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A novel frame shift mutation in the PQBP1 gene identified in a Tunisian family with X-linked mental retardation

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A novel frame shift mutation in the PQBP1 gene identified in a Tunisian family with X-linked mental retardation
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  Original article A novel frame shift mutation in the  PQBP1  gene identi 󿬁 edin a Tunisian family with X-linked mental retardation Imen Rejeb a , * , L amia Ben Jemaa a , b , Leila Abaied a , L ilia Kraoua b , Yoann Saillour c ,Faouzi Maazoul b , Jamel Chelly c , Habiba Chaabouni a , b a Laboratoire de Génétique Humaine, Faculté de Médecine de Tunis, 15 Jebel Lakhdhar LaRabta, Tunis 1007, Tunisia b Hôpital Charles Nicolle, Service des Maladies Héréditaires et Congénitales, Tunis, Tunisia c Institut Cochin, Université Paris Descartes, Inserm U567, UMR 8104, Paris, France a r t i c l e i n f o  Article history: Received 17 September 2010Accepted 20 January 2011Available online xxx Keywords: Lean bodyMutationMicrocephaly PQBP1 Short statureX-linked mental retardation a b s t r a c t Mental retardation (MR) is the most frequent cause of serious handicap in children and young adults.Despite recent progress, in most cases the molecular defects underlying this disorder remain unknown.Linkage studies followed by mutational analysis of known X-chromosomal genes related to mentalretardation (MRX genes) localized within de 󿬁 ned genetic intervals represent a rational strategy toidentify a genetic cause of the disorder.Here, we report a Tunisian family including 3 males with severe to mild mental retardation, shortstature, lean body and microcephaly; we mapped the disease to a unique interval encompassingXp21.1 e Xq21.33 (with a maximum LOD score of 0.90). Subsequent mutation analysis of genes locatedin this interval allowed us to identify a truncating mutation in the  PQBP1  gene. This mutation is aninsertion of an adenosine residue in exon 5 (c.631insA).This frameshift insertion causes premature stop codon at amino acid position 226. The observedmutation was found in all males with MR in this family.Together with previously reported observations, our data further con 󿬁 rm that  PQBP1  gene should betested for males showing mental retardation, short stature, lean body and microcephaly.   2011 Elsevier Masson SAS. All rights reserved. 1. Introduction X-linked mental retardation (XLMR) is a common cause of monogenic intellectual disability affecting mostly males, partlyaccounting for the higher prevalence of mental retardation (MR)among males relative to females[1]. This prevalence is estimatedto be 1 in 600 males and recent  󿬁 ndings have shown that theproportion of MR caused by X-linked factors in men is 10 e 12% [2].XLMR is classically divided into syndromic forms (MRXS) charac-terized by speci 󿬁 c clinical, biochemical, or neurological featuresassociated with mental retardation and non-syndromic forms(MRX) characterized by reduced mental capacities with no addi-tional features. However, if we take into account recent moleculardata, substantial overlaps between these two categories could bepointed out [3, 4]. Indeed, for several XLMR-related genes, muta-tions in the same gene are associated with both syndromic as wellas non-syndromic XLMR forms. Mutations in the polyglutamine-binding protein 1 ( PQBP1 ) supposed to interact with expandedpolyglutamine tracts of huntingtin, ataxin, and androgen receptor[5 e 7] were found in patients with non-syndromic MR as well as inpatients with syndromic MR and X-linked inheritance. In factmutations in the  PQBP1  gene located in Xp11.23 have been foundin patients with Renpenning syndrome [8], Sutherland-Haan syndrome [7], Hamel cerebropalatocardiac syndrome [7], Golabi- Ito-Hall syndrome [9], Porteous syndrome [10], and MRX55 [7], as well as in other XLMR families. The  PQBP1  gene comprises 6 exonsthat codes for a protein of 265 amino acids which contains severaldomains: a WW domain (characterized by two conserved tryp-tophans) encoded by the amino acid positions 47 e 78 that play animportant role in the regulation of transcriptional activity byinteracting with the carboxyl-terminal domain of the RNA poly-merase II [11], a polyglutamine-binding region (in the polar-amino-acid-rich domain: PRD) containing a DR/ER stretch whichis encoded by exon 4 and involved in transcriptional controlby binding to the polyQ region of the transcription factor Brn2[6,12], a nuclear localization signal (NLS), and a C-terminal regionshown to bind to a component of the nuclear pre-mRNA splicingmachinery [13,14]. *  Corresponding author. Tel.:  þ 216 24968503; fax:  þ 216 71570553. E-mail address:  imen_rejeb@yahoo.fr (I. Rejeb). Contents lists available at ScienceDirect European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg 1769-7212/$  e  see front matter    2011 Elsevier Masson SAS. All rights reserved.doi:10.1016/j.ejmg.2011.01.010 European Journal of Medical Genetics xxx (2011) 1 e 6 Please cite this article in press as: I. Rejeb, et al., A novel frame shift mutation in the  PQBP1  gene identi 󿬁 ed in a Tunisian family with X-linkedmental retardation, European Journal of Medical Genetics (2011), doi:10.1016/j.ejmg.2011.01.010  So far, ten mutations identi 󿬁 ed in this gene were reported in 24families: 3 small deletions [7,15 e 17], 2 missense mutation [9,16], 2 small insertions [7,15], and 3 gross deletions [18]. These mutations are resumed in Table 1. Recently Flynn et al. reported a 4.7 Mbduplication at Xp11.22 e p11.23 of the  PQBP1  gene leading toresembling Renpenning syndrome in a 47-year-old male [19].However it is essential to note that the c. 334 e 354del (21 bp) wasfound in controls[18]. Therefore, the 21 bp in-frame deletions reported by Cossée et al. may be non-pathogenic, or alternativelycould act subtly on  PQBP1  function [18].Here, we report the 11th mutation in this gene that was notpreviously reported. This insertion of one adenosine residue inexon 5 (c.631insA) is the second one located in the C-terminalregion. This frameshift insertion causes premature stop codon atamino acid position 226. The observed mutation was found in 3brothers with a phenotype comprising MR, short stature, lean bodyand microcephaly. 2. Methods  2.1. Case report  We report a Tunisian family including 3 males with severe andmildmentalretardation,shortstature,leanbodyandmicrocephaly.Threementallyretardedmales(II-1,II-2andII-3),theirmother(I-2)and their healthy brother (II-5) and sister (II-4) were availablefor this study (Fig. 1a). We reviewed their medical history andperformedpersonalinterviews,clinicalexaminationandmolecularstudies. Each patient had an uneventful pre- and perinatal history.The mother showed normal growth and mental function. For allpatients, FRAXA and FRAXE mutations and large molecular rear-rangements were excluded by conventional molecular approaches.Cytogenetic investigations, including high-resolution karyotype(700 band resolution), were also normal in all patients.  2.2. Molecular genetic studies Onthebasisofthepedigreeofthisfamily(Fig.1a),wesuspectedan X-Linked mental retardation (XLMR) transmission.Blood samples were obtained after written consent from alltested individuals and/or tutors. Genomic DNA was extracted fromperipheral blood lymphocytes using standard protocols for indi-viduals I-2, II-1, II-2, II-3, II-4 and II-5. For genetic mapping, a totalof 25 microsatellite markers distributed along the entire X chro-mosome were used. Lod scores for linkage between the diseaselocus and the genetic markers were calculated by the MLINK of theLINKAGE package (Lathrop and Laouel, 1984) (Fig.1b).For PQBP1  geneanalysis,the coding regions (sixexons, GenBankaccession number NM_005710) and  󿬂 anking intronic sequenceswere ampli 󿬁 ed by PCR using genomic DNA.Primer sequences and PCR conditions can be obtained fromauthors upon request. PCR products were directly sequenced withthe Big Dye Terminator ready reaction kit (PE Applied Biosystems)on an ABI-PRISM 3130 (PE Applied Biosystems). Base calling was  Table 1 Review of mutations reported in the  PQBP1  gene, the protein domain they involve and a resume of the most common features.Syndrome/family Mutation Domain Clinical features ReferencesRenpenning syndrome(srcinal family)c.641ins C C-terminal region MR, microcephaly, long face, short stature,lean body, small testesLenski et al., [2004] [15]Renpenning syndrome(K8600)c.459 e 462del AGAG DR/ER repeat MR, microcephaly, long face, lean body,small testesStevenson et al., [2005] [10]MRX55 c.459 e 462del AGAG DR/ER repeat MR, long face Kalscheuer et al., [2003] [7,27]N45 c.459 e 462del AGAG DR/ER repeat MR, microcephaly, long face, lean body,anal atresiaKalscheuer et al., [2003] [7]Sheen family c.459 e 462del AGAG DR/ER repeat MR, microcephaly, dysmorphic facies, hearingloss, short stature, periventricular heterotopiaSheen et al., [2010] [17]P family c.459 e 462del AGAG DR/ER repeat MR, microcephaly, dysmorphic facies,muscular atrophy lean body,Germanaud et al., [2010] [16]CB family c.459 e 462del AGAG DR/ER repeat MR, microcephaly, dysmorphic facies,muscular atrophy, lean bodyGermanaud et al., [2010] [16]S family c.459 e 462del AGAG DR/ER repeat MR, microcephaly, dysmorphic facies,lean bodyGermanaud et al., [2010] [16]B family c.459 e 462del AGAG DR/ER repeat MR, microcephaly, dysmorphic facies,muscular atrophy, lean bodyGermanaud et al., [2010] [16]AH family c.459 e 462del AGAG DR/ER repeat MR, microcephaly, dysmorphic facies,lean bodyGermanaud et al., [2010] [16]L family c.586 C / T NLS domain MR, microcephaly, dysmorphic facies,muscular atrophy, lean bodyGermanaud et al., [2010] [16]Hamel cerebropalatocardiac(N40)c. 461 e 462 del AG DR/ER repeat MR, microcephaly, short stature, long face,lean body, congenital heart defectKalscheuer et al., [2003] [7]Martinez-Garay family c. 461 e 462 del AG DR/ER repeat MR, microcephaly, short stature,microphthalmiaMartinez-Garay et al., [2006] [23]Sutherland-Haan syndrome c.463 e 464 dup AG DR/ER repeat MR, microcephaly, short stature, long face,lean body, small testes, anal stenosis or atresiaKalscheuer et al., [2003] [7]Golabi-Ito-Hall c.194 A / G WW domain MR, microcephaly, short stature, triangularface, lean body, spastic diplegiaLubs et al., [2006] [9]Porteous syndrome c.463 e 464 dup AG DR/ER repeat MR, long face, lean body Stevenson et al., [2005] [10]N09 c.463 e 464 dup AG DR/ER repeat MR, microcephaly, long face, lean body Kalscheuer et al., [2003] [7]K9008 c.575 e 576 del AG NLS domain MR, microcephaly, short stature long face,lean bodyLenski et al., [2004] [15]F 1 c.547 e 569del(23bp) After the DR/ER repeatMR, microcephaly, bilateral choanal atresiaand anal atresiaCossée et al., [2006] [18]F 2, F 3 and F 4 c.334 e 354del (21bp) PRD domain MR, behavioral anomalies Cossée et al., [2006] [18]F 5 c.393 e 413del (21bp) PRD domain MR, microcephaly, lower limbs spasticity Cossée et al., [2006] [18]Present study c.631 ins A C-terminal region MR, short stature, microcephaly, long face,lean bodyPresent study I. Rejeb et al. / European Journal of Medical Genetics xxx (2011) 1 e 6  2 Please cite this article in press as: I. Rejeb, et al., A novel frame shift mutation in the  PQBP1  gene identi 󿬁 ed in a Tunisian family with X-linkedmental retardation, European Journal of Medical Genetics (2011), doi:10.1016/j.ejmg.2011.01.010  ab 121212111212121112121111121212-inf -inf 0.900.000.900.900.900.000.900.900.000.000.90-inf -inf DXS1061DXS1214DXS8090DXS8102DXS8015DXS8080DXS8083DXS1055DXS1039DXS1216DXS1196DXS1217DXS8077DXS1059DXS8055I-2 II-1 II-2 II-3 II-4 II-5 Zmax111122112211221 / 21122112211221 / 21122112211221131322111111121111121212111312112111211111121 III 1212345 CtrlII-1 cd C-terminal WWNLS PRDDR/ER repeatc. 194 A → Gc.334-354del(21bp)c. 459–462 del AGAGc. 461–462 del AGc. 463–464 dup AGc. 575–576 del AG c.631 ins A c. 641ins CN-terminalc. 547–569 del(23bp)c.393-413del(21bp)c. 586 C → T Fig.1. a. Pedigree of the family  . Filled squares denote affected males and dotted circles denote carrier females. Asterisks indicate individuals for which DNA and phenotype datawere available.  b. Haplotypes of the Xp21.1-Xq21.33 region . Maximum Lod scores are indicated on the right.  c. Sequence chromatograms of a control individual and theaffected family member II.1 showing a partial sequence of exon 6 of   PQBP1  gene . Dot indicates the nucleotidic variation c.631insA.  d. Schematic representation of the proteinstructure of   PQBP1  protein with positions of the functional domains (WW domain, PRD domain, NLS domain and C terminal region as well as the position of previously described mutations  (c.194 A / G, c.334 e 354del(21bp), c.393 e 413del(21bp), c.459 e 462delAGAG, c.461 e 462delAG, c.463 e 464dupAG, c.547 e 569del(23bp), c.575 e 576delAG,c.586 C / T, c.641insC) and of the new c.631insA mutation describe in this report (underlined mutation). I. Rejeb et al. / European Journal of Medical Genetics xxx (2011) 1 e 6   3 Please cite this article in press as: I. Rejeb, et al., A novel frame shift mutation in the  PQBP1  gene identi 󿬁 ed in a Tunisian family with X-linkedmental retardation, European Journal of Medical Genetics (2011), doi:10.1016/j.ejmg.2011.01.010  performed byusing Sequencing analysis 5.2 software (Gene codes).Reference sequence was obtained from the UCSC Human GenomeBrowser (http://www.genom.ucsc.edu;  PQBP1 : NM_005710) andsequence analysis was performed by using SeqScape v2.5 (AppliedBiosystems). 3. Results  3.1. Molecular genetic   󿬁 ndings Segregation analysis using 25 polymorphic markers evenlydistributed on the X chromosome allowed us to exclude implica-tion in the MR phenotype of most regions of the X chromosome,except the region in Xp21.1 e Xq21.33 between the markersDXS8090 and DXS8077. At this region, affected male subjects werefound to share a haplotype spanning about 58 Mb and the highesttwo-point Lod-score within the interval was 0.90 (Fig. 1b). Thisregion contains more than 10 genes implicated in non-syndromicforms of XLMR. Because of phenotypic symptoms such as micro-cephalyand short stature and the signi 󿬁 cant overlap with reportedphenotypes of patients with mutations in  PQBP1  [17], we  󿬁 rstscreened the  PQBP1  gene for mutations by direct sequencing of the6 coding exons and their  󿬂 anking intronic sequences (GenBankaccession number. NM_005710).Mutation screening of the six exons of   PQBP1  revealed aninsertion of one adenosine residue in exon 5 (c.631insA). Thismutation was reported neither in the literature, nor in onlinedatabases as polymorphism (Fig. 1c). This frameshift insertioncauses premature stop codon at amino acid position 226. Weanalyzed the segregation of the mutation in the family andcon 󿬁 rmed that the mutation segregateswiththe phenotype, in factthe observed mutation was found in all affected males and in theirobligate carrier mother in this family.  3.2. Clinical features of the affected males The pedigree of the family is shown in Fig. 1a.Patient II.1 is a 30 year-old male with severe mental retardationand behavior disturbance.He was born by spontaneous delivery after an uneventfulpregnancy. The neonatal period was normal. The birth weight was2700 g. He experienced no signi 󿬁 cant childhood illness and had noseizures. The psychomotor development was normal. He was ableto walk independentlyat the age of 1 yearand did not have distinct Fig. 2.  Facial appearance of the 3 affected males. Photos of the affected males  II-1, II-2 , and  II-3  showing the dysmorphic features. Note the slender posture of individuals  II-2 , and II-3 . I. Rejeb et al. / European Journal of Medical Genetics xxx (2011) 1 e 6  4 Please cite this article in press as: I. Rejeb, et al., A novel frame shift mutation in the  PQBP1  gene identi 󿬁 ed in a Tunisian family with X-linkedmental retardation, European Journal of Medical Genetics (2011), doi:10.1016/j.ejmg.2011.01.010  speechuntiltheageof3years.Hestoppednormalschooleducationat the age of 6 years because of attention de 󿬁 ciency. Currently,he works with his father in the farm. He is mentally retarded,with an apparent normal speech but dif  󿬁 culties in reading andwriting. He is stubborn and talkative with slightly inarticulatespeech with a nasal voice. He presented with microcephaly (occi-pitofrontal circumference OC  ¼  52 cm  < 3rd centile) short stature(158cm < 3rdcentile)andleanbody(49000g < 3rdcentile).Hehasnormal tendon re 󿬂 exes and no spasticity. Dysmorphic featureswere noticed with long face, high sloping forehead, large cuppedears, beaked nose, long columella, highly arched palate, rethrog-nathism and a central balding. He had a lean body with slenderhands and feet (Fig. 2a).Patient II.2 is a 28 years old; he was born by spontaneousdelivery after an uneventful pregnancy. The birth weight was3000g.Intheneonatalperiodheexperiencedafeverepisodeattheage of 3 months. He experienced delayed motor and languagemilestones and attended special education throughout his schoolyears. He was able to walk independently at the age of 2 years anddid not have distinct speech until the age of 3 years. He performedpoorly in a regular school setting. Clinical history showed mildmentally retarded boy with normal speech and nasal voice, hewas able to write his name and write and read some numbers.He speaks unclearly and hastily and uses short sentences .Hisdeep tendon re 󿬂 exes are normal, there is no spasticity. He pre-sented with microcephaly (OC  ¼  51  < 3rd centile), short stature(158 cm  < 3rd centile), facial dysmorphism with long face, upslanting palpebral  󿬁 ssures, highly arched palate and a beginning of central balding. He had a lean body (46500 g  < 3rd centile) withslender hands and feet (Fig. 2b). He continues his education inaspecialized school.Brain imagingwithcomputerizedtomographywas normal for this patient.Patient II.3 is a 27 years old. He was born after normal preg-nancy. The psychomotordevelopment was normal according to hismotherwithwalkingatageof1yearandinitialspeechattheageof 2 years. He required special education throughout school. Clinicalexaminationshowedmicrocephaly(OC ¼ 51cm < 3rdcentile)shortstature (161 cm  < 3rd centile) and a lean body (45000 g  < 3rdcentile). He presented some dysmorphic features as up slantingpalpebral  󿬁 ssures, narrow sloping forehead, large cupped ears,highlyarched palate,beakednose, rethrognathism anda beginningof central balding. He had a lean body with slender hands and feet(Fig. 2c) .He worked as a street sweeper of municipality.Additional clinical features are listed in Table 2. 4. Discussion Our report describes the 24th family with mental retardationco-segregatingwith amutationin the  PQBP1 gene. This mutationisan insertion of one adenosine residue in exon 5 (c.631insA). Thisframeshift insertion causes a premature stop codon at amino acidposition 226. This c.631insA mutation was detected in all affectedmales as well as in the obligate carrier mother, but was absent inthe normal brother. Mutation described in this report is the secondinsertion mutation involving the C-terminal region. The  󿬁 rst onewasreportedbyLenskietal.[15]inthefamilyinitiallydescribedbyRenpenning and colleagues (1962) [8]. These two mutations cause a premature stop codon that can either lead to a truncated proteinor to a functional null-allele owing to non sense mediated mRNAdecay (NMD) [20]. A recent work of Musante et al. describes that inthepatientwiththec.641insC(p. Arg214fsX12)mutationin exon5,the expected truncated product of 30 kDa was present [21].All mutations identi 󿬁 ed so far in the  PQBP1  gene lead to thedisturbanceordeletionof theC-terminalregionintheprotein.Thisdomain has been shown to bind to U5-15 kDa [13] a component of the nuclear pre-mRNA splicing machinery [14]. A loss of functionalC-terminal region might, therefore, contribute to the pathogenesisof MRXS in all the families.In fact the two mutations, this one and the one found in thefamily of Renpenning and colleagues lead to the same truncatedprotein (226 aminoacid), and are associated with phenotypes thatsharesomecommonfeaturesspeci 󿬁 ctotheRenpenningsyndrome,such as mild to severe mental retardation, short stature, lean bodybuild, upslanting palpebrae and microcephaly (detailed compar-ison is provided in Table 3). Though some divergent features couldbe pointed out (i.e. testicular volume that was normal in 2 of theaffected males of our family, in fact only 1/3 presented withtesticular volume below 10 ml Table 2). In addition, in our familysevere mental retardation is noticed only in 2/3 of affected malestheir brother presented with mild mental retardation. These clin-ical  󿬁 ndings highlight the relative inter- and intrafamilial pheno-typic heterogeneity associated with mutations in  PQBP1 .  Table 2 Clinical features in the affected males.Patient and age II.1 (30 years) II.2 (28 years) II.3 (27 years)MR Severe (IQ  ¼ 40) Mild (IQ  ¼ 55) Severe (IQ  ¼ 40)Height 158.0 cm ( < 3rd) 158.0 cm ( < 3rd) 161.0 cm ( < 3rd),Headcircumference52.0 cm ( < 3rd) 51.0 cm ( < 3rd) 51.0 cm ( < 3rd)Birth weight 2700 g (10th) 3500 g ( > 10th) 3000 g ( > 10th)Weight 49,000 g ( < 3rd) 46,500 g ( < 3rd) 45,000 g ( < 3rd)Ear Protruding/Cupped Cupped Protruding/CuppedEar length 6 cm (50) 6.7 cm (70) 6.5 cm (55)Forehead High, sloping High, sloping Narrow, slopingEyes Normal Normal NormalInner Canthus 2.5 cm ( < 3rd) 3 cm (50) 3 cm (50)Interpupillary 5.5 cm (25 e 50) 6 cm ( > 75) 6 cm ( > 75)Nose Beaked nose Beaked nose Beaked noseMouth Normal Thin lips NormalPalate Highly arched Highly arched Highly archedHand/Palmlength17(25 e 50)/9cm (3rd)18(50 e 75)/9cm (3rd)17.5(50)/8 cm( < 3rd)Hair Central balding Beginning of baldnessBeginning of baldnessTesticular size Normal Normal  < 10 mlBehaviour andmentalfeaturesShort attentionspan, temperShort attentionspan,anxiety, clingingShort attentionspanSpeech Inarticulate Poor andinarticulateNormalVoice Nasal Nasal Nasal  Table 3 Comparison of manifestations in our family and the srcinal family of Renpenning[8].Renpenning et al.(1962) [8,15]This reportMutation c.641insC c.631insAAffected males 20 3Short stature ( < 3rd centile) 6/10 3/3Lean body build 3/4 3/3Microcephaly ( < 3rd centile) 13/14 3/3Upslanting palpebrae 4/4 3/3Large ears 1/10 3/3Cupped ears 0/5 3/3Short philtrum 5/5 0/3Small mouth 0/5 0/3Highly arched palate 0/5 3/3Central balding 3/5 1/3Small testes < 10 ml 4/9 1/3Slow development/mental retardationMild (IQ 50 e 70) 1/14 1/3Severe (IQ  < 50) 13/14 3/3 I. Rejeb et al. / European Journal of Medical Genetics xxx (2011) 1 e 6   5 Please cite this article in press as: I. Rejeb, et al., A novel frame shift mutation in the  PQBP1  gene identi 󿬁 ed in a Tunisian family with X-linkedmental retardation, European Journal of Medical Genetics (2011), doi:10.1016/j.ejmg.2011.01.010
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