Gene for Apparently Nonsyndromic X-LinkedMental Retardation (MRX32) Maps to an 18-MbRegion of Xp21.2-p22

Bernhard Hane,1 Roger E. Stevenson,1 J. Fernando Arena,2 Herbert A. Lubs,2 Richard J. Simensen,1and Charles E. Schwartz1*1J.C. Self Research Institute, Greenwood Genetic Center, Greenwood, South Carolina2University of Miami School of Medicine, Mailman Center for Child Development, Miami, Florida

We studied a family with 11 males havingX-linked mental retardation (XLMR) usingmicrosatellite markers. Aside from the men-tal retardation, the affected males do not ap-pear to differ from their unaffected brothersor uncles. The gene for this XLMR conditionhas been linked to DXS451 in Xp22.13 with alod score of 5.18 at u = 0. Recombination wasdetected at DXS992 (Xp21.3) and DXS1053(Xp22.2), thereby defining the limits of thelocalization. This family is considered tohave nonsyndromic XLMR and has been as-signed the designation MRX32. Am. J. Med.Genet. 85:271–275, 1999. © 1999 Wiley-Liss, Inc.

KEY WORDS: XLMR; MRX32; Xp22.13; link-age

INTRODUCTION

Since the first reports of linkage between nonsyn-dromic X-linked mental retardation (XLMR) and poly-morphic markers on the X chromosome by Suthers etal. [1988] and Arveiler et al. [1988], numerous addi-tional families have been regionally mapped and iden-tified by the acronym MRX followed by a progressivenumber. At present, over 50 families comprising anestimated 10 nonoverlapping linkage regions havebeen identified [Hane et al., 1996; Lubs et al., 1996;Gedeon et al., 1996a,b]. These linkage regions span theentire X chromosome and range in size from 5 to 18 cM.With the exception of MRX41 and MRX48 which aredue to mutations in the GDI gene [D’Adamo et al.,

1998], none of these disease-causing genes has beenisolated. It is to be anticipated that the linkage regionsrecognized at present will be further partitioned as ad-ditional nonsyndromic XLMR families are studied. Al-though Herbst and Miller [1980] estimated that 7 to 19genes on the X chromosome could be responsible fornonsyndromic XLMR, there are inadequate data at thistime to make a reliable revision of that estimate.

The designation MRX32 has been assigned to thefamily reported herein in which 11 males have mentalretardation. The kindred was published previously asfamily C by Howard-Peebles et al. [1979]. The genemaps to an 18-Mb region distal to the Duchenne mus-cular dystrophy locus in Xp21.

CLINICAL REPORT

A partial pedigree of kindred K8450 is shown as Fig-ure 1. Eleven males were affected in four generations.It was possible to perform clinical evaluations on fouraffected males; one was deceased, and six were notavailable for examination. Blood samples were ob-tained on three of the six who could not be examined.Five normal brothers were examined to permit pheno-type comparison (Table I). There were no morphomet-ric, craniofacial, skeletal, neurological, or other find-ings which distinguished the affected from the unaf-fected males.

Testicular volumes in 2 affected males were withinthe normal range. Affected males (n 4 4) scored 31 to67 on standardized IQ tests; nonaffected males (n 4 3)scored 97 to 107. High-resolution chromosome analysisand FMR1 mutational analysis were normal on oneaffected male. Creatine kinase was minimally elevatedin patient II-5 and several amino acids were minimallyelevated in his plasma amino acid analysis.

MATERIALS AND METHODS

Genomic DNA was isolated from peripheral blood us-ing a high-salt precipitation method [Schwartz et al.,1990]. Specific dinucleotide or trinucleotide polymor-phisms were generated as listed in the Genome Data-base (http://gdbwww.gdb.org). Forward primers for the

Contract grant sponsor: NICHD; Contract grant number:2R01HD26202; Contract grant sponsor: South Carolina Depart-ment of Disabilities and Special Needs (SCDDSN).

*Correspondence to: Charles E. Schwartz Ph.D., J.C. Self Re-search Institute of Human Genetics, Greenwood Genetic Center,One Gregor Mendel Circle, Greenwood, S.C. 29646. E-mail:schwartz@ggc.org

Received 22 August 1997; Accepted 4 March 1999

American Journal of Medical Genetics 85:271–275 (1999)

© 1999 Wiley-Liss, Inc.

polymerase chain reaction (PCR) primer pairs weresynthesized and labeled with fluorescein amidite (Fluo-rePrime, Pharmacia, Uppsala, Sweden) on a Beckman1000 DNA synthesizer and desalted through SephadexG-25 (NAP-10 columns, Pharmacia). The polymor-phisms were detected using an Automated Laser Fluo-rescent Sequencer (A.L.F., Pharmacia) in conjunctionwith Fragment Manager (Pharmacia) and the softwarepackage Automated Linkage Preprocessor (ALP)[Mansfield et al., 1994].

Two-point disease-to-marker linkage analysis wasconducted using the program MLINK of the LINKAGEpackage [Lathrop and Lalouel, 1984]. The mutationrate and gene frequency were set at 3 × 10−6 and 0.0001respectively.

RESULTSThe maximum expected lod score (ELS) for this fam-

ily was calculated to be 5.18 at u 4 0.00. Initial two-point lod scores for the disease locus and 16 markersspanning the X chromosome were computed usingMLINK. Significant linkage to Xp21.3 was noted forDXS992 (Z 4 4.74 at u 4 0.00, Table II). Once linkagewas established to this region additional markers wereused. Two-point analysis detected significantly highlod scores (>3) at zero recombination for numerous locispanning from DXS985 in Xp21.2 to DXS989 in Xp22.2(Table II). The ELS of 5.18 was observed at DXS451 inXp22.13. Recombination was detected at DXS1236(proximal) and DXS1053 (distal) in person IV-9 (Fig. 1).However, haplotype analysis indicated that a crossover

Fig. 1. Partial pedigree of family K8450 with X-linked mental retardation. Boxed haplotype identifies the “at risk” chromosome associated with theXLMR entity in the family (closed square, affected male).

272 Hane et al.

had occurred between DXS1218 and DXS992 in personIII-6 which only became apparent in her normal grand-son, V-2 (Fig. 1). Linkage analysis did not detect this asboth III-6 and her daughter IV-4 are homozygous forboth loci (Table II, Fig. 1). Likewise, IV-4 is homozy-gous for DXS985 allowing for recombination in V-2 toalso be undetected at this locus (Table II). Therefore,the region of linkage could be further narrowed by hap-lotype analysis to the area flanked by DXS1053 (distal)and DXS992 (proximal), which is about 18 Mb [Nelsonet al., 1995].

DISCUSSION

A growing list of families in which nonsyndromicXLMR has been mapped regionally through linkage

analysis gives the opportunity to study genes whichinfluence brain structure and/or function. No neuro-logical, imaging, or pathological findings suggest thepresence of structural anomalies of the brain in thesepatients. Likewise, there are no malformations, otherphysical anomalies, or identified biochemical distur-bances in these individuals.

Family K8450 described here meets the accepted cri-teria for nonsyndromic XLMR because the affectedmales do not have a consistent distinguishing pheno-type. Mapping to the region Xp21.3 to Xp22.2 qualifiesit for the designation MRX32.

The analysis in this family demonstrates the impor-tance of combining two-point linkage analysis withhaplotype analysis. The former indicated a broad re-

TABLE II. Two-Point Lod Scores Between MRX32 in K8450 and X Chromosome Loci

Locus Location

Recombination (u)

umax Zmax0.001 0.01 0.05 0.1 0.2 0.3

DXS996 Xp22.32 −8.21 −4.25 −1.63 −0.66 0.06 0.24 0.32 0.24DXS987 Xp22.31 0.97 1.91 2.36 2.34 1.95 1.37 0.07 2.38DXS1053 Xp22.2 0.22 1.18 1.69 1.76 1.58 1.21 0.09 1.76DXS999 Xp22.2 2.39 2.35 2.18 1.96 1.49 0.97 0.00 2.40DXS989 Xp22.13 4.92 4.85 4.52 4.10 3.18 2.17 0.00 4.93DXS451 Xp22.13 5.17 5.09 4.75 4.32 3.37 2.32 0.00 5.18DXS1202 Xp21.3 4.57 4.49 4.17 3.76 2.87 1.87 0.00 4.57DXS1218 Xp21.3 4.56 4.49 4.18 3.78 2.91 1.95 0.00 4.56DXS992 Xp21.3 4.74 4.68 4.38 3.99 3.13 2.16 0.00 4.75DXS985 Xp21.2 3.96 3.90 3.62 3.25 2.44 1.53 0.00 3.96DXS1236 Xp21.2 2.64 3.57 3.93 3.78 3.12 2.21 0.05 3.93DXS1237 Xp21.1 2.34 3.27 3.65 3.53 2.92 2.06 0.05 3.65DXS1242 Xp21.1 2.98 2.95 2.79 2.57 2.09 1.52 0.00 2.99DXS1110 Xp21.1 −5.16 −2.20 −0.29 0.36 0.74 0.69 0.23 0.75DXS993 Xp11.4 −8.33 −4.36 −1.69 −0.67 0.14 0.36 0.32 0.37MAOA Xp11.4 −5.03 −2.07 −0.16 0.50 0.87 0.80 0.23 0.88HumARA Xq12 −8.03 −4.06 −1.42 −0.41 0.34 0.51 0.30 0.51DXS986 Xq21.1 −7.16 −3.21 −0.65 0.24 0.79 0.78 0.24 0.82DXS995 Xq21.1 −8.59 −4.62 −1.96 −0.95 −0.14 0.13 0.36 1.62DXS101 Xq22.2 −1.82 0.13 1.29 1.58 1.51 1.12 0.13 1.61DXS1001 Xq24 −4.98 −2.03 −0.15 0.48 0.78 0.65 0.21 0.78HPRT Xq26.1 −9.02 −5.05 −2.40 −1.38 −0.54 −0.19 0.49 0.00DXS984 Xq27.1 −5.67 −2.71 −0.79 −0.10 0.37 0.43 0.28 0.44DXS1108 Xq28 −12.46 −7.48 −4.08 −2.68 −1.36 −0.67 0.50 0.00

TABLE I. Selected Measurements on Affected Males and Normal Brothers in Kindred 8450

Measurements

Affected males Normal sibs

II-5 III-2 IV-7 IV-9 II-6 II-9 III-3 III-7 IV-6

Age (years) 71 54 24 14 67 61 58 53 27Birth weight (kg)

(centile) —a 4.5 (>95) 4.0 (>95) 3.9 (>95) — — — — —Height (cm) (centile) 174 (42) 176.5 (55) 185.4 (93) 183 (>95) 176.5 (55) 188 (97) 169 (17) 174 (42) 178 (65)Head circumference

(cm) (centile) 59.1 (94) 55 (14) 58.9 (92) 55.5 (50) 58.2 (84) 57.6 (71) 58 (80) 57.5 (70) 54.9 (10)Weight (kg) (centile) 88.6 (83) 70.5 (25) 82 (65) 63.3 (75) 70.5 (25) 91 (87) 75 (40) 66 (13) 76 (44)Inner canthal (cm) 3.9 2.6 3.0 3.3 3.3 3.4 2.8 2.6 2.8Interpupillary (cm)

(centile) 7.1 (95) 6.0 (10) 5.9 (5) 6.4 (97) 6.9 (87) 6.5 (55) 5.9 (5) 6.3 (35) 5.7 (2)Outer canthal (cm) 10.0 8.8 9.5 9.8 9.4 9.2 8.8 8.9 8.5Ear length (cm)

(centile) 8.6 (>98) 7.3 (97) 6.8 (80) 6.8 (>75) 7.6 (>98) 8.0 (>98) 7.0 (90) 7.5 (99) 6.4 (45)Hand length (cm)

(centile) 19.4 (54) 19.2 (45) 21.2 (96) 19.6 (>95) 18.6 (20) 19.3 (50) 18.5 (19) 19.5 (59) 19.9 (76)Palm length (cm) 11.1 10.6 12.0 11.3 11 11.1 10.4 11.3 11.3Testes (ml) R 19.7 20.4 — — — — — — —

L 17.1 23 — — — — — — —IQ 67 31 34 52 107 — 97 101 —

aDash indicates measurement not available.

MRX32 Linked to Xp21.2-p22 273

gion of localization spanning from DXS1236 within theDMD gene (Xp21.2) to DXS1053 in Xp22.2. However,the latter indicated a crossover point between lociDXS992 and DXS1218, which is more distal thanDXS1236. The recombination could not be detected bylinkage analysis since it occurred in a maternal grand-mother (III-6) who was homozygous for alleles at lociDXS985, DXS992, and DXS1218 (Fig. 1). Thus, she be-came “uninformative” relative to her unaffected grand-son (V-2) at these loci. By utilizing haplotype analysis,the region of localization could be narrowed at theproximal boundary thereby eliminating the DMD genefrom consideration, and narrowing the area of localiza-tion from 22 cM to 18 Mb [Nelson et al., 1995].

Linkage data for MRX32 place it in a region contain-ing many other MRX entities. These include MRX10-13[Gedeon et al., 1996a; Kerr et al., 1992], MRX18-19[Gedeon et al., 1994; Donnelly et al., 1994], MRX21-22[Kozak et al., 1993; Passos-Bueno et at., 1993], andMRX29 [Hane et al., 1996]. Several syndromic XLMRentities also map to this same region: Partington syn-drome (MIM no. 309510), Coffin-Lowry syndrome(MIM no. 303600), Prieto syndrome [Watty et al.,1991], and Snyder-Robinson syndrome (MIM no.309583) [Arena et al., 1996]. Lastly, small deletions ofthis region have been associated with mental retarda-tion [Billuart et al., 1996; Raeymaekers et al., 1996].

The present ten MRX linkage regions are certain tobe further partitioned and the responsible gene(s) ineach region cloned and characterized [Hane et al.,1996; Lubs et al., 1996; Gedeon et al., 1996a]. For ex-ample, Xq28 contains at least three genes involved innonsyndromic XLMR conditions based on recent find-ings. D’Adamo et al. [1998] found disease-causing mu-tations in the GDI gene in MRX41 [Hamel et al., 1996]and MRX48 [des Portes et al., 1997] but not MRX3[Gedeon et al., 1991], MRX25 [Nordstrom et al., 1992],or MRX28 [Holinski-Feder et al., 1996]. This wouldmean that there are at least two MR genes, in additionto FRAXE [Knight et al., 1994; Gecz et al., 1996], lo-cated in the terminal band of Xq. Finally, several de-letions have been associated with nonsyndromicXLMR, but the deleted genes have not yet been iden-tified [May et al., 1995; Billuart et al., 1996; Raeymaek-ers et al., 1996].

ACKNOWLEDGMENTS

We wish to thank members of family K8450 for theircooperation. This work was supported in part byNICHD grant (2R01HD26202) to C.E.S. and H.A.L.and a grant from the South Carolina Department ofDisabilities and Special Needs (SCDDSN).

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