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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. edu). Since hemizygosity of CTNND2 gene due to large PCR products obtained by STR markers were run on 5p-tenninal deletion in a case with cri-du-chat syndrome 10% (19:1) polyacrylamide gels and the alleles were and severe mental retardation without congenital facia! genotyped independently by two co-workers. Marlcers in palsy was reported (Medina et al., 2000), the distal part of apparently homozygous individuals were also sequenced the critical region including CTNND2 gene and five of tlıe to detennine the presence of heterozygous SNPs that indi­ other genes was theoretically ex~luded. Among the remain­ cate the double doses of the Ioci. ing ten genes, BASP J was considered to be the candidate 254 Abdullah Uzumcu et al./ Journal ofGenetics and Genomics 36 (2009) 251-256

gene by functional relevancy. BASP 1 gene is expressed quence of NAP-22 protein contains PEST sequences predominantly in the brain and encodes for NAP-22 (Mosevitsky et al., l 997) that serve as proteolytic signals (CAP-23) protein. Protein NAP-22 is especially abundant in in proteins with high tumover rates (Rechsteiner and growth cones at axon tennini (Iino et al., 2004 ). When pro­ Rogers, 1996). Since NAP-22 protein is likely to be rapidly duced, NAP-22 protein undergoes N-myristoylation and degraded, its mRNA might be protected against non­ subsequently adjoins brain-derived membrane raft in a mediated mRNA decay via an interaction between BASP 1 cholesterol-dependent manner and functions in the and TP'l'g-BASP 1 on RNA level and thus, be continuously fonnation of cholesterol-rich domains in membranes (Khan translated. This speculation tempted us to select TP'l'g­ et al., 2003). NAP-22 co-localizes with functionally related BASP 1 as the candidate gene beside the BASP 1 gene. proteins GAP-43 and MARCKS, interacts with calmodulin lnterestingly, TP'l'g-BASP 1 is located on chromosome and protein kinase C at the plasmalemma, and regulates l 3q 12.12. Because of the limited resolution of GTG­ actin binding proteins and, thus, actin dynamics in regard to banding, del(l3)(ql2.I-ql2.2) and del(l3)(ql2.2-I2.3) extracellular signals (Maekawa et al., l 994; Frey et al., 2000; could not be distinguished clearly (Shaffer and Tommerup, Laux et al., 2000). Therefore, NAP-22 is considered to be 2005). Therefore, it is possible to think that the regions involved in neuronal sprouting, anatomical plasticity, and neighboring chromosome 13ql2.2-ql3 such as 13ql2.12 axonal pathfinding (Frey et al., 2000; Laux et al., 2000). might harbor the gene responsible for MBS. Baspl-ı-mice survived to adulthood exhibited enlarged Sequence analyses of BASP 1 covering the promoter re­ ventricles in the brain, axonal and synaptic abnonnalities in gion in the patient group did not reveal any alteration. the hippocampus and neo-cortex, as well as abnonnalities at Heterozygous genotypes for STR marker D5S2946 resid­ peripheral nerve tennini and neuromuscular endplates (Frey ing in the intron of BASP 1 excluded the hemizygosity in et al., 2000). Therefore, the causative gene in the mouse twelve patients, whereas seven patients were apparently model with abnonnal sprouting of facia! nerves mapped to homozygous {Table 2). Screening of the region 15qA2-qB2 might be different from Baspl. However, the containing TP'l'g-BASPJ revealed a de novo ALl57931: gain-of-function mutations or the mutations leading to ec­ g.8908_8909delAG variation in heterozygous state in topic expression of Baspl could not be excluded. MBS-1 (Fig. 1). The presence of SNP rs7489390 in het­ in silico analyses of BASP 1 gene sequence revealed a erozygous state in MBS-1 indicated that ALI 57931: transcribed processed pseudogene, TP'l'g-BASP 1 [Entrez g.8908_8909delAG variation occurred in the germline gene: LOC646201, UniGene: Hs.646643 {www.ncbi.nlm. cells of the father who was homozygous for SNP nih.gov); Ensembl gene: OTIHUMG00000016557 {www. rs7489390 {Fig. 1, B and C). Since this variation was not ensembl.org)], located on chromosome l3ql2.l2. Proc­ located in the predicted transcript [Entrez nucleotide: essed pseudogenes {P'Pgs) are intronless sequences de­ XR_016396 (www.ncbi.nlm.nih.gov); Ensembl transcript: rived from retrotransposition of predominantly spliced OTTHUMT00000044 I 43 (www.ensembl.org)], it was mRNAs with resistance to nonsense-mediated RNA decay considered to be a polymorphism. Heterozygosity for dif­ via reverse transcription and insertion into the nuclear ge­ ferent combinations of SNPs (rs7328194, rs932936, nome by the enzymatic machinery of LINEI non-LTR rs7489390, and rs9552762) indicated the double dose of retrotransposons {Pavlicek et al., 2006). A subset of P'Pgs TP'l'g-BASP 1 gene in thirteen patients {Table 2). Geno­ is capable of transcription to RNAs, which are or are not typing of STR marker Dl3S1863 in the remaining six pa­ further translated into proteins depending on the selection tients showed heterozygosity either by means of repeat pressure. Unlike retrogenes, transcribed processed pseu­ numbers on polyacrylamide gel electrophoresis or for both dogenes {TP'l'gs) encode ncRNAs {Yano et al., 2004; Har­ rs9510417 and rsl2583059 in sequence analyses and, tlıus, rison et al., 2005). Some ncRNAs were previously re­ excluded any possible gross deletions. ported to be involved in recognition oftarget nucleic acids According to the data obtained from tlıe study, we ex­ through complementary base pairing and to be associated cluded BASP 1 and TP'l'g-BASP 1 as tlıe causative genes for with the abnormalities in imprinting and dosage compen­ MBS. However, a small chance of deletion in the genomic sation mechanisms (Andersen and Panning, 2003; Yaz.gan region spanning BASP 1 gene could not be ruled out in and Krebs, 2007). Identifıcation of the role of an ncRNA MBS patients with no heterozygous genotypes. encoded by TP'Pg-Mkml in stabilization ofMkm1 mRNA Our study is the fırst molecular genetic study targeting a revealed the involvement of ncRNAs in post-transcriptional gene in the putative MBS locus on l 3q, as well as including regulation {Hirotsune et al., 2003). The amino acid se- the analysis of another gene in a locus different from tlıe •

Abdullah Uzumcu et al./Journal o/Genetics and Genomics 36 (2009) 25 /-256 255

Table2 Genotypes of MBS patients in BASP I and TP'Pg-BASP I genes and STR markers D5S2946 and D 13S 1863 MBSMBSMBSMBSMBSMBSMBSMBSMBSMBSMBSMBSMBS MBS MBS MBS MBS MBS MBS Gene/STR -1 -2 -3 -4 -5 -6 -7 -8 -9 -10 -11 -12 -13 -14 -15 -16 -17 -18 -19 BASPI Sequence WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT D582946 PAGE Het Het Hom Het Hom Het Het Het Hom Het Het Hom Hom Het Hom Hom Het Het Het Sequence NS NS WT NS WT NS NS NS WT NS NS WT WT NS WT WT NS NS NS TP'l'g-BASPI Sequence 159552762 WT WT WT WT Het Het WT WT WT WT WT WT WT WT Het Het WT Het Het rs7489390 Het WT WT WT Het Het WT WT WT WT WT WT WT WT WT WT WT WT WT ALl57931: Het WT WT WT WT WT WT WT WT WT g.8908_8909de!AG WT WT WT WT WT WT wr wr wr 15932936 WT WT WT Het Het Het WT WT Het wr Het Het wr Het Het Het WT Het Het rs7328194 wr WT WT Het Het Het Het WT Het WT Het Het WT Het Het Het wr Hct Het D13Sl863 PAGE NG Het Het NG NG NG NG Het NG Het NG NG Het NG NG NG Hom NG NG Sequence 159510417 NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS Hct NS NS rsl2583059 NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS Het NS NS

Het, heterozygous; Hom, homozygous; NG. not genotyped; NS, not sequenced; PAGE, polyacrylarnide gel electroplıoresis; STR., short tandem repeat.

A AOAOO AOAOAOAOAAAGO AOAOOAOAO A040AOAAAAGAT. TO AGAOAGGOG AO ACTGTT T TAAOTGGT AT TAT TTT T

1:

!)li !· -'N0f~Nv·

B AGAGGAOAOAGAAAOGAGAGGAOAGAGAGAOAAGAGATT04GAAAGGGOAGACTGTTTTAAGTGGTATTATTTTT.

f: h

C M--'ıllde A1JAAAOG AG AOGAGAOAGAG AOAAAAG,\ T TGAOAGAOOOG AOAC TG TT T TAAOTOOT AT TAT TTT T AOAOG AO AOAO AAAGOAOAGOAG AOAG AO.\OAAOAG.\ TTOAGAOAGGGG-\0,\CTOTT T TA !\OT GOT ATTA TTT T TCC Pllfflıllalelo

Fig. ı. Analyses of partial sequence of TP'Pg-BASP I gene in mother (A), father (B), and index patient (C) of MBS-1 family. The position of de now, variation in patient MBS-1, AL 157931: g.8908 _ 8909delAG. was indicated by red box in ( C). • the position of SNP rs7489390. 256 Abdullah Uzumcu et al./ Journa/ ofGenetics and Genomics 36 (2009) 251-256

HCFPI locus. Our results showed that BASP1 and (2000). GAP43, MARCKS, and CAP23 modulate Pl(4,5)P(2) at TP'l'g-BASP J genes were not involved in the etiology of plasmalemmal rafts, and regulate celi cortex actin dynamics througlı a common mechanism. J. Celi Biol. 149: 14S5-1472. MBS in our patient group. ünce the causative gene resid­ Legum, C., Godel, V., and Ntmet, P. ( 1981 ). Heterogeneity and plei­ ing in the murine locus for abnormal sprouting of the facial otropism in the Moebius syndrome. Clin. Genet. 20: 254-2S9. nerves is identifıed, it will be worthwhile to be screened in Maekawa, S., Murofusbi, H., and Nakamun, S. (1994). Inhibitory effect of calmodulin on phosphorylation of NAP-22 with protein MBS patients. Functional analyses are needed to verify kinase C. J. Biol. Chem. 269: 19462-1946S. whether and how BASPJ and TP'l'g-BASPI interact with Mar, L, Rivkiu, E., Kim, D.Y., Yu, J.Y., aad Cordes, S.P. (200S). 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