American Journal of Medical Genetics 100:62±65 (2001)

Linkage Mapping of a Nonspeci®c Form of X-LinkedMental Retardation (MRX53) in a Large PakistaniFamily

Wasim Ahmad,1,2 Sara Noci,1 Mohammad Faiyaz ul Haque,2 Tiziana Sarno,1 Paolo Aridon,1

M. Maqbool Ahmad,2 Muhammad Amin-ud-din,2 Muhammad Arshad Ra®q,2 Saeed ul Haque,2

Maurizio De Fusco,1 Andrea Ballabio,1 Brunella Franco,1 and Giorgio Casari1,3*1Telethon Institute of Genetics and Medicine, Milan, Italy2Department of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan3San Raffaele Scienti®c Institute, Milan, Italy

Nonspeci®c X-linked mental retardation is anonprogressive, genetically heterogeneouscondition that affects cognitive function inthe absence of other distinctive clinicalmanifestations. We report here linkage dataon a large Pakistani family affected by aform of X-linked nonspeci®cmental retarda-tion. X chromosome genotyping of familymembers and linkage analysis allowed theidenti®cation of a newdisease locus,MRX53.The de®ned critical region spans approxi-mately 15 cM between DXS1210 andDXS1047 in Xq22.2±26. A LOD score valueof 3.34 at no recombination was obtainedwith markers DXS1072 and DXS8081. ß 2001

Wiley-Liss, Inc.

KEY WORDS: Nonspeci®c mental retarda-tion; X chromosome; linkagemapping

INTRODUCTION

X-linked mental retardation (XLMR) is the mostcommon cause of mental retardation in males. XLMRcan be classi®ed into speci®c (MRXS) and nonspeci®c(MRX) forms [Neri et al., 1991]. Mental retardation isapparently the only clinical feature of MRX forms.Several XLMR loci have already been mapped bylinkage analysis. Lubs et al. [1999] reviewed 178 XLMRconditions, of which 120 are speci®c (MRXS) and 58nonspeci®c. An increasing number of MRX familieshave also been reported more recently (75 listed at theNinth International Workshop on Fragile X-Syndrome

and X-Linked Mental Retardation, Strasbourg, France,1999).

The genetic causes of XLMR are heterogeneous andthe pathogenesis of the disease is not clear. So far, 21genes responsible for MRXS and 7 genetic determi-nants for MRX have been cloned. In the present study,we describe a large Pakistani family in which MRsegregates as a recessive X-linked trait. Linkageanalysis, using a collection of highly polymorphicmicrosatellite markers spanning the whole X chromo-some, localizes the gene responsible for this form ofmental retardation to Xq22.2±Xq26.

MATERIALS AND METHODS

The family pedigree (partially reported in Ahmad etal. [1997]) is shown in Figure 1. The eight affectedmales show mental retardation, with IQ between 45and 50. All female members of the family, includingobligate carriers, were free of mental impairment.Neurological examination of the affected individualsshowed no abnormality; in particular, gross motordevelopment was normal and they never receivedphysiotherapeutic treatment. No other consistent clin-ical signs were detected in the affected members of thefamily. Their behavior was normal and no one wasdescribed as dif®cult to control.

Genotype Analysis and Linkage Analysis

DNAwas extracted from peripheral blood samples bystandard methods [Sambrook et al., 1989]. All geneticmarkers used in the present analysis are microsatel-lites. Initially, a set of ¯uorescence-labeled markers(DXS1060, DXS987, DXS1226, DXS1202, DXS1214,DXS1068, DXS993, DXS1055, DXS991, DXS986,DXS990, DXS1106, DXS1001, DXS1047, DXS1227)was used for primary screening on an automatedsequencer ABI-PE 377 (Perkin-Elmer). Once primarylinkage was determined, further re®nement of thelocalization was carried out by locally increasing themarker map density. a-32P-dCTP was incorporated in

Grant sponsor: Telethon Institute of Genetics and Medicine.

*Correspondence to: Dr. Giorgio Casari, Human MolecularGenetics Unit, San Raffaele Scienti®c Institute, Via Olgettina 58,20132 Milan, Italy. E-mail: casari.giorgio@hsr.it

Received 15 September 2000; Accepted 28 December 2000

Published online 8 March 2001

ß 2001 Wiley-Liss, Inc.

PCR reactions of the analyzed marker. Ampli®cationwas carried out with 30 cycles of 1 min at 948C, 1 min at558C, and 1 min at 728C in a ®nal volume of 20 ml.Subsequently, labeled fragments were separated on 6%denaturing polyacrylamide gels and visualized byautoradiography. Repeat expansions of CGG tripletsin the FMR-1 gene [Oostra and Verkerk, 1992] wereexcluded by PCR analysis according to the methoddescribed by Brown et al. [1996].

Two-point linkage analysis between each marker andthe disease locus was performed using the computerprogram LINKAGE 5.1 [Lathrop et al., 1984] andassuming X-linked recessive inheritance. The fre-quency of the disease allele was chosen arbitrarily as0.00001. Genetic and physical distances of the markerloci and map localization were adapted fromMarsh®eldMap (Marsh®eld Medical Center, http://www.marsh-med.org/genetics) and from UDB Map (Uni®ed Data-base for Human Genome Mapping at WeizmannInstitute of Science, http://bioinformatics.weizmann.a-c.il/udb/).

RESULTS

Two-point LOD scores between the disease locusMRX53 and the polymorphic markers spreading overthe entire X chromosome are presented in Table I. Oncethe primary linkage was observed with markerDXS1001, additional neighboring markers were usedto re®ne localization. A signi®cant LOD score has beenobtained with ®ve adjacent markers (DXS1059,

DXS1072, DXS8081, DXS8055, and DXS1001) withknown regional localization in Xq23±Xq26. The max-imum LOD score value of 3.34 has been obtained withmarkers DXS1072 and DXS8081 in the absence ofrecombination. A recombination event between mar-kers DXS1210 and DXS1059 has been observed in thehealthy subject III-20 (Fig. 1), thus de®ning thecentromeric boundary of the candidate region. Norecombination was observed between DXS1059 andDXS1001, while the telomeric end of the candidateregion is de®ned by the recombination observed inthree affected members (III-3, III-8, III-12) betweenmarkers DXS1001 and DXS1047. The identi®ed criticalregion spans a gene-poor chromosomal area of 15 cM (9Mb) in Xq22.2±q26.

DISCUSSION

The MRX53 family described in this study includeseight moderately affected members and represents anadditional nonspeci®c XLMR form. Linkage analysismaps the gene responsible for MRX53 to Xq22.2±Xq26between markers DXS1210 and DXS1047, in a regionspanning 15 cM. Other forms of nonspeci®c MRX(MRX23, MRX27, MRX35, MRX42, MRX46, MRX57[Lubs et al., 1999]; MRX30 and MRX47 due to PAK3mutation [Allen et al., 1998; Bienvenu et al., 2000]) mapin regions overlapping our locus, as well as severalspeci®c forms. Early predictions on the number ofdistinct MRX genes [Gedeon et al., 1996], based onnonoverlapping regions identi®ed by linkage analysis,

Fig. 1. MRX53 Pedigree. Haplotypes for selected markers in Xq22.2±Xq26. Markers DXS1210, DXS1072, DXS8081, DXS8055, DXS1001, andDXS1047 are ordered pter±qter. Arrows indicate the recombination events delimiting the critical region.

Linkage Mapping of MRX53 63

led to a minimum estimate of eight. More recently,MRX families mapping in the same region have notshown mutations in the same gene. Therefore, Gecz[2000] speculated that the actual number of MRX genesmay be higher. However, to date, the possibility that asingle gene is responsible for all MRX families localiz-ing to the same interval cannot be ruled out.

The UDB map reports 21 genes in our critical region,3 of which are potential candidates for MR. Thedoublecortin gene (DCX), mapping in Xq22.3±q23,participates in signaling pathway crucial for neuronalinteraction. Mutations in DCX cause X-linked lissence-phaly (MIM 300067), a severe neuronal migrationdisorder leading to cortical dysgenesis [des Portes etal., 1998; Gleeson et al., 1998]. TRPC5, a genehomologous to the murine brain receptor±activatedcapacitative Ca2� entry channel, is exclusivelyexpressed in developing and adult brain [Sossey-Alaouiet al., 1999]. It has also been proposed as candidate fornonsyndromic MR that maps to Xq23 [Gu XX et al.,1996; des Portes et al., 1998]. The glutamate receptor 3(GRIA3), mapping in Xq25±Xq26, is a major player inpostsynaptic signaling via Ca2�/calmodulin-dependentpathway [Gecz et al., 1999]. However, GRIA3 has beenexcluded as a possible cause of MRX27 [Gecz et al.,1999].

To date, seven genes responsible for MRX have beencloned: FMR2, a putative transcription factor [Gecz etal., 1996; Gu Y et al., 1996]; GDI1, a Rab GDB±dissociation inhibitor [D'Adamo et al., 1998];OFHN1, aRho GTPase±activating protein [Billuart et al., 1998];PAK3, a p21-activated kinase [Allen et al., 1998];RPS6KA3, a growth factor±regulated kinase [Meri-enne et al., 1999]; IL1RAPL, a homolog to interleukinereceptor accessory protein-like [Carrie et al., 1999]; andTM4SF2, an integrin-associated protein [Zemni et al.,2000]. With the exception of FMR2, all these genes are

involved in various stages of intracellular signalingpathways. Chelly [1999] underlined how these genesare required for morphological changes to establishfunctional connections in the nervous system, whichare necessary for the development of cognitive func-tions. On the basis of these observations and consider-ing their action in neuronal interaction, signaltransmission, and brain development, we proposeDCX, TRPC5, and GRIA3 as strong candidates forMRX53.

ACKNOWLEDGMENTS

We gratefully acknowledge the families included inthis study.W.A. was supported by a postdoctoral fellow-ship (205/bs) from the Italian Telethon Foundation andby the Quaid-I-Azam University Research Fund.

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TABLE I. Two-Point LOD Scores for MRX53 Spanning the X Chromosome, From pter (Top) to qter

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