 41 affected individuals) afflicted with Nager syndrome were available for study (Figure 1 and Table 1, Table 2, Table 3). These families consisted of 28 simplex families, including one in which the parents were consanguineous, and seven multiplex families: two with mother-to-son transmissions, one with a father-to-daughter transmission, two with mother-to-daughter transmissions, one consisting of an affected mother and her two affected sons, and one with a pair of affected monozygotic twins (Table S1, available online). An experienced clinical geneticist was responsible for each diagnosis of Nager syndrome, each participant provided informed consent, and studies were approved by the institutional review boards of the University of Calgary, the University of Washington, and Seattle Children's Hospital.Table 1Clinical Features of Nager-Syndrome Patients A-1 through H-1 with SF3B4 MutationsA-1B-1C-1D-1E-1F-1F-2G-1H-1Inheritancesporadicsporadicsporadicsporadicsporadicsporadic; father of F-2AD; daughtersporadicsporadicGenderfemalefemalemalefemalefemalemalefemalefemalefemaleAge (years)5422423381125Downslanted palpebral fissures++++++ND–NDAbsent lower eyelashessparseNDND+–NDND–+Midface retrusion+++++NDNDNDNDMicrognathia+++++++++Ankylosis of TM jointNDND––NDND+NDAbnormal palatecleft soft palate–cleft–––cleftcleftcleftTracheotomy–+++––++NDAbnormal ears++++mild–NDNDNDHearing loss+++–+++Radial ray abnormality–+absent bilateral––NDND++Abnormal thumbsstiff bilateral+absent bilateralsmall right thumb and slender left thumbhypoplasiaNDNDabsent bilateral+Radioulnar synostosisbilateral+–unilateralbilateral+++NDDevelopmentnormalnormaldelayednormalspeech delay onlynormalNDNDNDOther malformations–VSD and diaphragmatic herniafused 1st and 2nd right metacarpals and bilateral foot deformitiesabnormal teeth, partial absence of left fingers 3–5, slender haluces, and hallux valgusasymmetric face, VPI, and cervical ribsvalgus laxity of knees and anklessubglottic stenosis––SF3B4 mutationc.1A>GaConfirmed de novo mutation.c.864delTaConfirmed de novo mutation.c.1147delCaConfirmed de novo mutation.c.913+1G>Ac.1148dupAc.1A>GbConfirmed familial mutation.c.1A>GbConfirmed familial mutation.c.827dupCc.1232delCThe following abbreviations are used: TM, temporomandibular; ND, not determined; VPI, velopharyngeal insufficiency; AD, autosomal dominant; and VSD, ventriculoseptal defect.a Confirmed de novo mutation.b Confirmed familial mutation. Open table in a new tab Table 2Clinical Features of Nager-Syndrome Patients I-1 through M-3 with SF3B4 MutationsI-1J-1J-2K-1L-1M-1M-2M-3N-1InheritancesporadicAD; sonsporadic; mother of J-1sporadicsporadicsporadic; father of M-2, M-3AD; sonAD; sonSporadicGenderfemalemalefemalefemalefemalefemalemalemalefemaleAge (years)28175615449201713Downslanted palpebral fissures+++ND+++++Absent lower eyelashessparse+–ND+NDsparse++Midface retrusion++NDNDND++++Micrognathia+++ND+–+++Ankylosis of TM jointNDNDNDNDNDNDNDND+Abnormal palate–NDNDND–cleft–+high arched; cleft soft palateTracheotomyND+NDND––+–+Abnormal ears++NDND–cuppedcupped; narrow canalscupped; narrow canals+Hearing loss++NDND+++++Radial ray abnormalityNDNDNDND+––––Abnormal thumbssmall+NDNDabsent left thumb and fused right DIPsmallabsent right thumb and small left thumbsmall right thumb compared to leftabsent right thumb and small and stiff left thumbRadioulnar synostosisNDNDNDND+–right > leftleft+DevelopmentNDnormalNDNDNDNDdelayed speech and fine motordelayeddelayedOther malformations––––limited ROM in all extremities at birth, arachnodactly, and small 5th fingers–short stature (3–5%), dacryostenosis, and small first toes and first metatarsalsdacryostenosis, short first metatarsals, and sandal gaplimited ROM in elbows and shoulders, camptodactyly, left clubfoot, renal abnormalities, VSD, and dacryostenosisSF3B4 mutationc.1060dupCaConfirmed de novo mutation.c.1A>GbConfirmed familial mutation.c.1A>GbConfirmed familial mutation.c.452C>Ac.836_837 insGGGTATGaConfirmed de novo mutation.c.1199delCbConfirmed familial mutation.c.1199delCbConfirmed familial mutation.c.1199delCbConfirmed familial mutation.c.88delTThe following abbreviations are used: TM, temporomandibular; ND, not determined; AD, autosomal dominant; ROM, range of motion; VSD, ventriculoseptal defect; and DIP, distal interphalangeal joint.a Confirmed de novo mutation.b Confirmed familial mutation. Open table in a new tab Table 3Clinical Features of Nager-Syndrome Patients O-1 through T-1 with SF3B4 MutationsO-1O-2P-1Q-1R-1R-2S-1T-1InheritanceAD; sonsporadic; mother of O-1sporadicsporadicAD; daughterAD; mothersporadicsporadicGendermalefemalemalefemalefemalefemalefemalemaleAge (years)128217ND42Downslanted palpebral fissures+++ND+ND++Absent lower eyelashesNDNDdecreasedminimalNDNDNDNDMidface retrusionNDND++++++Micrognathia+++++ND++Ankylosis of TM jointNDNDNDNDNDNDNDNDAbnormal palateabnormal soft palateabnormal soft palateabnormal soft palateabnormal soft palate+NDhigh arched+TracheotomyNDNDNDNDNDND+NDAbnormal ears+++++ND++Hearing loss+++++NDND+Radial ray abnormalityNDND++shortND++Abnormal thumbsabsent right thumb and small left thumbsmallproximally placed and stiffabsent bilateralstiff bilateralstiff bilateral++Radioulnar synostosis–bilateralright–NDNDND+DevelopmentNDND–NDNDNDNDIDOther malformations–––hair extension on cheek, strabismus, mitral valve prolapse, and limited ROM in elbows––short stature and bilateral syndactyly of the 4th and 5th toes–SF3B4 mutationc.1147dupCaConfirmed familial mutation.c.1147dupCaConfirmed familial mutation.c.769delAc.625C>Tc.1252_1258delCTTCGAGnot testedc.796dupAc.661_664dupCCCAThe following abbreviations are used: ID, intellectual disability; TM, temporomandibular; ND, not determined; AD, autosomal dominant; and ROM, range of motion.a Confirmed familial mutation. Open table in a new tab The following abbreviations are used: TM, temporomandibular; ND, not determined; VPI, velopharyngeal insufficiency; AD, autosomal dominant; and VSD, ventriculoseptal defect. The following abbreviations are used: TM, temporomandibular; ND, not determined; AD, autosomal dominant; ROM, range of motion; VSD, ventriculoseptal defect; and DIP, distal interphalangeal joint. The following abbreviations are used: ID, intellectual disability; TM, temporomandibular; ND, not determined; AD, autosomal dominant; and ROM, range of motion. Exome sequencing of five independent simplex cases was performed by the FORGE Canada team at the McGill University and Genome Québec Innovation Centre according to the manufacturer's (Illumina) standard protocols for the Agilent SureSelect 50 Mb exome enrichment kit, and captured targets were sequenced on a HiSeq2000 sequencer. Reads were aligned to a human reference (hg19) with the Burrows-Wheeler Aligner (BWA),14Li H. Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (26648) Google Scholar and indel realignment was performed with the GATK.15McKenna A. Hanna M. Banks E. Sivachenko A. Cibulskis K. Kernytsky A. Garimella K. Altshuler D. Gabriel S. Daly M. DePristo M.A. The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data.Genome Res. 2010; 20: 1297-1303Crossref PubMed Scopus (14776) Google Scholar Duplicate reads were then marked with Picard and excluded from downstream analyses. Coverage of consensus coding sequence (CCDS) bases was assessed with GATK, which showed that all samples had >90% coverage of CCDS bases and a depth of at least 10×. For each sample, single-nucleotide variants (SNVs) and short insertions and deletions (indels) were called with the use of SAMtools pileup16Li H. Handsaker B. Wysoker A. Fennell T. Ruan J. Homer N. Marth G. Abecasis G. Durbin R. 1000 Genome Project Data Processing SubgroupThe Sequence Alignment/Map format and SAMtools.Bioinformatics. 2009; 25: 2078-2079Crossref PubMed Scopus (31559) Google Scholar with the extended base alignment quality (BAQ) adjustment (-E), and they were then quality filtered so that at least 20% of the reads supported the variant call. We annotated variants by using both Annovar17Wang K. Li M. Hakonarson H. ANNOVAR: Functional annotation of genetic variants from high-throughput sequencing data.Nucleic Acids Res. 2010; 38: e164Crossref PubMed Scopus (7872) Google Scholar and custom scripts to identify whether they affected protein-coding sequence and whether they were previously observed in dbSNP132, 1000 Genomes Project data (Nov. 2011), or ∼300 exomes previously obtained at the center. At the University of Washington, exome sequencing was performed on four simplex cases with Nager syndrome and three unrelated individuals from multiplex families; these three latter individuals included one affected child born to an affected parent and two affected parents, each of whom had at least one affected child. In brief, we performed exome capture on a shotgun library created from 1 μg of genomic DNA by using the ∼62 Mb target from Roche Nimblegen SeqCap EZ v2.0 (∼300,000 exons and flanking sequence). Exome capture was followed by massively parallel sequencing on a HiSeq 2000 sequencer (Illumina). BAM files were also aligned to a human reference (hg19) with the BWA,14Li H. Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (26648) Google Scholar and duplicate reads were removed with Picard. Variant detection and genotyping were performed with the UnifiedGenotyper tool from GATK (refv1.529). Exome completion was defined as having >90% of the exome target at >8× coverage. Typically, this requires that the target have a mean coverage of 60–80×. An automated pipeline, the SeattleSeq Annotation Server, was used for the annotation of variants. Novel variants are defined as those not observed in dbSNP version 134 or in 1,200 exomes drawn from a subset of samples from the National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project (ESP). Because the mode of inheritance of Nager syndrome for most cases was unclear, discrete filtering for novel variants in the same gene shared among cases was performed under both autosomal-dominant and autosomal-recessive models. In neither cohort was a gene found to be shared among all the unrelated Nager-syndrome cases under a recessive mode of inheritance, i.e., those who were compound heterozygous for two novel variants or homozygous for a rare novel variant. This suggested that if Nager syndrome were an autosomal-recessive disorder, it would have high locus heterogeneity. Under an autosomal-dominant model, one gene, TTN (MIM 188840), in the FORGE cohort and two genes, MUC12 (MIM 604609) and MUC16 (MIM 606154), in the UW cohort had novel variants shared among all unrelated cases. These large genes are frequent sources of false-positive calls and were excluded from consideration. In both cohorts, several genes had novel or rare variants shared among two, three, or four cases with Nager syndrome, and prioritizing these candidates was not possible. We further filtered the gene list from the FORGE cohort by assigning higher priority to variants predicted to have a greater deleterious impact on protein function (i.e., nonsense, frameshift, initiation-codon, and splice-site variants) and then reanalyzed the list. A single gene, SF3B4 (MIM 605593), which encodes a component of the U2SNP of the major spliceosome, was found to have different novel variants in two unrelated cases of Nager syndrome (Table S2). In the UW cohort, subsequent discrete filtering limited to only genes with novel variants shared among the three familial-Nager-syndrome cases identified three candidate genes, including SF3B4. In two of these cases, the variant identified (c.1A>G [p.M1?]) was identical to one of the SF3B4 variants found in the FORGE cohort. Sanger sequencing subsequently confirmed that all four novel variants were present in SF3B4 and that the variants were de novo in the FORGE simplex cases and inherited from an affected parent (one case) and transmitted to an affected child (one case) in the UW families. Further evidence supporting the hypothesis that mutations in SF3B4 cause Nager syndrome was provided by a recent finding that mutations in EFTUD2 (MIM 603892), which also encodes a component of the spliceosome, cause a mandibulofacial dysostosis with microcephaly (MFDM [MIM 610536]) whose craniofacial findings overlap the symptoms of Nager syndrome.18Lines M.A. Huang L. Schwartzentruber J. Douglas S.L. Lynch D.C. Beaulieu C. Guion-Almeida M.L. Zechi-Ceide R.M. Gener B. Gillessen-Kaesbach G. et al.FORGE Canada ConsortiumHaploinsufficiency of a spliceosomal GTPase encoded by EFTUD2 causes mandibulofacial dysostosis with microcephaly.Am. J. Hum. Genet. 2012; 90: 369-377Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar Furthermore, an individual had previously been reported to have an unclassifiable AFD and a 1q21.2 deletion spanning the region containing SF3B4.19Waggoner D.J. Ciske D.J. Dowton S.B. Watson M.S. Deletion of 1q in a patient with acrofacial dysostosis.Am. J. Med. Genet. 1999; 82: 301-304Crossref PubMed Scopus (23) Google Scholar Collectively, these results strongly suggest that mutations in SF3B4 cause Nager syndrome. To determine the extent to which SF3B4 mutations explain cases of Nager syndrome, we used Sanger sequencing to screen SF3B4 in a validation cohort of 23 additional unrelated families affected by Nager syndrome. We also screened the eight individuals who underwent exome sequencing but in whom no SF3B4 mutation was found. Collectively, between the initial study cohort and our validation studies, SF3B4 mutations were found in 20 of the 35 (57%) families, including five of the seven families with more than one individual affected by Nager syndrome. For simplex cases, 15 of 28 (54%) had mutations in SF3B4, and in each of the five simplex cases for which DNA was available from both parents, the SF3B4 mutation was confirmed to have arisen de novo. No SF3B4 mutation was identified in the single consanguineous family in our cohort. Overall, 25 out of 41 (61%) Nager-syndrome-affected individuals in our cohort had mutations in SF3B4 (Table 1, Table 2, Table 3 and Table S1). A total of 18 unique mutations were identified, and these include 14 frameshift, two nonsense, one splicing, and one missense mutation (c.1A>G [p.Met1?]), the last of which is predicted to abolish the methionine initiation codon and was the only recurrent (i.e., in three unrelated families) mutation found (Table 4 and Figure 2). Four of the Nager-syndrome cases with a SF3B4 variant that was missed by exome sequencing were found to have indels, and manual inspection of the BAM files revealed that they all occurred in regions with relatively low coverage (i.e., 10,800 chromosomes sequenced as part of the NHLBI-ESP (see Web Resources). Indeed, only 22 rare (minor allele frequency of G1p.Met1?de novo (A) and familial (F and J)loss of initiator methionineNc.88delT2p.Trp30Glyfs∗10NAframeshiftKc.452C>A3p.Ser151∗NAnonsenseQc.625C>T3p.Gln209∗NAnonsenseTc.661_664dupCCCA3p.Asn222Thrfs∗265NAframeshiftPc.769delA4p.Ile257Tyrfs∗63NAframeshiftSc.796dupA4p.Met266Asnfs∗220NAframeshiftGc.827dupC4p.Ser277Ilefs∗209NAframeshiftLc.836_837insGGGTATG4p.Thr280Glyfs∗208de novoframeshiftBc.864delT4p.His288Glnfs∗32de novoframeshiftDc.913+1G>Aintron 4NANAspliceIc.1060dupC5p.Arg354Profs∗132de novoframeshiftCc.1147delC6p.His383Metfs∗75de novoframeshiftOc.1147dupC6p.His383Profs∗103familialframeshiftEc.1148dupA6p.His383Glnfs∗103NAframeshiftMc.1199delC6p.Pro400Leufs∗58familialframeshiftHc.1232delC6p.Pro411Glnfs∗47NAframeshiftRc.1252_1258delCTTCGAG6p.Leu418Alafs∗38familialaMother's sample was not available for testing.frameshiftThe following abbreviation is used: NA, parental DNAs were unavailable for the confirmation of inheritance.a Mother's sample was not available for testing. Open table in a new tab The following abbreviation is used: NA, parental DNAs were unavailable for the confirmation of inheritance. All of the SF3B4 variants identified are predicted to encode a truncated polypeptide chain or an elongated polypeptide with an altered 3′ end in the absence of nonsense-mediated RNA decay (Figure 2C). Together with the observation that a deletion of chromosomal region 1q12–q21.1 or q21.3, which encompasses SF3B4, has been reported in a child with characteristics of Nager syndrome,19Waggoner D.J. Ciske D.J. Dowton S.B. Watson M.S. Deletion of 1q in a patient with acrofacial dysostosis.Am. J. Med. Genet. 1999; 82: 301-304Crossref PubMed Scopus (23) Google Scholar our findings suggest that Nager syndrome probably results from haploinsufficiency of SF3B4. SF3B4 encodes SAP49, a spliceosomal protein that is one of seven core proteins of the mammalian SF3B complex and is highly conserved with two RNA recognition motifs (RRMs) followed by a proline-glycine rich domain (Figure 2B). During assembly of the U2SNP prespliceosomal complex, SAP49 binds to the pre-mRNA just upstream of the branch point sequence but also interacts specifically with other U2 snRNPS, particularly SAP145, suggesting that SAP49 plays a crucial role in tethering the U2 snRNP to the branch site.20Champion-Arnaud P. Reed R. The prespliceosome components SAP 49 and SAP 145 interact in a complex implicated in tethering U2 snRNP to the branch site.Genes Dev. 1994; 8: 1974-1983Crossref PubMed Scopus (95) Google Scholar Our discovery adds Nager syndrome to an emerging group of disorders caused by mutations in genes that encode subunits of the spliceosome. Mutations in splicing factors were initially reported in patients with autosomal-dominant retinitis pigmentosa.21McKie A.B. McHale J.C. Keen T.J. Tarttelin E.E. Goliath R. van Lith-Verhoeven J.J. Greenberg J. Ramesar R.S. Hoyng C.B. Cremers F.P. et al.Mutations in the pre-mRNA splicing factor gene PRPC8 in autosomal dominant retinitis pigmentosa (RP13).Hum. Mol. Genet. 2001; 10: 1555-1562Crossref PubMed Scopus (228) Google Scholar, 22Tanackovic G. Ransijn A. Ayuso C. Harper S. Berson E.L. Rivolta C. A missense mutation in PRPF6 causes impairment of pre-mRNA splicing and autosomal-dominant retinitis pigmentosa.Am. J. Hum. Genet. 2011; 88: 643-649Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 23Vithana E.N. Abu-Safieh L. Allen M.J. Carey A. Papaioannou M. Chakarova C. Al-Maghtheh M. Ebenezer N.D. Willis C. Moore A.T. et al.A human homolog of yeast pre-mRNA splicing gene, PRP31, underlies autosomal dominant retinitis pigmentosa on chromosome 19q13.4 (RP11).Mol. Cell. 2001; 8: 375-381Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 24Zhao C. Bellur D.L. Lu S. Zhao F. Grassi M.A. Bowne S.J. Sullivan L.S. Daiger S.P. Chen L.J. Pang C.P. et al.Autosomal-dominant retinitis pigmentosa caused by a mutation in SNRNP200, a gene required for unwinding of U4/U6 snRNAs.Am. J. Hum. Genet. 2009; 85: 617-627Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar Subsequently, biallelic mutations in RNU4ATAC (MIM 601428), a component of the minor spliceosome required for excision of the U12 class of introns, were reported to cause lethal microcephalic osteodysplastic primordial dwarfism type I (MIM 210710).25He H. Liyanarachchi S. Akagi K. Nagy R. Li J. Dietrich R.C. Li W. Sebastian N. Wen B. Xin B. et al.Mutations in U4atac snRNA, a component of the minor spliceosome, in the developmental disorder MOPD I.Science. 2011; 332: 238-240Crossref PubMed Scopus (171) Google Scholar, 26Nagy R. Wang H. Albrecht B. Wieczorek D. Gillessen-Kaesbach G. Haan E. Meinecke P. de la Chapelle A. Westman J. Microcephalic osteodysplastic primordial dwarfism type I with biallelic mutations in the RNU4ATAC gene.Clin. Genet. 2011; (Published online August 4, 2012)https://doi.org/10.1111/j.1399-0004.2011.01756.xCrossref PubMed Scopus (31) Google Scholar Most recently, mutations in EFTUD2 have been reported in individuals with MFDM, a disorder with many features that overlap those of Nager syndrome.18Lines M.A. Huang L. Schwartzentruber J. Douglas S.L. Lynch D.C. Beaulieu C. Guion-Almeida M.L. Zechi-Ceide R.M. Gener B. Gillessen-Kaesbach G. et al.FORGE Canada ConsortiumHaploinsufficiency of a spliceosomal GTPase encoded by EFTUD2 causes mandibulofacial dysostosis with microcephaly.Am. J. Hum. Genet. 2012; 90: 369-377Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar Although the limbs are typically normal in MFDM, the fact that some individuals have thumb abnormalities suggests that Nager syndrome and MFDM could be confused with one another.18Lines M.A. Huang L. Schwartzentruber J. Douglas S.L. Lynch D.C. Beaulieu C. Guion-Almeida M.L. Zechi-Ceide R.M. Gener B. Gillessen-Kaesbach G. et al.FORGE Canada ConsortiumHaploinsufficiency of a spliceosomal GTPase encoded by EFTUD2 causes mandibulofacial dysostosis with microcephaly.Am. J. Hum. Genet. 2012; 90: 369-377Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar To this end, we screened EFTUD2 in all of the UW cohort Nager-syndrome individuals who did not have mutations in SF3B4, and we identified a single individual with a novel nonsense mutation (c.2495C>G [p.Tyr832∗]). This mutation affects the amino acid residue adjacent to an EFTUD2 mutation (c.2493C>A [p.Tyr831∗]) found in one family with MFDM.18Lines M.A. Huang L. Schwartzentruber J. Douglas S.L. Lynch D.C. Beaulieu C. Guion-Almeida M.L. Zechi-Ceide R.M. Gener B. Gillessen-Kaesbach G. et al.FORGE Canada ConsortiumHaploinsufficiency of a spliceosomal GTPase encoded by EFTUD2 causes mandibulofacial dysostosis with microcephaly.Am. J. Hum. Genet. 2012; 90: 369-377Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar In retrospect, this individual also had microcephaly, suggesting that MFDM rather than Nager syndrome is the appropriate diagnosis. It is clear that SAP49 and U5-116KD, encoded by EFTUD2, are critical components of the major spliceosome, but their role in the pathogenesis of defects in craniofacial and limb development is unknown. The mouse homolog of SF3B4, SAP49 (mSAP49), is highly conserved and shows broad expression in adult tissues.27Ruiz-Lozano P. Doevendans P. Brown A. Gruber P.J. Chien K.R. Developmental expression of the murine spliceosome-associated protein mSAP49.Dev. Dyn. 1997; 208: 482-490Crossref PubMed Scopus (17) Google Scholar, 28Nishanian T.G. Waldman T. Interaction of the BMPR-IA tumor suppressor with a developmentally relevant splicing factor.Biochem. Biophys. Res. Commun. 2004; 323: 91-97Crossref PubMed Scopus (22) Google Scholar Whole-mount in situ hybridization shows high expression at day 11 after conception in limbs and somites, as well as dynamic patterns of expression in the developing heart.27Ruiz-Lozano P. Doevendans P. Brown A. Gruber P.J. Chien K.R. Developmental expression of the murine spliceosome-associated protein mSAP49.Dev. Dyn. 1997; 208: 482-490Crossref PubMed Scopus (17) Google Scholar These results suggest that mSAP49 expression during development varies in both a time- and tissue-specific manner. Whereas limb malformations are frequently seen in Nager syndrome, the fact that only 2 out of 20 (10%) SF3B4-mutation carriers had cardiac defects suggests that SF3B4 haploinsufficiency has only minor effects on human heart development. Spliceosomes are not only critical for mediating intron splicing but are also key regulators of alternative splicing29Johnson T.L. Vilardell J. Regulated pre-mRNA splicing: The ghostwriter of the eukaryotic genome.Biochim Biophys Acta. 2012; (Published online January 9, 2012)Google Scholar, 30Blaustein M. Pelisch F. Srebrow A. Signals, pathways and splicing regulation.Int. J. Biochem. Cell Biol. 2007; 39: 2031-2048Crossref PubMed Scopus (75) Google Scholar and, as such, play an important role in the control of gene-expression pathways. Furthermore, alternative splicing of mRNA is a major source of protein diversity, and tissue-specific alternative splicing further increases the diverse cellular functions of proteins. Spliceosomes might directly regulate developmental genes via the control of splicing or tissue specificity. Thus, the defects observed in individuals with Nager syndrome could be due to aberrant splicing of genes involved in craniofacial and limb development. However, this hypothesis is not supported by evidence from sf3b1+/−-heterozygous-null mice. Isono et al.31Isono K. Mizutani-Koseki Y. Komori T. Schmidt-Zachmann M.S. Koseki H. Mammalian polycomb-mediated repression of Hox genes requires the essential spliceosomal protein Sf3b1.Genes Dev. 2005; 19: 536-541Crossref PubMed Scopus (96) Google Scholar reported that Sf3b1+/− heterozygous mice exhibited skeletal malformations concomitant with ectopic Hox expression. Because polycomb group (PcG) proteins are required for the stable repression of Hox, Isono et al. measured the transcript levels for five PcG genes and three Hox genes.31Isono K. Mizutani-Koseki Y. Komori T. Schmidt-Zachmann M.S. Koseki H. Mammalian polycomb-mediated repression of Hox genes requires the essential spliceosomal protein Sf3b1.Genes Dev. 2005; 19: 536-541Crossref PubMed Scopus (96) Google Scholar Despite Sf3b1 expression levels that were reduced by half in the sf3b1+/− mice, transcript levels of the PcG and Hox genes were similar to those of the controls. Thus, the homeotic transformations in sf3b1+/− mice appear to be independent of alterations in overall gene transcription. Further evidence that mutations in SF3B4 might cause Nager syndrome via a mechanism unrelated to its role in mediating splicing is provided by studies linking SAP49 to bone morphogenetic protein (BMP) signaling. BMP proteins are multifunctional growth factors with roles in early embryogenesis and skeletogenesis. BMP2 and BMP4 are the main sources of BMP signaling in the developing limbs and also play a critical role in chondrogenesis.32Robert B. Bone morphogenetic protein signaling in limb outgrowth and patterning.Dev. Growth Differ. 2007; 49: 455-468Crossref PubMed Scopus (70) Google Scholar To find molecules specific to BMP-mediated signal transduction, Watanabe et al.33Watanabe H. Shionyu M. Kimura T. Kimata K. Watanabe H. Splicing factor 3b subunit 4 binds BMPR-IA and inhibits osteochondral cell differentiation.J. Biol. Chem. 2007; 282: 20728-20738Crossref PubMed Scopus (39) Google Scholar identified SAP49 by using BMPR-IA, a BMP receptor, as a bait protein in a yeast two-hybrid screen. Coimmunoprecipitation and immunoblot analysis confirmed their interaction in mammalian cells.33Watanabe H. Shionyu M. Kimura T. Kimata K. Watanabe H. Splicing factor 3b subunit 4 binds BMPR-IA and inhibits osteochondral cell differentiation.J. Biol. Chem. 2007; 282: 20728-20738Crossref PubMed Scopus (39) Google Scholar SAP49 is also found in cell-membrane fractions, and overexpression of SAP49 inhibits BMP-2-mediated osteogenic and chondrocytic differentiation. These findings suggest that SAP49, in addition to its role in mRNA splicing, might also specifically inhibit BMP-mediated osteochondral cell differentiation33Watanabe H. Shionyu M. Kimura T. Kimata K. Watanabe H. Splicing factor 3b subunit 4 binds BMPR-IA and inhibits osteochondral cell differentiation.J. Biol. Chem. 2007; 282: 20728-20738Crossref PubMed Scopus (39) Google Scholar In summary, we applied exome sequencing across 12 unrelated cases to discover that mutations in SF3B4 cause Nager syndrome and explain ∼60% of the cases in our overall cohort, including five out of seven multiplex families and 54% of simplex cases. The clinical characteristics of individuals with Nager syndrome caused by SF3B4 mutations were indistinguishable from those without SF3B4 mutations. Although partial or whole-gene deletions or mutations in noncoding regulatory regions of SF3B4 might explain some of the SF3B4 mutation-negative cases in our cohorts, this observation is strong evidence that Nager syndrome is genetically heterogeneous. Our findings also indicate that the phenotypic overlap between AFD Nager syndrome and MFDM is probably due in part to a shared defect in the same biological process and suggests that similar clinical phenotypes for which the cause has yet to be identified might be due to variants in genes that have a similar role. We thank the families for their participation and the Foundation for Nager and Miller Syndromes for their support. Our work was supported in part by grants from the National Institutes of Health (NIH) National Human Genome Research Institute (1U54HG006493 to M.B., D.N., and J.S.; 1RC2HG005608 to M.B., D.N., and J.S.; and 5RO1HG004316 to H.T.), the Life Sciences Discovery Fund (2065508 and 0905001), the Washington Research Foundation, and the NIH National Institute of Child Health and Human Development (1R01HD048895 to M.B.). S.B.N. is supported by the Agency for Science Technology and Research, Singapore. The FORGE (Finding of Rare Disease Genes) Steering Committee includes Kym Boycott (leader; University of Ottawa), Jan Friedman (coleader; University of British Columbia), Jacques Michaud (coleader; Université de Montréal), Francois Bernier (University of Calgary), Michael Brudno (University of Toronto), Bridget Fernandez (Memorial University), Bartha Knoppers (McGill University), Mark Samuels (Université de Montréal), and Steve Scherer (University of Toronto). FORGE CANADA was funded by the Government of Canada through Genome Canada, the Canadian Institutes of Health Research (CIHR), and the Ontario Genomics Institute (OGI-049). Additional funding was provided by Genome Québec and Genome British Columbia. O.C. and H.R. are supported by the CIHR Training Program in Genetics, Child Development, and Health. G.A. is a Clinician Scientist of the CIHR and is supported by the Fondation Nussia et André Aisenstadt. Download .pdf (.05 MB) Help with pdf files Document S1. Tables S1 and S2 and supplemental acknowledgments The URLs for data presented herein are as follows:Exome Variant Server, http://evs.gs.washington.edu/EVS/FASTX Toolkit, http://hannonlab.cshl.edu/fastx_toolkit/GATK, http://www.broadinstitute.org/gsa/wiki/Human Genome Varation, http://www.hgvs.org/mutnomen/Online Mendelian Inheritance in Man (OMIM), http://www.omim.org/Picard Tools, http://picard.sourceforge.net/SAMtools, http://samtools.sourceforge.net/SeattleSeq, http://snp.gs.washington.edu/SeattleSeqAnnotation/ The RefSeq accession number for the SF3B4 sequence reported in this paper is NM_005850.4.