Mapping of a Gene (MRXS9) for X-Linked MentalRetardation, Microcephaly, and Variably ShortStature to Xq12-q21.31

Antony E. Shrimpton,1* Kathleen M. Daly,2 and Joe J. Hoo2

1Department of Clinical Pathology, State University of New York, Health Science Center, Syracuse, New York2Department of Pediatrics, State University of New York, Health Science Center, Syracuse, New York

Three boys from two families were identi-fied as having a syndrome of X-linked men-tal retardation (XLMR) with microcephalyand short stature, clinically resemblingRenpenning syndrome but with normal sizeof testicles in affected men. When the effortto map the gene for the above condition wasinitiated, it was realized that the two fami-lies were actually related to each other.Over 50 polymorphic markers of known lo-cations along the X chromosome werescored in this family in a study to map thedisease gene. Nine affected and four unaf-fected males were genotyped to produce amaximum LOD score of 4.42 at zero recom-bination with markers in proximal Xq. Theresults indicate that the gene responsiblefor this disorder is located in the cytoge-netic Xq12 to Xq21.31 interval of the X chro-mosome within a section of chromosome ofabout 17 cM between the AR and DXS1217loci over some 25 mb. Since the gene for theX-linked mental retardation from the origi-nal Saskatchewan family described by Ren-penning [Renpenning et al., 1962: Can MedAssoc J 87:954–956; Fox and Gerrard, 1980:Am J Med Genet 7:491–495] was recentlymapped to a different nonoverlapping re-gion [Stevenson et al., 1998: Am J HumGenet 62:1092–1101] this would appear to bea separate disorder. Am. J. Med. Genet. 84:293–299, 1999. © 1999 Wiley-Liss, Inc.

KEY WORDS: X-linked mental retardation;microcephaly; short stature;MRXS9

INTRODUCTION

Mental retardation is found in approximately 1 in500 male births. A conservative estimate is that thereare at least seven nonspecific X-linked recessive men-tal retardation (XLMR) genes defined by nonoverlap-ping regional localization [Gedeon et al., 1996; Lubs etal., 1996]. In addition there are X-linked mental retar-dation syndromes (XLMRS) genes, some of which couldconceivably be indicative of allelic heterogeneity. Theavailability of excellent microsatellite marker linkagemaps enable accurate and precise mapping of the re-combination events that define the chromosomal sec-tions containing each of these genes.

The present study presents the results of a linkagestudy performed on 19 members of a single three-generation pedigree segregating for an XLMR syn-drome with microcephaly and variably short stature.Over 50 microsatellite primer pairs were used and twoand three point linkage analyses were performed oninformative results.

CLINICAL REPORTS

Family P and Family T are of Caucasian origin andwere ascertained independently. When gene mappingefforts were initiated, it turned out that they were re-lated (Fig. 1).

Family P

B.P. (IV-5) and R.P. (IV-6) were referred to the Ge-netic Clinic for evaluation of developmental delay andmicrocephaly (Fig. 2 and Table I). Both boys had nor-mal pre- and perinatal history. Their weight, length,and occipitofrontal circumference (OFC) at birth werenormal, but their OFC started to drop below the 5thcentile early in infancy, and their height growth wasbehind their weight growth. Their gross motor devel-opment was normal, but there was delay in fine motordevelopment and severe delay in speech development.B.P. had only 10–15 single words at 5 1/2 years andwas not toilet trained. R.P. had no speech at all at 3 1/2years, he had spastic diplegia in infancy and wastreated with physical therapy, and he had bilateralstrabismus surgery. Both boys were hyperactive butfriendly. Results of karyotype analysis, DNA fragile-X

Contract grant sponsor: Hendrick Fund of SUNY Health Sci-ence Center.

*Correspondence to: Antony E. Shrimpton, Department ofClinical Pathology, 750 E. Adams St., Syracuse, NY 13210. E-mail: Shrimpta@mailbox.hscsyr.edu

Received 22 August 1997; Accepted 8 February 1999

American Journal of Medical Genetics 84:293–299 (1999)

© 1999 Wiley-Liss, Inc.

study, and urine amino acid and organic acid analysiswere normal on both children.

Mrs. P. (III-10) and her older sister (III-6) grew up ina foster home and had learning difficulty in school, es-pecially reading. Mrs. P. knew that she had severalretarded male cousins from her mother’s side of thefamily, but was unable to state the exact kinship.

Mrs. P.’s two older brothers, J.S. (III-7) and R.S. (III-8), both in their thirties, were microcephalic and se-verely mentally retarded. J.S. was very quiet. R.S. wasfriendlier and tried to engage in conversation, but hisspeech was severely limited. He has a right esotropia.They were not significantly shorter than their unaf-fected brothers. Further physical studies could not bedone.

Family T

J.T. (IV-2) was the second child of his parents (Fig. 3and Table I). His older sister is healthy and mentallynormal. The pre- and perinatal history was uneventful.His birth weight and length were normal. The OFC atbirth was 32 cm (at the 5th centile), but then dropped

below the 2nd centile. His early gross motor develop-ment was normal. Magnetic resonance imaging scan at16 months showed normal brain myelination, struc-ture, and skull sutures. At 26 months cognitive, finemotor, speech, and social skills were around 9–15month levels, with the speech/communicative skills be-ing most delayed. At 4 years he had only a few singlewords and he was not toilet trained. His karyotype,DNA fragile-X, and plasma amino acids were normal.

T.T. (IV-3) was born at term with a birth weight of3,875 g (between 90th and 95th centile) and length of53 cm (at the 90th centile), and OFC of 33 cm (betweenthe 5th and 10th centile). Aside from the relativelysmall head size, especially in comparison to his weightand length, no other physical abnormality was noted.At 3 months his length (61.5 cm) dropped to the 50thcentile, his OFC (38 cm) was at the 2nd centile, whilehis weight (7.5 kg) remained at the 95th centile.

Mrs. T. (III-2) was mentally normal and she hadbeen instrumental in helping us collect the bloodsamples from the extended family members. Herbrother, G.S. (III-5), was severely mentally retarded.

Fig. 1. MRXS9 pedigree showing microsatellite marker haplotypes located on proximal Xq.

294 Shrimpton et al.

His expressive language consisted of only one or twowords, but his receptive language skills appeared to behigher. He had the habit of biting his finger nails, andshowed occasional self-injurious behavior. Aside frommicrocephaly and right esotropia, no obvious minoranomalies were noted. His testes were of normal size.

The family pedigree indicated that there were sev-eral other males with mental retardation, and we wereable to obtain the DNA samples of two other males, II-1and II-6. Both were severely retarded, in their fifties,and lived in institutions.

MATERIALS AND METHODS

Genomic DNA was extracted from peripheral bloodusing inorganic extraction kits (Oncor, Gaithersburg,MD). Polymerase chain reaction (PCR) [Saiki et al.,1988] was performed using the primers flanking X-linked microsatellite markers obtained from either Re-search Genetics Inc. (Huntsville, AL) or synthesized byGenosys Biotechnologies, Inc. (The Woodlands, TX)PCR conditions were in 100-mL reactions containing500 ng genomic DNA, 0.2 mM each dNTP, 1.5 mMMgCl2, 0.5 mM each oligodeoxynucleotide primer, 100mg/mL gelatin, 10 mM Tris-HCl pH 8.3, 50 mM KCl2. Asmall wax pellet was added to each PCR reaction mixwhich was then heated at 95°C for 5 min, and cooled on

ice prior to the addition of 1.5 U AmpliTaq Taq DNAPolymerase (Perkin-Elmer Cetus, Norwalk, CT) in 10mL. PCR cycling was performed on a Hybaid (Tedding-ton, England) Omnigene thermocycler using the follow-ing parameters: one cycle of 2 1/2 min at 94°C, 1 min at55°C, and 1 min at 72°C, followed by 34 cycles of 1 minat 94°C, 1 min at 55°C, and 1 min at 72°C (with theaddition of 1 sec per cycle), followed by 10 min at 72°C.

Single-stranded PCR products were analyzed on 8 Murea polyacrylamide (29:1 acrylamide to bis-acrylamide) gel by running PCR product on a 40-cmlong 1-mM thick gel. Five microliters of PCR productdiluted 1:3 with 95% formamide loading buffer plus dye(20 mL total volume loaded) was heated at 85°C for 3min, and cooled rapidly on ice and then loaded on gels.Gels were run at 50°C for between 5,000 and 15,000Vhrs depending on the fragment sizes being detected.PCR amplified products were detected by Silver stain-ing the gel essentially as indicated by Wallace et al.[1993].

Initially two-point disease to marker analysis wasconducted using the MLINK and ILINK programs ofthe LINKAGE package [Lathrop and Lalouel, 1984].Microsatellite markers were simplified to having fourequally frequent alleles. Three-point disease to mark-ers analysis was performed subsequently using LINK-MAP [Lathrop et al., 1985]. A mutation rate and dis-ease gene allele frequency of 1 × 10−6 was used.

RESULTSFragile X syndrome (FMR1) analysis was performed

by standard Southern blot analysis and no abnormalityin the p(CGG)n triplet repeat was detected. G bandedkaryotyping was also performed and no abnormalitiesdetected.

Nineteen members of what turned out to be one pedi-gree were recruited into this study to map the generesponsible for the mental retardation syndrome seg-regating in this family. Results were obtained for over50 microsatellite markers, including 28 informativemarkers located on average every 10 cM along the Xchromosome. The largest gap between informativemarkers was 14 cM (Fig. 4 and Table II). The chance ofthe disease gene being located outside proximal Xq wasreduced to less than 2%. The average distance betweeninformative markers was 9 cM and the probability ofthe disease gene being located between them less than1%, whereas the LOD score associated with DXS1111and DXS1197 was 4.42. By convention a negative LODscore of less than minus two is required to exclude aregion while a LOD score greater than three on anautosome or two on the X chromosome is required toshow a significant association. Two-point and three-

TABLE I. Pertinent Clinical Features of Affected Family Members

II-1 II-6 III-5 III-7 III-8 IV-2 IV-3 IV-5 IV-6

Age (years) >50 >50 27 35 32 4 3 Months 5 1/2 3 1/2Mental retardation Severe Severe Severe Severe Severe Severe ? Severe SevereOFC centile ? ? <2nd <2nd <2nd <2nd <2nd <2nd <2ndHeight centile ? ? 5th <2nd 5th 5–10th 50th 5th <2ndOther anomalies ? ? Strabismus None Strabismus None None None Spastic diplegia,

strabismus

Fig. 2. Photographs of four affected males in Family P.

Mapping MRXS9 to Xq12-q21.3 295

point linkage studies indicated a significant negativeLOD score (i.e., a LOD score of less than minus three atzero recombination) with all markers except thosemarkers in the Xq12 to Xq21interval (Figs. 1 and 4,Table II). The crossover event that defines the most

proximal position of MRXS9 can be seen to have oc-curred between AR and DXS1111 in the gamete thatgave rise to III-2. The distal boundary is defined by acrossover event that occured between DXS1196 andDXS1217 in the gamete of III-10 that gave rise to IV-4(Fig. 1). The flanking markers that define the bound-aries that contain MRXS9 are thus AR and DXS1217.This gap contains approximately 17 cM and 25 mb ofDNA within the Xq12-q21 interval.

DISCUSSION

The family presented here shows segregation of anXLMR syndrome with microcephaly and variably shortstature. While R.P. (IV-6) is obviously short, his olderbrother B.P. (IV-5) remains at the 5th centile and hissecond cousin J.T. (IV-2) is at between the 5th and 10thcentile. Furthermore, the affected adults are at or be-low the 5th centile, but they are not significantlyshorter than their unaffected brothers. It should benoted though that the paternity of the S. brothers is notcertain, thus a comparison should be done with reser-vation. The testicle and penis size of the three affectedboys are normal for age. The testicle and penis size ofthe 27-year-old G.S. (III-5) is normal for an adult. Al-though the microcephaly and short stature are compa-rable with the findings in the original Renpenning fam-ily, the normal size of testes is not.

While we were finishing the gene mapping work,Mrs. T. notified us about her pregnancy with T.T. (IV-3). Cord blood was collected and was processed for link-age study. The results of the molecular study suggested

Fig. 3. Photographs of three affected males in Family T.

Fig. 4. Sequential three-point LINKMAP LOD scores of microsatellite markers distributed along the length of the X chromosome.

296 Shrimpton et al.

that T.T. is affected. This is consistent with the clinicalfindings based on his relatively small head circumfer-ence. Obviously this preliminary prediction needs to beconfirmed by future evaluation of T.T.’s development.

Herbst and Miller [1980] found mental retardationin 1.83/1,000 live male births and based on certain as-sumptions estimated that there are 7 to 19 genes caus-ing nonspecific mental retardation on the X chromo-some. At least some of these families turned out tosegregate the fragile X syndrome. Gedeon et al. [1996]discussed the likely number of XLMR genes and con-cluded that there must be at least seven discrete MRXgenes as defined by nonoverlapping critical regions.Since this study, there has been a doubling of MRXdesignations, of 32 to 64 (Genome Database), which in-cludes seven syndromic designations (MRXS1-7).

The original family with Renpenning syndrome(RENS1, MRXS8) was mapped to Xp11 [Stevenson etal., 1998]. Thus it would appear that, since the twocritical regions of the present family and the originalRenpenning family do not overlap, these are two sepa-rate entities and that there is locus heterogeneity forRenpenning-type syndrome, and the gene for the cur-rent disease has been given the designation MRXS9.

Overlapping Mental Retardation Disorders,Nonsyndromic (MRX)

Other mental retardation loci mapping to this sec-tion include according to Lubs et al. [1996] MRX1,MRX4, MRX5, MRX7, MRX8, MRX9, MRX13, MRX14,MRX17, MRX20, MRX26, and MRX31 (Table III). The

TABLE II. Two-point LOD Scores (Z) Between X Chromosome Loci and Disease Gene (MRXS9)*

Position Locus

Recombination (Q)

ZMAX with QMAX0.0 0.05 0.10 0.20 0.30 0.40

Xpter DXS6796 −14.3 −5.09 −3.63 −1.84 −0.90 −0.34 0Xp22.3 DXS996 −9.95 −4.47 −1.59 −0.79 −0.37 −0.13 0Xp22.3 KAL −3.89 −2.71 −0.05 0.09 0.09 0.05 0.1 at 26 cMXp22.2 DXS207 −9.81 −2.71 −1.85 −1.03 −0.06 −0.03 0Xp22.13 DXS999 −4.14 −1.21 −0.71 −0.30 −0.13 −0.06 0Xp22.11 DXS989 −9.75 −1.72 −1.16 −0.64 −0.36 −0.17 0Xp21.3 DXS992 −15.05 −2.16 −1.33 −0.58 −0.23 −0.05 0Xp11.4 DXS1058 −4.85 −2.35 −0.02 0.10 0.11 0.06 0.12 at 25 cMXp11.4 DXS993 −4.07 0.06 0.25 0.31 0.23 0.12 0.32 at 17 cMXp11.3 DXS1003 −4.07 0.06 0.25 0.31 0.23 0.12 0.32 at 17 cMXp11.21 DXS991 −4.07 0.06 0.25 0.31 0.23 0.12 0.32 at 17 cMXq12 AR −2.09 2.32 2.31 1.95 1.41 0.75 2.23 at 7 cMXq12 DXS1111 4.34 3.98 3.62 2.83 1.97 1.02 4.34 at 0 cMXq21.1 DXS1197 4.34 3.98 3.62 2.83 1.97 1.02 4.34 at 0 cMXq21.3 DXS1196 3.91 3.6 3.27 2.56 1.78 0.92 3.91 at 0 cMXq21.3 DXS1066 2.83 2.48 2.15 1.74 1.19 0.60 2.83 at 0 cMXq21.3 DXS1217 −2.09 2.04 2.06 1.75 1.26 0.66 2.08 at 8 cMXq21.33 DXS990 −4.85 −0.24 −0.02 0.10 0.11 0.06 0.12 at 12 cMXq22.2 DXS1106 −10.06 −1.15 −0.63 −0.22 −0.06 0.00 0.01 at 44 cMZq24 DXS424 −10.06 −1.34 −0.89 −0.39 −0.15 −0.04 0Xq24 DXS1001 −4.33 0.23 0.40 0.42 0.32 0.17 0.43 at 16 cMXq25 DXS994 −4.63 −0.51 −0.28 −0.10 −0.04 −0.01 0Xq27 DXS984 −5.24 −0.57 −0.31 −0.08 0.00 0.02 0.02 at 40 cMXq27 DXS8106 −15.11 −2.20 −1.38 −0.65 −0.29 −0.10 0Xq27.3–q28 DXS1113 −10.33 −1.32 −0.81 −0.37 −0.17 −0.06 0Xq28 F8C −10.49 −1.16 −0.63 −0.18 −0.01 0.04 0.04 at 40 cMXqter DXS8087 −5.75 −0.97 −0.62 −0.30 −0.15 −0.06 0

*Text in bold indicates the MRXS9 critical region.

TABLE III. MRX, Apparently Nonsyndromic

Gene Location Reference Phenotype

MRX1 Xp11.3–q12 Suthers et al. [1988]MRX4 Xq13 Hu et al. [1994]MRX5 Xp11.4–q21.2 Sammans et al. [1991]MRX7 Xq21.31 Jedele et al. [1992] Mild MR onlyMRX8 Xq21 Schwartz et al. [1992] Impaired intellect and small headMRX9 Xp21.1–q12 Willems et al. [1993]MRX13 Xp22.3–q21.22 Kerr et al. [1991] Mild handicap, mild MR in carriersMRX14 Xp11.3–q13.2 Gendrot et al. [1994]MRX17 Xp11.23–q12 Gedeon et al. [1996]MRX20 Xq21 Lazzarini et al. [1995]MRX22 Xp21.1–q21.31 Passos-Bueno et al. [1993]MRX26 Xp11.4–q12 Robledo et al. [1996]MRX31 Xp11.23–q13.3 Donnelly et al. [1996]? Xq21.1 May [1995] MR gene near CHM gene

van Brokhoven et al. [1997]

Mapping MRXS9 to Xq12-q21.3 297

phenotype of the current family seems very similar tothat of MRX8, and it seems reasonable that they couldbe alleles of the same gene. Another MRX gene wasmapped to this region by van Brokhoven et al. [1997]who have defined a 500-kb MRX-containing critical re-gion in Xq21 just distal to the ZNF6 gene.

Overlapping Mental Retardation Disorders,Syndromic (MRXS)

Allan-Herndon syndrome (AHS) was mapped to nearDXYS21 in Xq21 [Schwartz et al., 1990] (Table IV);however, since this syndrome is characterized by se-vere mental retardation, muscle hypoplasia, spasticparaplagia and ataxia, elongated face and normal headcircumference, it appears unlikely to be a case of allelicheterogeneity and locus heterogeneity seems morelikely. Another disorder, Wieacker-Wolff syndrome,characterized by mild mental retardation and congen-ital foot deformities, also seems an unlikely candidatefor allelic heterogeneity [Wieacker et al., 1985, 1987].Similarly, Carpenter [1988] described a syndrome,Carpenter-Waziri syndrome, of severe mental retarda-tion, asymmetric face, hypogonadism, joint hypermo-bility, and digital arches but which does include mico-cephaly. This is an allelic form of a-thalassemia/mentalretardation syndrome, nondeletion type (ATRX). Milesand Carpenter [1991] described members of a family(MCS, MRXS4) with severe XLMR mapping to proxi-mal Xq, with short stature and microcephaly. However,they had characteristic asymmetric facial appearances,exotropia, hypogonadism, joint hypermobility, rockerbottom feet, and 10 low digital arches, with prominentlips, bushy eyebrows, depressed nasal bridge with wid-ening of the tip of the nose, and widely spaced teeth aswell as brachydactyly and broadening of the distal pha-langes. The lack of facial, hand, and foot anomalies inthe present family make allelic rather than locus het-erogeneity unlikely. Members of an Australian family(WTS/MRXS6) of Wilson et al. [1991] have moderatemental retardation, obesity, gynecomastia, tapering

fingers, and small feet, but normal height and headcircumferences. Sutherland et al. [1988] described afamily (MRXS3/SRS) in which males had a syndrome ofmental retardation, short stature, microcephaly,brachycephaly, spastic diplegia, small testes, and pos-sibly intrauterine growth retardation. Of significance,spastic diplegia was a finding in IV-6. FG syndrome,which exhibits locus heterogeneity, has one locus at acritical region that partially overlaps the MRXS9 criti-cal region [Briault et al., 1997]. FG syndrome is char-acterized by mental retardation, hypotonia, macro-cephaly, anal anomalies, and characteristic facies. Ah-mad et al. [1997] mapped a gene (MRXS7) in a familywith mental retardation, obesity, and hypogonadism.

Other Genes Within Critical Region

A potentially interesting gene known to map to Xq21is the aforementioned zinc finger gene (ZNF6, Affara etal. [1991]) and may deserve screening for disease-causing mutations in MRX overlapping families. A sec-ond gene of potential interest which may map to thisregion is the fibroblast growth factor (FGF13, alsocalled FHF2) gene. Fibroblast growth factor (FGF) ho-mologous factors have been implicated in nervous sys-tem development [Smallwood et al., 1996] and given itsinvolvement in the central nervous system (CNS) andthe association between other FGF genes and diseases,it would make a good candidate. However, there is adiscrepancy between its location at Xq21 according toLovec et al. [1997] and Xq26 according to Smallwood etal. [1996] which needs to be resolved.

ACKNOWLEDGMENTS

This study was partly supported by the HendrickFund of SUNY Health Science Center.

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TABLE IV. MRXS, Syndromic MRX Genes

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Mapping MRXS9 to Xq12-q21.3 299