- Email: [email protected]
Nonsyndromic X-linked mental retardation: where are the missing mutations?
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Opinion
TRENDS in Genetics Vol.19 No.6 June 2003
Nonsyndromic X-linked mental retardation: where are the missing mutations? Hans-Hilger Ropers1, Maria Hoeltzenbein1, Vera Kalscheuer1, Helger Yntema2, Ben Hamel2, Jean-Pierre Fryns3, Jamel Chelly4, Michael Partington5, Jozef Gecz6 and Claude Moraine7 1
Max-Planck Institut fu¨r Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany Department of Human Genetics, University Hospital Nijmegen, Geert Grooteplein 20, NL-6500 HB Nijmegen, The Netherlands 3 Center for Human Genetics, University Hospital Leuven, U.Z. Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium 4 INSERM U129-ICGM, Faculte´ de Me´de´cine Cochin, 24 rue du Faubourg Saint-Jacques, F-75014 Paris, France 5 Hunter Genetics and University of Newcastle, PO Box 84, Waratah, New South Wales, Australia 2298 6 Department of Cytogenetics and Molecular Genetics, Women’s and Children’s Hospital 72, King William Road, North Adelaide, SA 5006, Australia 7 Service de Ge´ne´tique, CHU Bretonneau, INSERM U316 2 Boulevard Tonnelle´, F-37044 Tours Ce´dex, France 2
Analysis of linkage intervals from 125 unrelated families with nonsyndromic X-linked mental retardation (NS-XLMR) has revealed that the respective gene defects are conspicuously clustered in defined regions of the human X-chromosome, with approximately 30% of all mutations being located on the proximal Xp. In 83% of these families, underlying gene defects are not yet known. Our observations should speed up the search for mutations that are still missing and pave the way for the molecular diagnosis of this common disorder. Mental retardation (MR) occurs significantly more often in males than in females, and there is now ample evidence [1,2] to support the idea that this male bias is largely due to the involvement of X-linked genes that play a role in brain differentiation and function. As deduced by Herbst and Miller [3] from the absolute and relative frequency of MR in males and females, 1.8 males per 1000 carry a gene defect leading to X-linked mental retardation (XLMR). Approximately a third of these patients have syndromic forms of XLMR, where MR is associated with recognizable clinical signs such as skeletal abnormalities or dysmorphic facial features. The underlying gene defect has been identified in 30 forms of these disorders ([4]; see also http://xlmr.interfree.it/home.htm). MR with fragile X (FRAXA), the most common form of XLMR known to date, is now also considered to be syndromic, although there are often no specific recognizable features, particularly in young children, and it has become apparent that its incidence is much lower than previously thought [5,6]. Finding the molecular causes of nonsyndromic (NS)XLMR has turned out to be much more difficult because of Corresponding author: Hans-Hilger Ropers ([email protected]).
genetic heterogeneity, which precludes pooling of linkage data from different families. Therefore, mapping intervals have remained comparatively wide. Until 1998, only a single gene, FMR2, could be isolated, because of its association with another fragile site, FRAXE [7,8]. Analogous to the mechanism leading to inactivation of FMR1 in patients with FRAXA, transcriptional silencing of FMR2 was found to be caused by the expansion and subsequent hypermethylation of a CCG trinucleotide repeat in the 50 noncoding region of the gene. In the past five years, the search for NS-XLMR genes has gained considerable momentum, particularly through the formation of international consortia uniting clinical and molecular expertise. The European XLMR Consortiump, created in 1996, has collected 350 families with established or probable XLMR, and its resources and those of associated groups† have been instrumental in identifying 8 of the 13 genes presently known to play a role in NS-XLMR (Table 1). Most of these genes were cloned by studying mentally retarded patients with X-chromosomal rearrangements, notably balanced translocations and microdeletions, and their identity was subsequently confirmed by mutation screening in families with NS-XLMR. Positional and functional candidate gene approaches have also been successful, this being the search for mutations in brain-expressed genes mapping to defined linkage intervals or in X-linked genes with a known role in brain function or development. Mutations were also found in several genes that had been implicated previously in syndromic forms of XLMR, such as MECP2 and RSK2 [9,10]. These and other observations [11] suggest that there is no molecular * J. Chelly et al., Paris; J-P. Frijns et al., Leuven; B.C.J. Hamel et al., Nijmegen; C. Moraine et al., Tours, and H.H. Ropers et al., Berlin. † G. Turner ([email protected]), M. Partington, Newcastle, and J. Gecz, Adelaide.
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Table 1. Genes involved in nonsyndromic X-linked mental retardation Location
Gene name
Gene symbol
Potential function
Refs
Xp22.3 –22.1 Xp22.1 Xp22.1 –p21.3
RPS6KA3/RSK2 ARX IL1RAPL1
Serine/threonine kinase Transcription factor Unknown
[10] [11,16] [31]
TM4SF2
Xq22
p21 (CDKN1A)-activated kinase
PAK3
Xq22.3
FACL4
Xq28 Xq28
Fatty acid coenzyme A ligase, long chain 4 Angiotensin II receptor, type 2 Rho guanine nucleotide exchange factor 6 Fragile X mental retardation 2 GDP-dissociation inhibitor
Xq28 Xq28
Methyl CpG-binding protein Solute carrier family 6, member 8
MECP2 SLC6A8
Tetraspanin protein, interacts with integrins, control of neurite outgrowth? RhoGAP involved in regulating actin cytoskeleton dynamics and neuronal morphogenesis Rac/Cdc42 effector involved in regulating actin cytoskeleton dynamics and neuronal morphogenesis Involved in vesicle transport, membrane fusion and gene expression Unknown Role in integrin-mediated signaling, regulates Rho-GTPases Rac1/Cdc42 Transcription factor? Rab GDP-dissociation inhibitor involved in synaptic vesicle fusion and neuronal morphogenesis Chromatin remodelling and gene silencing Creatine transporter
[32]
Xq12
Ribosomal protein S6 kinase Aristaless-related homeobox gene Interleukin-1 receptor accessory protein-like 1 Transmembrane 4 superfamily member 2 Oligophrenin 1
Xp11.4
Xq24 Xq26
OPHN1
AGTR2 ARHGEF6 FMR2/FRAXE GDI1
basis for strictly separating syndromic and nonsyndromic forms of XLMR. In most of the genes presently known to play a role in NS-XLMR, mutations have turned out to be very rare (reviewed in Ref. [12]). Extrapolation of these findings suggests that close to 100 different genes might be involved in nonspecific XLMR [13], 5 –10 times more than previously thought [3,14,15]. By contrast, mutations in the recently isolated ARX gene, the human homologue of the Drosophila gene aristaless [11,16], seem to be relatively common in NS-XLMR and various syndromic forms such as XLMR with epilepsy (West syndrome; [11,17]), or dystonic movements of the hands (Partington syndrome; see OMIM entry #309510 at http://www.ncbi.nlm.nih.gov/ htbin-post/Omim/dispmim?309510l). This observation has opened up the possibility that there are still other, hitherto unrecognized, genes that play a major role in the aetiology of NS-XLMR. To find out how these missing mutations are distributed on the X-chromosome and whether they are clustered in specific regions, we have now compiled and analysed linkage data from all published and numerous unpublished families with NS-XLMR. The results are described here. Material and methods Since 1988, 78 large families with XLMR and LOD scores of .2.0 have been published and given separate MRX numbers by the HUGO nomenclature committee (one of these numbers, MRX40, refers to a shortest region of overlap between microdeletions and has been disregarded). Our analysis was based on linkage information from a total of 125 families with NS-XLMR, including 47 unpublished families from the European MRX Consortium and from Australia. X-linkage was assumed when there were at least two affected males in different sibships that were connected through healthy or mildly affected females; families in which only brothers were affected were excluded. http://tigs.trends.com
[33]
[25]
[27] [28] [26,34] [8,19] [20]
[9,21,22] [23]
To increase the resolution of the analysis and to link genetic intervals to genes that have been physicallymapped, all markers used for linkage studies were mapped in silico using a recent version of the X-chromosome sequence (June 2002 freeze, Human Genome Browser, http://genome.ucsc.edu/). When necessary, information from the Genome database (http://www.ncbi.nlm.nih.gov/) or from other sources was included. No discrepancy was detected either between the order of markers thus obtained or their order in previously published genetic maps (e.g. [18] and http://research.marshfieldclinic.org/ genetics/). This had not been the case for earlier versions of the Genome Browser sequence, which contained various inverted segments and other obvious errors. The regional distribution of gene defects in NS-XLMR was inferred from the distribution of the linkage intervals of all families analysed. Each of these intervals, irrespective of their lengths, provides information about the map position of one mutation (or the defect of one gene) and should have the same impact on the curve describing the distribution of mutations. This was achieved by representing all linkage intervals as bars with identical surfaces, or weights, but with varying thickness to compensate for their different lengths. The curves shown in Fig. 1 result from superposition of these bars, or rectangles, and the surface below a segment of these curves reflects the probability that this segment harbours XLMR mutations. To validate these data, computer simulations were performed whereby bars corresponding in size and shape to the 125 weighted linkage intervals were distributed randomly along the length of the X-chromosome in 1000 independent Monte Carlo experiments. A curve representing the mean of the resulting 1000 curves was calculated, and standard deviations from the mean were calculated as the sum of the squares of the deviations from the mean for each marker position. Subsequently, analogous calculations were carried out for the curve resulting from
Opinion
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TRENDS in Genetics Vol.19 No.6 June 2003
Sum of weighted linkage intervals
FMR2 GDM SLO6A8 MECP2
ARHGEF6
FACL4 PAK3 AGTR2
OPHN1
RSK2 ARX
70
ILRAPL
(a)
TM4SF2
(b)
60
50
40
30
20
10
0 q28
q27
q26.3
q25
1000 1100 1200 1300 1400 1500 0.1 Mb q23
900 q21.3
800 q21.1
700 q13.3
600 q13.1
500
p11.22 Xcen q12
400 p11.4
300 p21.3
200 p22.11
100 p22.2 p22.13
0
TRENDS in Genetics
Fig. 1. Regional distribution of mutations in families with nonsyndromic X-linked mental retardation (NS-XLMR) (a) and gene density on the human X-chromosome (b). (a) Upper curve: all families analysed; lower curve: families with known mutations analysed. The surface under these curves corresponds to the sum of ‘weighted’ linkage intervals for individual families, each of which is represented by bars of different height to compensate for different lengths of these intervals. Triangles indicate the map positions of known NS-XLMR genes. (b) Distribution of genes on the human X-chromosome (adapted from http://www.ensembl.org/Homo_sapiens/mapview?chr ¼ X) Blue bars, known genes; red bars, other genes.
superposition of all 125 weighted linkage intervals in their original location. The deviation from the mean was found to be 12.1 times above the standard deviation obtained by randomizing the map position of these linkage intervals. This indicates that this curve cannot have arisen by chance, and that mutations underlying NS-XLMR are not randomly distributed on the X chromosome (see http://www.molgen.mpg.de/research/ropers/ for a complete list of markers, families analysed and linkage intervals, and for further details of this analysis). Results and discussion The results of this analysis are illustrated in Fig. 1. It is apparent that mutations that give rise to NS-XLMR are not evenly distributed along the length of the X-chromosome; instead, they seem to cluster in specific regions. In particular, there is a very high concentration of NS-XLMR genes at Xq28, which is close to the telomere of the long X-chromosome arm. In line with this, at least four genes have already been identified in this region: FMR2, GDI1, MECP2 and SLC6A8 [10,19– 23]. It is conceivable that there are several other genes for NS-XLMR at Xq28 that are still waiting to be discovered, but several of the families used for this analysis might also have as-yet-undetected http://tigs.trends.com
mutations in already identified MR genes. It is of note that the height and steepness of the Xq28 peak is significantly influenced by data from one single family with a very small linkage interval and a hitherto unknown mutation (MRX72 [24]). Without these data, the height of the peak would be about half its present value. Another peak is located at Xp22.1 – p21.3.1, distal to the gene for Duchenne muscular dystrophy, which includes ARX as well as the IL1RAPL1 gene. However, in absolute terms, the proximal short arm of the X-chromosome seems to harbour even more mutations than the Xq28 band, as judged from the surface under the respective portion of the curve (including the centromeric region, see below). Moreover, the irregular shape of the curve, with peaks at Xp11.22 and Xp11.3 –p11.4, argue for the involvement of several different genes in this region. On the long arm, there is a broad ‘hill’ extending from Xq23 to Xq26, the shape of which suggests that this region carries several different genes for NS-XLMR. Indeed, four genes have been identified so far in this interval: PAK3, ARHGEF6, FACL4 and AGTR2 [25 – 28]. Mutations in the AGTR2 gene might be more common than mutations in other known genes of this interval, but this still has to be confirmed.
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TRENDS in Genetics Vol.19 No.6 June 2003
Before taking the results of this analysis at face value, a few cautionary remarks on the possibilities and limitations of this approach may be appropriate. First, using the map positions of flanking markers instead of true crossover breakpoints will systematically overestimate the size of linkage intervals. The resolution of this analysis is confined by the limited number of families for which linkage data were available, and the shape of the curve depends on the number and regional distribution of crossovers. Their frequency varies widely between different parts of the X-chromosome; in particular, crossovers are extremely scarce in the pericentric region. Consequently, linkage intervals around a gene defect in the pericentric region will tend to include the centromere, and the respective peaks will become broader than in other regions of the X-chromosome. This is illustrated by the shape of the highest peak on proximal Xp, which has an extended shoulder at the centromere and falls off steeply at the other side of Xcen. By contrast, there is no reason for assuming that the selection of families has influenced the shape of the curve since we have been careful to include linkage data from all families with NS-XLMR for which results of linkage studies (i.e. markers flanking the linkage intervals) were available. Particular care was taken not to include linkage intervals that were related to syndromic forms of XLMR. For this reason, several families with mutations in the ARX gene were excluded because, in retrospect, the index cases in these families did not have truly NS-XLMR. According to the same criteria, only a single family with a mutation in RSK2, the gene for Coffin – Lowry syndrome [10], was included in this study. Thus, despite the abovementioned limitations, the curve shown in Fig. 1 should quite accurately reflect the regional distribution of mutations in NS-XLMR. The most conspicuous result of our analysis is the prediction that approximately 30% of all mutations underlying NS-XLMR are located in the Xp11.2 –Xp11.3 interval on the proximal short arm of the X chromosome, and that no single gene for NS-XLMR has been identified yet in this region. However, this might soon change, given the fact that several mentally retarded patients with balanced translocations or inversions and breakpoints in the relevant interval are being studied in the laboratories of the European XLMR Consortium and elsewhere (see http://www.molgen.mpg.de/research/ropers/). Moreover, in view of the limited size of the respective interval and the improving possibilities for high-throughput mutation detection, brute-force mutation screening of all relevant genes is another option. Our consortium, with its unrivalled resources, including cell lines from almost 200 unrelated XLMR families and . 350 families in total, is very well equipped for such efforts. Indeed, an ongoing study of this kind has already led to the identification of a novel gene for syndromic and NS-XLMR in the Xp11.2 region (V. Kalscheuer et al., unpublished). From the clinical and diagnostic point of view, the most important question is how many genes are involved in NS-XLMR, and how many of these have to be screened to diagnose 90% or 95% of the total number of mutations. It is of note that, in regions carrying many of the missing http://tigs.trends.com
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mutations, the gene density also tends to be very high (Fig. 1). In particular, this holds true for the Xq28 and the Xp11.2 – p11.3 region, where the high frequency of gene defects leading to MR could simply reflect the high number of genes. However, this might not be true for the proximal part of the Xp21.3 – p22.1 interval, characterized by a relatively high density of mutations and comparatively few genes. A significant portion of the peak in this region can be ascribed to mutations in the ARX gene, which was mutated in 6 out of 125 families included in this analysis. This is also illustrated by the shape of the lower curve in Fig. 1, which represents the sum of weighted linkage intervals for families with known mutations. Hitherto undetected mutations in the neighbouring IL1RAPL1 gene might also contribute to the height of the peak at Xp21.3 – p22 and, thus, it might well be that IL1RAPL1 is a more important cause of NS-XLMR than previously thought. To date, only a fraction of the families for which linkage data are available have been systematically screened for mutations in all relevant known genes for NS-XLMR. Mutations have only been found in 21 out of 125, or 17%, of the families included in this study. Therefore, it is likely that screening of all families with overlapping linkage intervals will reveal additional mutations in these genes and, thus, known genes might account for more than 17% of the mutations that give rise to NS-XLMR. By contrast, it is also clear that defects in many other, hitherto unknown, genes play a role in this disorder and, thus, the bulk of the work still lies ahead. Knowing where on the X-chromosome the missing mutations are located should set the stage for large-scale, systematic mutation screening in candidate genes that map to these regions. With an incidence of 0.5%, severe MR, characterized by an IQ of 50 or below, is all but rare, and NS-XLMR might account for approximately 20 – 25% of these cases. Therefore, the identification of the genes underlying NS-XLMR will have a major impact on the early diagnosis and prevention of mental handicaps. Moreover, it is conceivable that allelic variants of these genes are major determinants of intelligence [29,30]; thus, their functional characterization might also give us new insight into the molecular mechanisms of cognition. Acknowledgements We thank A. Beck and C. Wei (Berlin) for performing computer simulations, A. Gedeon and J. Mulley (Adelaide) for providing linkage information on unpublished Australian families that were included in this study, and C. Scharff (Berlin) for carefully reading the manuscript. This work was supported by the German National Genome Research Network.
References 1 Turner, G. et al. (1970) Renpenning syndrome – X-linked mental retardation. Lancet 2, 365 – 366 2 Lehrke, R.A. (1972) A theory of X-linkage of major intellectual traits. Am. J. Ment. Defic. 76, 611 – 619 3 Herbst, D.S. and Miller, J.R. (1980) Nonspecific X-linked mental retardation II: the frequency in British Columbia. Am. J. Med. Genet. 7, 461 – 469 4 Chiurazzi, P. et al. (2001) XLMR genes: update 2000. Eur. J. Hum. Genet. 9, 71 – 81 5 Turner, G. et al. (1996) Prevalence of fragile X syndrome. Am. J. Med. Genet. 64, 196 – 197 6 de Vries, L.B. et al. (1998) A large diagnostic program for the fragile X
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10 11
12 13 14 15
16
17
18
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syndrome among the mentally handicapped. I. An epidemiologic survey. Ned. Tijdschr. Geneeskd. 142, 1666 – 1671 Knight, S.J. et al. (1994) Triplet repeat expansion at the FRAXE locus and X-linked mild mental handicap. Am. J. Hum. Genet. 55, 81 – 86 Ge´cz, J. et al. (1996) Identification of the gene FMR2, associated with FRAXE mental retardation. Nat. Genet. 13, 105 – 108 Meloni, I. et al. (2000) A mutation in the Rett syndrome gene, MECP2, causes X-linked mental retardation and progressive spasticity in males. Am. J. Hum. Genet. 67, 982 – 985 Merienne, K. et al. (1999) A missense mutation in RPS6KA3 (RSK2) responsible for non-specific mental retardation. Nat. Genet. 22, 13 – 14 Stromme, P. et al. (2002) Mutations in the human ortholog of Aristaless cause X-linked mental retardation and epilepsy. Nat. Genet. 30, 441 – 445 Chelly, J. and Mandel, J-L. (2001) Monogenic causes of X-linked mental retardation. Nat. Rev. Genet. 2, 669– 678 Ge´cz, J. and Mulley, J. (2000) Genes for cognitive function: developments on the X. Genome Res. 10, 157 – 163 Gedeon, A.K. et al. (1996) How many X-linked genes for non-specific mental retardation (MRX) are there? Am. J. Med. Genet. 64, 158– 162 Claes, S. (1997) Localization of genetic factors for nonspecific and syndromic X-linked mental retardation. Thesis. Acta Biomedica Lovaniensia 160, Leuven University Press Bienvenu, T. et al. (2002) ARX, a novel Prd-class-homeobox gene highly expressed in the telencephalon, is mutated in X-linked mental retardation. Hum. Mol. Genet. 11, 981 – 991 Scheffer, I.E. et al. (2002) X-linked myoclonic epilepsy with spasticity and intellectual disability: mutation in the homeobox gene ARX. Neurology 59, 348– 356 Broman, K.W. et al. (1998) Comprehensive human genetic maps: individual and sex-specific variation in recombination. Am. J. Hum. Genet. 63, 861 – 869 Gecz, J. et al. (1996) Identification of the gene FMR2, associated with FRAXE mental retardation. Nat. Genet. 13, 105 – 108 D’Adamo, P. et al. (1998) Mutations in GDI1 are responsible for X-linked non-specific mental retardation. Nat. Genet. 19, 134 – 139
21 Couvert, P. et al. (2001) MECP2 is highly mutated in X-linked mental retardation. Hum. Mol. Genet. 10, 941 – 946 22 Yntema, H.G. et al. (2002) Low frequency of MECP2 mutations in mentally retarded males. Eur. J. Hum. Genet. 10, 487– 490 23 Hahn, K.A. et al. (2002) X-linked mental retardation with seizures and carrier manifestations is caused by a mutation in the creatinetransporter gene (SLC6A8) located in Xq28. Am. J. Hum. Genet. 70, 1349– 1356 24 Russo, S. et al. (2000) Mapping to distal Xq28 of nonspecific X-linked mental retardation MRX72: linkage analysis and clinical findings in a three-generation Sardinian family. Am. J. Med. Genet. 94, 376– 382 25 Allen, K.M. et al. (1998) PAK3 mutation in nonsyndromic X-linked mental retardation. Nat. Genet. 20, 25 – 30 26 Kutsche, K. et al. (2000) Mutations in ARHGEF6, encoding a guanine nucleotide exchange factor for Rho GTPases, in patients with X-linked mental retardation. Nat. Genet. 26, 247 – 250 27 Meloni, I. et al. (2002) FACL4, encoding fatty acid-CoA ligase 4, is mutated in nonspecific X-linked mental retardation. Nat. Genet. 30, 436– 440 28 Vervoort, V.S. et al. (2002) AGTR2 mutations in X-linked mental retardation. Science 296, 2401– 2403 29 Turner, G. and Partington, M.W. (1991) Genes for intelligence on the X chromosome. J. Med. Genet. 28, 429 30 Nokelainen, P. and Flint, J. (2002) Genetic effects on human cognition: lessons from the study of mental retardation syndromes. J. Neurol. Neurosurg. Psychiatry 72, 287 – 296 31 Carrie´, A. et al. (1999) A new member of the IL-1 receptor family highly expressed in hippocampus and involved in X-linked mental retardation. Nat. Genet. 23, 25 – 31 32 Zemni, R. et al. (2000) A new gene involved in X-linked mental retardation identified by analysis of an X;2 balanced translocation. Nat. Genet. 24, 167 – 170 33 Billuart, P. et al. (1998) Oligophrenin-1 encodes a rhoGAP protein involved in X-linked mental retardation. Nature 392, 923 – 926 34 Rosenberger, G. et al. (2003) Interaction of aPIX (ARHGEF6) with b-parvin (PARVB) suggests an involvement of aPIX in integrinmediated signaling. Hum. Mol. Genet. 12, 155 – 167
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Nonsyndromic X-linked mental retardation: where are the missing mutations? Hans-Hilger Ropers1, Maria Hoeltzenbein1, Vera Kalscheuer1, Helger Yntema2, Ben Hamel2, Jean-Pierre Fryns3, Jamel Chelly4, Michael Partington5, Jozef Gecz6 and Claude Moraine7 1
Max-Planck Institut fu¨r Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany Department of Human Genetics, University Hospital Nijmegen, Geert Grooteplein 20, NL-6500 HB Nijmegen, The Netherlands 3 Center for Human Genetics, University Hospital Leuven, U.Z. Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium 4 INSERM U129-ICGM, Faculte´ de Me´de´cine Cochin, 24 rue du Faubourg Saint-Jacques, F-75014 Paris, France 5 Hunter Genetics and University of Newcastle, PO Box 84, Waratah, New South Wales, Australia 2298 6 Department of Cytogenetics and Molecular Genetics, Women’s and Children’s Hospital 72, King William Road, North Adelaide, SA 5006, Australia 7 Service de Ge´ne´tique, CHU Bretonneau, INSERM U316 2 Boulevard Tonnelle´, F-37044 Tours Ce´dex, France 2
Analysis of linkage intervals from 125 unrelated families with nonsyndromic X-linked mental retardation (NS-XLMR) has revealed that the respective gene defects are conspicuously clustered in defined regions of the human X-chromosome, with approximately 30% of all mutations being located on the proximal Xp. In 83% of these families, underlying gene defects are not yet known. Our observations should speed up the search for mutations that are still missing and pave the way for the molecular diagnosis of this common disorder. Mental retardation (MR) occurs significantly more often in males than in females, and there is now ample evidence [1,2] to support the idea that this male bias is largely due to the involvement of X-linked genes that play a role in brain differentiation and function. As deduced by Herbst and Miller [3] from the absolute and relative frequency of MR in males and females, 1.8 males per 1000 carry a gene defect leading to X-linked mental retardation (XLMR). Approximately a third of these patients have syndromic forms of XLMR, where MR is associated with recognizable clinical signs such as skeletal abnormalities or dysmorphic facial features. The underlying gene defect has been identified in 30 forms of these disorders ([4]; see also http://xlmr.interfree.it/home.htm). MR with fragile X (FRAXA), the most common form of XLMR known to date, is now also considered to be syndromic, although there are often no specific recognizable features, particularly in young children, and it has become apparent that its incidence is much lower than previously thought [5,6]. Finding the molecular causes of nonsyndromic (NS)XLMR has turned out to be much more difficult because of Corresponding author: Hans-Hilger Ropers ([email protected]).
genetic heterogeneity, which precludes pooling of linkage data from different families. Therefore, mapping intervals have remained comparatively wide. Until 1998, only a single gene, FMR2, could be isolated, because of its association with another fragile site, FRAXE [7,8]. Analogous to the mechanism leading to inactivation of FMR1 in patients with FRAXA, transcriptional silencing of FMR2 was found to be caused by the expansion and subsequent hypermethylation of a CCG trinucleotide repeat in the 50 noncoding region of the gene. In the past five years, the search for NS-XLMR genes has gained considerable momentum, particularly through the formation of international consortia uniting clinical and molecular expertise. The European XLMR Consortiump, created in 1996, has collected 350 families with established or probable XLMR, and its resources and those of associated groups† have been instrumental in identifying 8 of the 13 genes presently known to play a role in NS-XLMR (Table 1). Most of these genes were cloned by studying mentally retarded patients with X-chromosomal rearrangements, notably balanced translocations and microdeletions, and their identity was subsequently confirmed by mutation screening in families with NS-XLMR. Positional and functional candidate gene approaches have also been successful, this being the search for mutations in brain-expressed genes mapping to defined linkage intervals or in X-linked genes with a known role in brain function or development. Mutations were also found in several genes that had been implicated previously in syndromic forms of XLMR, such as MECP2 and RSK2 [9,10]. These and other observations [11] suggest that there is no molecular * J. Chelly et al., Paris; J-P. Frijns et al., Leuven; B.C.J. Hamel et al., Nijmegen; C. Moraine et al., Tours, and H.H. Ropers et al., Berlin. † G. Turner ([email protected]), M. Partington, Newcastle, and J. Gecz, Adelaide.
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Table 1. Genes involved in nonsyndromic X-linked mental retardation Location
Gene name
Gene symbol
Potential function
Refs
Xp22.3 –22.1 Xp22.1 Xp22.1 –p21.3
RPS6KA3/RSK2 ARX IL1RAPL1
Serine/threonine kinase Transcription factor Unknown
[10] [11,16] [31]
TM4SF2
Xq22
p21 (CDKN1A)-activated kinase
PAK3
Xq22.3
FACL4
Xq28 Xq28
Fatty acid coenzyme A ligase, long chain 4 Angiotensin II receptor, type 2 Rho guanine nucleotide exchange factor 6 Fragile X mental retardation 2 GDP-dissociation inhibitor
Xq28 Xq28
Methyl CpG-binding protein Solute carrier family 6, member 8
MECP2 SLC6A8
Tetraspanin protein, interacts with integrins, control of neurite outgrowth? RhoGAP involved in regulating actin cytoskeleton dynamics and neuronal morphogenesis Rac/Cdc42 effector involved in regulating actin cytoskeleton dynamics and neuronal morphogenesis Involved in vesicle transport, membrane fusion and gene expression Unknown Role in integrin-mediated signaling, regulates Rho-GTPases Rac1/Cdc42 Transcription factor? Rab GDP-dissociation inhibitor involved in synaptic vesicle fusion and neuronal morphogenesis Chromatin remodelling and gene silencing Creatine transporter
[32]
Xq12
Ribosomal protein S6 kinase Aristaless-related homeobox gene Interleukin-1 receptor accessory protein-like 1 Transmembrane 4 superfamily member 2 Oligophrenin 1
Xp11.4
Xq24 Xq26
OPHN1
AGTR2 ARHGEF6 FMR2/FRAXE GDI1
basis for strictly separating syndromic and nonsyndromic forms of XLMR. In most of the genes presently known to play a role in NS-XLMR, mutations have turned out to be very rare (reviewed in Ref. [12]). Extrapolation of these findings suggests that close to 100 different genes might be involved in nonspecific XLMR [13], 5 –10 times more than previously thought [3,14,15]. By contrast, mutations in the recently isolated ARX gene, the human homologue of the Drosophila gene aristaless [11,16], seem to be relatively common in NS-XLMR and various syndromic forms such as XLMR with epilepsy (West syndrome; [11,17]), or dystonic movements of the hands (Partington syndrome; see OMIM entry #309510 at http://www.ncbi.nlm.nih.gov/ htbin-post/Omim/dispmim?309510l). This observation has opened up the possibility that there are still other, hitherto unrecognized, genes that play a major role in the aetiology of NS-XLMR. To find out how these missing mutations are distributed on the X-chromosome and whether they are clustered in specific regions, we have now compiled and analysed linkage data from all published and numerous unpublished families with NS-XLMR. The results are described here. Material and methods Since 1988, 78 large families with XLMR and LOD scores of .2.0 have been published and given separate MRX numbers by the HUGO nomenclature committee (one of these numbers, MRX40, refers to a shortest region of overlap between microdeletions and has been disregarded). Our analysis was based on linkage information from a total of 125 families with NS-XLMR, including 47 unpublished families from the European MRX Consortium and from Australia. X-linkage was assumed when there were at least two affected males in different sibships that were connected through healthy or mildly affected females; families in which only brothers were affected were excluded. http://tigs.trends.com
[33]
[25]
[27] [28] [26,34] [8,19] [20]
[9,21,22] [23]
To increase the resolution of the analysis and to link genetic intervals to genes that have been physicallymapped, all markers used for linkage studies were mapped in silico using a recent version of the X-chromosome sequence (June 2002 freeze, Human Genome Browser, http://genome.ucsc.edu/). When necessary, information from the Genome database (http://www.ncbi.nlm.nih.gov/) or from other sources was included. No discrepancy was detected either between the order of markers thus obtained or their order in previously published genetic maps (e.g. [18] and http://research.marshfieldclinic.org/ genetics/). This had not been the case for earlier versions of the Genome Browser sequence, which contained various inverted segments and other obvious errors. The regional distribution of gene defects in NS-XLMR was inferred from the distribution of the linkage intervals of all families analysed. Each of these intervals, irrespective of their lengths, provides information about the map position of one mutation (or the defect of one gene) and should have the same impact on the curve describing the distribution of mutations. This was achieved by representing all linkage intervals as bars with identical surfaces, or weights, but with varying thickness to compensate for their different lengths. The curves shown in Fig. 1 result from superposition of these bars, or rectangles, and the surface below a segment of these curves reflects the probability that this segment harbours XLMR mutations. To validate these data, computer simulations were performed whereby bars corresponding in size and shape to the 125 weighted linkage intervals were distributed randomly along the length of the X-chromosome in 1000 independent Monte Carlo experiments. A curve representing the mean of the resulting 1000 curves was calculated, and standard deviations from the mean were calculated as the sum of the squares of the deviations from the mean for each marker position. Subsequently, analogous calculations were carried out for the curve resulting from
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TRENDS in Genetics Vol.19 No.6 June 2003
Sum of weighted linkage intervals
FMR2 GDM SLO6A8 MECP2
ARHGEF6
FACL4 PAK3 AGTR2
OPHN1
RSK2 ARX
70
ILRAPL
(a)
TM4SF2
(b)
60
50
40
30
20
10
0 q28
q27
q26.3
q25
1000 1100 1200 1300 1400 1500 0.1 Mb q23
900 q21.3
800 q21.1
700 q13.3
600 q13.1
500
p11.22 Xcen q12
400 p11.4
300 p21.3
200 p22.11
100 p22.2 p22.13
0
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Fig. 1. Regional distribution of mutations in families with nonsyndromic X-linked mental retardation (NS-XLMR) (a) and gene density on the human X-chromosome (b). (a) Upper curve: all families analysed; lower curve: families with known mutations analysed. The surface under these curves corresponds to the sum of ‘weighted’ linkage intervals for individual families, each of which is represented by bars of different height to compensate for different lengths of these intervals. Triangles indicate the map positions of known NS-XLMR genes. (b) Distribution of genes on the human X-chromosome (adapted from http://www.ensembl.org/Homo_sapiens/mapview?chr ¼ X) Blue bars, known genes; red bars, other genes.
superposition of all 125 weighted linkage intervals in their original location. The deviation from the mean was found to be 12.1 times above the standard deviation obtained by randomizing the map position of these linkage intervals. This indicates that this curve cannot have arisen by chance, and that mutations underlying NS-XLMR are not randomly distributed on the X chromosome (see http://www.molgen.mpg.de/research/ropers/ for a complete list of markers, families analysed and linkage intervals, and for further details of this analysis). Results and discussion The results of this analysis are illustrated in Fig. 1. It is apparent that mutations that give rise to NS-XLMR are not evenly distributed along the length of the X-chromosome; instead, they seem to cluster in specific regions. In particular, there is a very high concentration of NS-XLMR genes at Xq28, which is close to the telomere of the long X-chromosome arm. In line with this, at least four genes have already been identified in this region: FMR2, GDI1, MECP2 and SLC6A8 [10,19– 23]. It is conceivable that there are several other genes for NS-XLMR at Xq28 that are still waiting to be discovered, but several of the families used for this analysis might also have as-yet-undetected http://tigs.trends.com
mutations in already identified MR genes. It is of note that the height and steepness of the Xq28 peak is significantly influenced by data from one single family with a very small linkage interval and a hitherto unknown mutation (MRX72 [24]). Without these data, the height of the peak would be about half its present value. Another peak is located at Xp22.1 – p21.3.1, distal to the gene for Duchenne muscular dystrophy, which includes ARX as well as the IL1RAPL1 gene. However, in absolute terms, the proximal short arm of the X-chromosome seems to harbour even more mutations than the Xq28 band, as judged from the surface under the respective portion of the curve (including the centromeric region, see below). Moreover, the irregular shape of the curve, with peaks at Xp11.22 and Xp11.3 –p11.4, argue for the involvement of several different genes in this region. On the long arm, there is a broad ‘hill’ extending from Xq23 to Xq26, the shape of which suggests that this region carries several different genes for NS-XLMR. Indeed, four genes have been identified so far in this interval: PAK3, ARHGEF6, FACL4 and AGTR2 [25 – 28]. Mutations in the AGTR2 gene might be more common than mutations in other known genes of this interval, but this still has to be confirmed.
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Before taking the results of this analysis at face value, a few cautionary remarks on the possibilities and limitations of this approach may be appropriate. First, using the map positions of flanking markers instead of true crossover breakpoints will systematically overestimate the size of linkage intervals. The resolution of this analysis is confined by the limited number of families for which linkage data were available, and the shape of the curve depends on the number and regional distribution of crossovers. Their frequency varies widely between different parts of the X-chromosome; in particular, crossovers are extremely scarce in the pericentric region. Consequently, linkage intervals around a gene defect in the pericentric region will tend to include the centromere, and the respective peaks will become broader than in other regions of the X-chromosome. This is illustrated by the shape of the highest peak on proximal Xp, which has an extended shoulder at the centromere and falls off steeply at the other side of Xcen. By contrast, there is no reason for assuming that the selection of families has influenced the shape of the curve since we have been careful to include linkage data from all families with NS-XLMR for which results of linkage studies (i.e. markers flanking the linkage intervals) were available. Particular care was taken not to include linkage intervals that were related to syndromic forms of XLMR. For this reason, several families with mutations in the ARX gene were excluded because, in retrospect, the index cases in these families did not have truly NS-XLMR. According to the same criteria, only a single family with a mutation in RSK2, the gene for Coffin – Lowry syndrome [10], was included in this study. Thus, despite the abovementioned limitations, the curve shown in Fig. 1 should quite accurately reflect the regional distribution of mutations in NS-XLMR. The most conspicuous result of our analysis is the prediction that approximately 30% of all mutations underlying NS-XLMR are located in the Xp11.2 –Xp11.3 interval on the proximal short arm of the X chromosome, and that no single gene for NS-XLMR has been identified yet in this region. However, this might soon change, given the fact that several mentally retarded patients with balanced translocations or inversions and breakpoints in the relevant interval are being studied in the laboratories of the European XLMR Consortium and elsewhere (see http://www.molgen.mpg.de/research/ropers/). Moreover, in view of the limited size of the respective interval and the improving possibilities for high-throughput mutation detection, brute-force mutation screening of all relevant genes is another option. Our consortium, with its unrivalled resources, including cell lines from almost 200 unrelated XLMR families and . 350 families in total, is very well equipped for such efforts. Indeed, an ongoing study of this kind has already led to the identification of a novel gene for syndromic and NS-XLMR in the Xp11.2 region (V. Kalscheuer et al., unpublished). From the clinical and diagnostic point of view, the most important question is how many genes are involved in NS-XLMR, and how many of these have to be screened to diagnose 90% or 95% of the total number of mutations. It is of note that, in regions carrying many of the missing http://tigs.trends.com
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mutations, the gene density also tends to be very high (Fig. 1). In particular, this holds true for the Xq28 and the Xp11.2 – p11.3 region, where the high frequency of gene defects leading to MR could simply reflect the high number of genes. However, this might not be true for the proximal part of the Xp21.3 – p22.1 interval, characterized by a relatively high density of mutations and comparatively few genes. A significant portion of the peak in this region can be ascribed to mutations in the ARX gene, which was mutated in 6 out of 125 families included in this analysis. This is also illustrated by the shape of the lower curve in Fig. 1, which represents the sum of weighted linkage intervals for families with known mutations. Hitherto undetected mutations in the neighbouring IL1RAPL1 gene might also contribute to the height of the peak at Xp21.3 – p22 and, thus, it might well be that IL1RAPL1 is a more important cause of NS-XLMR than previously thought. To date, only a fraction of the families for which linkage data are available have been systematically screened for mutations in all relevant known genes for NS-XLMR. Mutations have only been found in 21 out of 125, or 17%, of the families included in this study. Therefore, it is likely that screening of all families with overlapping linkage intervals will reveal additional mutations in these genes and, thus, known genes might account for more than 17% of the mutations that give rise to NS-XLMR. By contrast, it is also clear that defects in many other, hitherto unknown, genes play a role in this disorder and, thus, the bulk of the work still lies ahead. Knowing where on the X-chromosome the missing mutations are located should set the stage for large-scale, systematic mutation screening in candidate genes that map to these regions. With an incidence of 0.5%, severe MR, characterized by an IQ of 50 or below, is all but rare, and NS-XLMR might account for approximately 20 – 25% of these cases. Therefore, the identification of the genes underlying NS-XLMR will have a major impact on the early diagnosis and prevention of mental handicaps. Moreover, it is conceivable that allelic variants of these genes are major determinants of intelligence [29,30]; thus, their functional characterization might also give us new insight into the molecular mechanisms of cognition. Acknowledgements We thank A. Beck and C. Wei (Berlin) for performing computer simulations, A. Gedeon and J. Mulley (Adelaide) for providing linkage information on unpublished Australian families that were included in this study, and C. Scharff (Berlin) for carefully reading the manuscript. This work was supported by the German National Genome Research Network.
References 1 Turner, G. et al. (1970) Renpenning syndrome – X-linked mental retardation. Lancet 2, 365 – 366 2 Lehrke, R.A. (1972) A theory of X-linkage of major intellectual traits. Am. J. Ment. Defic. 76, 611 – 619 3 Herbst, D.S. and Miller, J.R. (1980) Nonspecific X-linked mental retardation II: the frequency in British Columbia. Am. J. Med. Genet. 7, 461 – 469 4 Chiurazzi, P. et al. (2001) XLMR genes: update 2000. Eur. J. Hum. Genet. 9, 71 – 81 5 Turner, G. et al. (1996) Prevalence of fragile X syndrome. Am. J. Med. Genet. 64, 196 – 197 6 de Vries, L.B. et al. (1998) A large diagnostic program for the fragile X
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16
17
18
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syndrome among the mentally handicapped. I. An epidemiologic survey. Ned. Tijdschr. Geneeskd. 142, 1666 – 1671 Knight, S.J. et al. (1994) Triplet repeat expansion at the FRAXE locus and X-linked mild mental handicap. Am. J. Hum. Genet. 55, 81 – 86 Ge´cz, J. et al. (1996) Identification of the gene FMR2, associated with FRAXE mental retardation. Nat. Genet. 13, 105 – 108 Meloni, I. et al. (2000) A mutation in the Rett syndrome gene, MECP2, causes X-linked mental retardation and progressive spasticity in males. Am. J. Hum. Genet. 67, 982 – 985 Merienne, K. et al. (1999) A missense mutation in RPS6KA3 (RSK2) responsible for non-specific mental retardation. Nat. Genet. 22, 13 – 14 Stromme, P. et al. (2002) Mutations in the human ortholog of Aristaless cause X-linked mental retardation and epilepsy. Nat. Genet. 30, 441 – 445 Chelly, J. and Mandel, J-L. (2001) Monogenic causes of X-linked mental retardation. Nat. Rev. Genet. 2, 669– 678 Ge´cz, J. and Mulley, J. (2000) Genes for cognitive function: developments on the X. Genome Res. 10, 157 – 163 Gedeon, A.K. et al. (1996) How many X-linked genes for non-specific mental retardation (MRX) are there? Am. J. Med. Genet. 64, 158– 162 Claes, S. (1997) Localization of genetic factors for nonspecific and syndromic X-linked mental retardation. Thesis. Acta Biomedica Lovaniensia 160, Leuven University Press Bienvenu, T. et al. (2002) ARX, a novel Prd-class-homeobox gene highly expressed in the telencephalon, is mutated in X-linked mental retardation. Hum. Mol. Genet. 11, 981 – 991 Scheffer, I.E. et al. (2002) X-linked myoclonic epilepsy with spasticity and intellectual disability: mutation in the homeobox gene ARX. Neurology 59, 348– 356 Broman, K.W. et al. (1998) Comprehensive human genetic maps: individual and sex-specific variation in recombination. Am. J. Hum. Genet. 63, 861 – 869 Gecz, J. et al. (1996) Identification of the gene FMR2, associated with FRAXE mental retardation. Nat. Genet. 13, 105 – 108 D’Adamo, P. et al. (1998) Mutations in GDI1 are responsible for X-linked non-specific mental retardation. Nat. Genet. 19, 134 – 139
21 Couvert, P. et al. (2001) MECP2 is highly mutated in X-linked mental retardation. Hum. Mol. Genet. 10, 941 – 946 22 Yntema, H.G. et al. (2002) Low frequency of MECP2 mutations in mentally retarded males. Eur. J. Hum. Genet. 10, 487– 490 23 Hahn, K.A. et al. (2002) X-linked mental retardation with seizures and carrier manifestations is caused by a mutation in the creatinetransporter gene (SLC6A8) located in Xq28. Am. J. Hum. Genet. 70, 1349– 1356 24 Russo, S. et al. (2000) Mapping to distal Xq28 of nonspecific X-linked mental retardation MRX72: linkage analysis and clinical findings in a three-generation Sardinian family. Am. J. Med. Genet. 94, 376– 382 25 Allen, K.M. et al. (1998) PAK3 mutation in nonsyndromic X-linked mental retardation. Nat. Genet. 20, 25 – 30 26 Kutsche, K. et al. (2000) Mutations in ARHGEF6, encoding a guanine nucleotide exchange factor for Rho GTPases, in patients with X-linked mental retardation. Nat. Genet. 26, 247 – 250 27 Meloni, I. et al. (2002) FACL4, encoding fatty acid-CoA ligase 4, is mutated in nonspecific X-linked mental retardation. Nat. Genet. 30, 436– 440 28 Vervoort, V.S. et al. (2002) AGTR2 mutations in X-linked mental retardation. Science 296, 2401– 2403 29 Turner, G. and Partington, M.W. (1991) Genes for intelligence on the X chromosome. J. Med. Genet. 28, 429 30 Nokelainen, P. and Flint, J. (2002) Genetic effects on human cognition: lessons from the study of mental retardation syndromes. J. Neurol. Neurosurg. Psychiatry 72, 287 – 296 31 Carrie´, A. et al. (1999) A new member of the IL-1 receptor family highly expressed in hippocampus and involved in X-linked mental retardation. Nat. Genet. 23, 25 – 31 32 Zemni, R. et al. (2000) A new gene involved in X-linked mental retardation identified by analysis of an X;2 balanced translocation. Nat. Genet. 24, 167 – 170 33 Billuart, P. et al. (1998) Oligophrenin-1 encodes a rhoGAP protein involved in X-linked mental retardation. Nature 392, 923 – 926 34 Rosenberger, G. et al. (2003) Interaction of aPIX (ARHGEF6) with b-parvin (PARVB) suggests an involvement of aPIX in integrinmediated signaling. Hum. Mol. Genet. 12, 155 – 167
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