7 Available online at www.sciencedirect.com -,, JOURNALOF ·:;- ScienceDirect GENETICS AND GENOMICS EI..SEVIER J. Genet. Genomics 36 (2009) 251-256 • www.jgenetgenornics.org Mutational screening of BASP 1 and transcribed processed pseudogene TP'Pg-BASP 1 in patients with Möbius syndrome 8 Abdullah Uzumcu 8, Sukru Candan , Guven Toksoy a, h, Z. Oya Uyguner 8, Birsen Karaman 8, Hacer Eris 8, Burak Tatli C, Hulya Kayserili 8, Adnan Yuksel d, Bilge Geckinli h, Memnune Yuksel-Apak 8, Seher Basaran a, • 'Department of Medica/ Genetics, /stanbul Medica/ Faculty, lstanbul University, lstanbul 34390, Turkey b Department ofGenetics, Zeynep Kamil Gyneco/ogic and Pediatric Training and Research Hospital, /stanbul 34668, Turkey • Division ofNeuro/ogy, Department ofPediatrics, /sıanbul Medica/ Facu/ty, lstanbul University, Istanbul 34390, Turkey d Department ofMedical Genetics, Ce"ahpasa Medica/ Faculty, lstanbul University, lstanbul 34098, Turkey Received for publication 11 July 2008; revised 30 December 2008; accepted 21 January 2009 Abstract Möbius syndrome is a rare disorder primarily characterized by congenital facia! palsy, frequently accompanied by ocular abduction anomalies and occasionally associated with orofacial, limb and musculoskeletal malfonnations. Abnormal development of cranial nerves V through XII underlines the disease pathogenesis. Although a genetic etiology for Möbius syndrome was proposed, molecular genetic studies to identify the causative gene(s) are scarce. in this study, we selected two candidate genes. One is BASPJ residing in a hwnan chromosome 5pl5.l-pl5.2, syntenic to mouse chromosome 15qA2-qB2, to which a mouse model with facia! nerve anomalies was mapped. The other is transcribed processed pseudogene TP'l'g-BASP 1, which is located on chromosome 13q flanking the putative locus for Möbius syndrome and might be involved in the regulation of the transcripts encoded by BASP 1. Mutation analyses in nineteen pa­ tients excluded these genes as being candidates for Möbius syndrome. Keywords: Möbius syndrome; facia! palsy; candidate gene; BASPI; transcribed processed pseudogene; non-coding RNA; mutation screening Introduction of the syndrome, whereas abducens nerve (CN VI) and hypoglossal nerve (CN XII) involvements are relatively Möbius syndrome (MBS) is a rare genetic disorder common (Strömland et al., 2002; Verzijl et al., 2003; Ames characterized by congenital facial palsy, frequently ac­ et al., 2005; Briegel, 2006). Although not yet completely companied by ocular abduction anomalies and occasion­ established, it has been suggested that the pathologicaJ ally associated with orofacial, limb and musculoskeletal mechanism underlying MBS is consistent with rhomben­ malformations. Abnormal development of cranial nerves cephalic maldevelopment involving predominantly motor (CNs) V through XII might be involved in the disease nuclei and axons, as well as traversing Iong tracts (Verzijl pathogenesis, either unilaterally or bilaterally. Primarily, et al., 2003). The etiology of MBS remains elusive. Vas­ abnormality in facia! nerve (CN VII) is the typical feature cular, teratogenic and genetic etiologies were proposed. Theories of vascular etiologies are mainly associated with • Corresponding author. Tel: +90-212-631 1363; Fax: +90-212-260 2283. disruption ofblood flow in the basilar artery (Samat, 2004), E-mail address: [email protected] premature regression of the primitive trigeminal arteries DOi: I0.1016/Sl673-8527(08)60112-5 ~------------~J l 252 Abdullah Uzumcu et al./ Journal o/Genelics and Genomics 36 (2009) 251-256 (Huerva et al., 1992), or disruption ofthe subclavian-artery mapped in a mouse model (Mar et al., 2005). TP'l'g­ supply (St Charles et al., 1993), alt of which interrupt the BASP I, which encodes for a non-coding RNA (ncRNA), embryonic blood supply. Strong association between ma­ was selected due to both relevancy of its location to the temal use of misoprostol and MBS was reported (Pas­ putative MBS locus on l 3q and its hypothesized role in the tuszak et al., 1998). Ergotamine (Smets et al., 2004) and regulation of BASP I transcripts. cocaine (Puvabanditsin et al., 2005) exposures during early fetal development were also suggested to be among the possible causes ofMBS. Material and methods A reciprocal translocation between chromosomes 1p34 and 13q 13 cosegregating with congenital facial diplegia Clinical data and flexion finger contractures in seven family members over three generations (Ziter et al., 1977) and a deletion of Nineteen patients with MBS were clinically evaluated chromosome l 3q 12.2 in a 2.5-year-old gir! with MBS with the collaboration between Department of Medical were reported (Slee et al., 1991). Therefore, it was sug­ Genetics, and Department of Pediatric Neurology, Istanbul gested that the gene responsible for MBS might be located Medical Faculty, Istanbul University, Turkey; Department on chromosome 13ql2.2-ql3. of Medical Genetics, Cerrahpasa Medical Faculty, lstanbul in this study, we screened two candidate genes, BASP I University; Department of Genetics, Zeynep Kamil Gyne­ and transcribed processed pseudogene BASP I (TP'l'g­ cologic and Pediatric Training and Research Hospital, BASP J}, in nineteen patients diagnosed with MBS. BASPI Turkey. Clinical findings of the patients are presented was selected because its orthologous gene is located within in Table 1. AII the patients had cytogenetically normal the region where the causative gene responsible for ab­ karyotype, except for MBS-12 who was found normal sprouting or branching of the facia! nerve was 46, XX.[13]/47, XXX:[19] mosaic. Table 1 Clinical findings ofMöbius syndrome patients included in the present study ~S~S~S~S~S~S~S~S~S~S~S~S~S~S~S~S~SMBSMBS Patienls -1 -2 -3 -4 -5 -o -7 -8 -9 -10 -11 -12 -13 -14 -15 -16 -17 -18 -19 Gender F F M F M M M F F M F F F F M M F M M Age(years+ 4+o 3+3 1+3 2+1 2+7 5+7 5+10 3+o 0+10 0+7 1+6 1+7 7+o 3+8 8+8 3+7 9+o O+IO 13+7 montlıs) CN VIl palsy +/+ -/+ +/- +/- +/+ +/+ +/+ +/- +/+ +/- +/+ +/+ +/+ -/+ +/+ -/+ -/+ -/+ +/+ (R/L) CNVIpalsy +/+ -/+ +/+ +/+ +/+ +/+ +{- +!+ - +{+ +{+ +{+ - +/+ (R/L) CN XII palsy +/+ - +/- -/+ -1+ +{+ +{+ (R/L) CN lII palsy +!+ +!- +/+ +/- - +{+ +{+ +{+ (R/L) CN IVpalsy +{+ - (R/L) CNVpalsy + + + CNIX/X + + + + + palsies Orofııcial + + + + + + + + + + + + + + + + + + + dysmorphisın Limb + + + + + + + + + + + + + + defonnities Poland + + + + anomaly Hypogonadism + CN, cranial neıve; F, female; M, male; R, right; L, left. Abdullah Uzumcu et al./Journal ofGenetics and Genomics 36 (2009) 251-256 253 DNA iso/ation Sequencing Peripheral blood samples were collected following Sequence analyses were carried out using an automated the approval of the informed consents by the family sequencer (Applied Biosystems 3730xl, Macrogen ine., members and genomic DNA was isolated using standard Seoul, Korea). procedures. Amp/ification ofgenomic sequences Results and discussion Two mechanisms are suggested for the pathogenesis of Both coding (chr5:17328224-17329420) and non-coding MBS: 1) the interruption of the vascular supply of the exons of BASP 1 gene including putative promoter region brainstem by environmental, mechanical, and genetic juxtaposing the transcription start site ( chr5: l 7270004- causes, which would result in ischaemia predominantly 17271027), the genomic region containing TP'Pg-BASP 1 disrupting the CN VII (St Charles et al., 1993; Graf and (chrl3:22368979-22370339), and STR markers D5S2946 Shepard, 1997; Pastuszak et al., 1998; Sarnat, 2004; Smets (chr5:17293088-17293313) and D13Sl863 (chrB:22374359- et al., 2004); 2) rhombomeric maldevelopment including 22374579, UCSC Genome Browser, www.genome.ucsc. motor nuclei and axons due to a genetic defect (Verzijl et edu) were amplified from genomic DNA by touch-down al., 2003). Families with MBS showing either autosomal PCR. A final volume of 25 µL reaction mixture containing and gonosomal inheritance pattems were described 100 ng of genomic DNA, 1 x PCR butfer with (Nl4) SO , 2 4 (Krueger and Friedrich, 1963; Legum et al., 1981; Dotti et 1.5 mmol/L MgClı, 200 µmol/L dNTP, 400 µmol/L of al., 1989; Joumel et al., 1989). A putative locus for MBS each primer, 0.75 U Taq polymerase (MBI Fermentas, was reported to be located on chromosome 13ql2.2-ql3 in Elips, Turkey), and 4%-10% DMSO (AppliChem, Dateks, regards to chromosomal abnormalities identified in two Turkey) with an initial denaturation at 94°C for 4 min fol­ studies (Ziter et al., 1977; Slee et al., 1991). However, up lowed by 34-38 cycles with denaturation at 94°C for 30 s, to date, only PLXNDJ gene, located in hereditary con­ annealing between 62°C and 52°C for 30 s, and extension genital facia! paresis l (HCFPl) locus at 3q21-q22, was at 70°C for 60 s, and a final extension at 70°C for 7 min screened and excluded in isolated MBS patients (van der using a DNA Engine PTC-200 Thermal Cycler (BioRad, Zwaag et al., 2004). Medtek, Turkey). Primer sequences used in this study were A locus responsible for abnormal sprouting or branch­ as follows: BASPl-lF, 5'-GAGGTAGCGGAATCGTAGT ing of the facial and acoustic nerves, severely reduced CG-3'; BASPl-lR, 5'-CGCTGCATGAAGGGAGAG-3'; lower jaw, smaller head, and paucity of blood was mapped BASP1-2F, 5'-CCCAGAACCCCTTGCTTT-3'; BASP1-2R, within the region tlanked by Dl 5MIT280 and Dl 5MITI il 5' -CATCACTCCCAACTCCCATT-3'; PseudoBASPl-F, on mouse chromosome 15qA2-qB2 (Mar et al., 2005), 5' -GGTGGCAGTAGTGGCAGCT-3'; PseudoBASPl-R, which is syntenic to human chromosome 5pl5.l-pl52 5'-GTTGTITAGAGTCATTGGTCAGGC-3'; D5S2946-F, where human cri-du-chat syndrome locus resides. The 5'-CCACCAAAATTCCTACGTTG-3'; D5S2946-R, 5'-CT phenotype of those mice basically resembles the clinical CACCCCAGCTTCAAACT-3'; D13Sl863-F, 5'-GCAT features of MBS and the mutant gene might thus be hy­ GGACTGAACTAACAGA-3'; Dl3Sl863-R, 5'-CCTA pothesized to underlie the syndrome. Computational analyses revealed that the comparable GGTGACAGTGCGA-3'. human critical region was of ~7 Mb in length and located between CCT5 and BASP 1 genes, where a total of sixteen Polyacry/amide gel e/ectrophoresis genes reside (UCSC Genome Browser, www.genome.ucsc.