Cloning and characterization of DXS6673E, a candidate gene for X-linked mental retardation in Xq13.1
Cloning and characterization of DXS6673E , a candidate gene for X-linked mental retardation in Xq13.1Silvère M. van der Maarel1,2,*, Inge H. J. M. Scholten1, Irene Huber1, Christophe Philippe3, Ron F. Suijkerbuijk1, Simone Gilgenkrantz4, Juha Kere5,+, Frans P. M. Cremers1 and Hans-Hilger Ropers1,2
1Department of Human Genetics, University Hospital Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands, 2Max-Planck-Institut für Molekulare Genetik, Ihnestraße 73, D-14195 Berlin, Germany, 3The Wellcome Trust Centre for Human Genetics, Oxford, UK, 4Centre Hospitale Universitaire de Nancy, Nancy, France and 5Washington University School of Medicine, St. Louis, USA
Received February 20, 1996;Revised and Accepted April 17, 1996
In several families with non-specific X-linked mental retardation (XLMR) linkage analyses have assigned the underlying gene defect to the pericentromeric region of the X chromosome, but none of these genes have been isolated so far. Here, we report on the cloning and characterization of a novel gene, DXS6673E, that maps to Xq13.1, is subject to X-inactivation and is disrupted in the 5' untranslated region by a balanced X;13 translocation in a mentally retarded female. The DXS6673E gene is highly conserved among vertebrates and its expression is most abundant in brain. It encodes a hydrophilic protein of 1358 amino acids (aa) that does not show sequence homology to other known proteins. A segment of this protein consisting of neutral and hydrophobic aa with a proline residue in every second position may represent a transmembrane domain. Almost complete sequence identity was found between the 3' end of the DXS6673E gene and two expressed sequence tags (ESTs) and between the 5' end of the DXS6673E gene and a third EST. Moreover, weaker sequence similarity was observed between coding regions and two other ESTs.
It has long been recognized that the X chromosome contributes disproportionally to the total number of loci that are involved in mental retardation (MR). Population studies have revealed that the prevalence of X-linked mental retardation (XLMR) in males varies from 1:300 to about 1:500 and have suggested that X-linked genes account for approximately 20-50% of all cases with MR. On the basis of data it has been estimated that at least seven to 19 X chromosomal genes play a major part in MR (1 -5 ). Recently, several gene defects underlying specific forms of XLMR were identified, such as the fragile X syndrome (FraXA), MASA syndrome (mental retardation, aphasia, shuffling gait and adducted thumbs) and mental retardation/[alpha]-thalassaemia (ATR-X) syndrome (6 -9 ). In contrast to these disorders with specific recognizable phenotypes or karyotypes, there are many forms of non-specific XLMR without additional clinical features. So far, the genetic heterogeneity of non-specific XLMR has prevented its molecular elucidation and for the majority of cases with this condition, neither carrier detection nor early diagnosis is possible.
Linkage analyses in individual families have revealed that there are at least six genes on the X chromosome that play a part in non-specific XLMR (10 ,11 ). For all of these genes however, linkage intervals are still too wide to allow reliable genetic counselling and the identification of causative gene defects by positional cloning. Cytogenetically recognizable chromosomal rearrangements such as X-autosome translocations have greatly facilitated the positional cloning of disease-associated gene defects (12 ). Balanced X-autosome translocations and inversions have also been described in several patients with MR (13 -16 ) and the characterization of the relevant breakpoint regions on the X chromosome is therefore a promising strategy to identify candidate genes for XLMR.
Here, we report on the identification and characterization of an X chromosomal gene, DXS6673E, that is disrupted by a balanced X;13 translocation involving the Xq13.1 band in a mentally retarded female. Apart from mental retardation, scoliosis and spotty abdominal hypopigmentation are the only other conspicuous clinical features in this patient (15 ,17 ).
A hybrid cell line containing the derivative chromosome 13 as its only human X-chromosomal component (17 ) was used to regionally map the X chromosomal breakpoint by Southern blot analysis and PCR amplification of markers from the Xq13-q21 region. The breakpoint could be assigned to a small segment of Xq13.1 between the gene encoding the gap junction protein connexin 32 (GJB1) (18 ,19 ) and the cell cycle gene CCG1 (20 ) (data not shown). Four YAC clones, two of which were identified with DXS348 (21 ,22 ) and two with CCG1, were hybridized in situ to metaphase chromosomes of the patient. One of the YACs identified with CCG1, clone yWXD940, spans the breakpoint on the X chromosome (Fig. 1 ). Apart from the two X-centromeric hybridization signals, three signals were seen representing the hybridization of the YAC to the normal X chromosome, the derivative X-chromosome and the derivative chromosome 13 [der(13)], respectively.
By subcloning YAC yWXD940, a partial cosmid contig was generated. Employing Southern blot analysis a cosmid, L2, could be identified that crossed the breakpoint. This cosmid is located approximately 150 kb proximal to the cell cycle gene CCG1 as estimated from pulsed field gel electrophoresis (data not shown).
The human insert of cosmid L2 was hybridized to a fetal brain cDNA library which resulted in the isolation of 17 cDNA clones. Further screening of the same library with these cDNAs led to the isolation of over 45 homologous, partially overlapping cDNA clones. Hybridization of the clones 50:11 and 50:17 representing the full length cDNA (Fig. 3 ) to a Southern blot with PvuII digested DNA from the patient, the hybrid cell line carrying the der(13), as well as several controls, clearly showed that a gene represented by these cDNA clones is disrupted by the translocation (Fig. 2 ). In the DNA of the patient, two aberrant restriction fragments were seen. One of these fragments was also seen in the DNA from the human-hamster hybrid cell line containing the der(13 ) chromosome. Aberrant bands were also obtained by hybridization of the same probe to a Southern blot containing DNA from the patient and a female control digested with several other enzymes (data not shown). The DXS6673E gene, from which these cDNAs are derived, hybridizes to three adjacent EcoRI fragments of 6, 10 and 6 kb. The most distal 6 kb EcoRI fragment is disrupted by the translocation in the patient (Fig. 3 ).
The positional cloning of numerous disease genes has been facilitated by the presence of chromosome aberrations that are associated with specific disorders (12 ). The incidence of balanced chromosome rearrangements among newborns is approximately 1:3000 and 6% of these rearrangements are associated with abnormal phenotypes (13 ). In 50% of these cases, the aberrant clinical phenotype may be causally related to the karyotypic changes. This proportion may even be higher for X-autosome translocations because in males truncation of X-chromosomal genes will result in a functional nullisomy. Usually, the same is true for females with balanced X-autosome translocations because of preferential inactivation of the normal X (13 ,14 ,27 ). The molecular characterization of balanced X chromosomal rearrangements associated with MR therefore, is a promising strategy for the identification of new candidate genes for XLMR.
Here, we have identified and characterized an X chromosomal gene, DXS6673E, that is disrupted in its 5' non-coding region by an X;13 translocation in a mentally retarded female. The gene encodes two mRNAs of 6126 bp and 5538 bp, respectively and consists of 25 exons spread over approximately 22 kb of genomic DNA. It contains two alternative first exons both encoding a 5' untranslated region, which may be under the control of different tissue-specific promotors. Analysis of cDNA clones and RT-PCR experiments demonstrated that these sequences are part of the gene. Moreover, the splice junctions of both exons fit the consensus sequence. There is a 13 bp discrepancy between the cDNA clone and the RT-PCR product of the first exon from various tissues and cell lines at the splice donor site. As both variants show convincing similarity with the consensus splice donor site, again this may be due to alternative splicing. For these variants an open reading frame of 1358 aa was identified that starts in exon 2 and continues through all downstream exons. There are two putative AUG start codons at position 726 and 807, respectively (Fig. 4 ). The second AUG fits the Kozak consensus sequence better than the first which lacks a purine residue at position -3 (28 ). Therefore, we believe that the second AUG is the translation initiation site. Identity was found with five ESTs when comparing the DXS6673E gene to the database, three of which (HS04328, HS448109 and HS21F9F) encode the same gene. Interestingly, HS21F9F was isolated by using a methylated DNA binding column that selects for CpG-like sequences (29 ). Moreover, this region of identity colocalizes with the breakpoint region. However, a CpG plot (GCG package) did not reveal the presence of a CpG island in exon 1A (24 ,30 ). In contrast, a 300 bp sequence starting in intron 1A and extending through exon 1B into intron 1B (Fig. 3 ) was identified that meets all criteria for a CpG island (data not shown). The two other ESTs may be derived from homologous domains of related genes. One of these ESTs, HS071103, is similar to the putative transmembrane domain of the DXS6673E protein which exhibits highly conserved stretches of neutral and hydrophobic residues with a proline residue on every second position. The two ESTs are derived from different tissues; HS365200 from fetal liver and spleen and HS0711023 from postnatal whole brain, respectively. Still, they may represent different parts of one gene.
Northern blots containing poly(A)+ RNA from fetal tissues only show one hybridization signal which corresponds to the length of the cDNA. As most of the clones from the fetal brain cDNA library contained exon 1B, we assume that this signal represents splice variant 1B and that variant 1A is expressed at a low level during fetal development. Moreover, the apparent lack of a CpG island associated with exon 1A and the presence of an exon 1B associated CpG island, supports the ubiquitous expression of variant 1B and a tissue-specific expression of variant 1A as all housekeeping genes and only 40% of all tissue-restricted genes exhibit 5' CpG islands (30 -32 ). In contrast to fetal RNA, poly(A)+ RNA from adult tissues yielded multiple hybridization signals which may represent different splice variants or homologous genes. The presence of different cDNA variants, the identity with two ESTs and a faint autosomal restriction fragment seen on the Southern blot (Fig. 2 ), can be reconciled with both possibilities. Although the expression of the gene seems to be ubiquitous, the highest expression is found in brain and striated muscles. Hybridization of the DXS6673E gene to DNA from various animal species, indicates that the gene is highly conserved among vertebrates and suggests that it plays an important part.
In the great majority of females with balanced X-autosome translocations, the non-rearranged X chromosome is preferentially inactivated (14 ,33 ). The same holds true for this patient as judged from replication studies on PBLs. Our RT-PCR experiments have shown that no products were generated when DXS6673E specific primers were used from both sides of the breakpoint, which is consistent with a complete inactivation of the non-rearranged DXS6673E allele. However, when using primer combinations located proximally from the breakpoint, normal gene products were identified suggesting that variant 1A of the DXS6673E gene is under the control of chromosome 13 sequences. This may also be true for variant 1B, as exon 1B is located approximately 600 bp downstream from the breakpoint. Therefore, the phenotype of the patient may be due to aberrant temporal and spatial expression or silencing of the DXS6673E gene. Alternatively, the translocation may directly disrupt a gene on chromosome 13, may place a chromosome 13 gene under the control of X chromosomal sequences, or indirectly interfere with proper expression of other genes on chromosome 13 as a result of spreading of X-inactivation into the autosomal segment of the der(13). If a gene on chromosome 13 is disrupted by the breakpoint, the phenotype of the patient could be the result of haploinsufficiency (27 ), or if the other allele is mutated, the translocation may unmask its heterozygosity (34 ). Large deletions resulting in partial chromosome 13 monosomies have been described in a number of patients with `13q'-syndrome (reviewed in refs. 35 and 36 ) and association of these deletions with disease phenotypes may support the presence of genes in this region that are sensitive to haploinsufficiency. This syndrome includes MR as well as various malformations and the major manifestations in this syndrome seem to be associated with a deletion of the 13q32 band.
The breakpoint on chromosome 13 has been mapped between the loci D13S755 and D13S747 by PCR analysis of the der(13 ) cell line (data not shown). This segment is located up to 3 centimorgans (cM) proximal to the endothelin receptor B (EDNRB) gene, which was found to be mutated in patients with Hirschsprung disease (HSCR2) (37 ,38 ). Although hypopigmentation is also one of the clinical features of HSCR2, an effect of the translocation on the expression of the EDNRB gene expression is not very likely. Likewise, inactivation of the EDNRB locus cannot be ascribed to spreading of X inactivation into the chromosome 13 since the X inactivation centre (39 ) is located on the der(13) and the EDNRB gene on the der(X). The observed cutaneous spotting may indicate that there is a small proportion of cells in which the der(X) is inactivated instead of the normal X (40 ). Although we cannot exclude the possibility that the MR in this patient is due to haploinsufficiency resulting from disruption of a gene on chromosome 13, the disruption of the DXS6673E locus remains by far the most plausible cause for MR in this patient. A recent overview by Neri et al. (10 ) lists eight families with non-syndromic MR where the mapping interval includes the Xq13 band (MRX1, 4, 5, 7, 8, 12, 13 and 17). Also multiple syndromic types of XLMR have been mapped to the pericentromeric region of the X-chromosome. Mutation screening in these families should clarify the role of the DXS6673E gene in the aetiology of XLMR.
Patient PMI is a mentally retarded girl carrying a de novo balanced X;13 translocation. She was the second of healthy non-consanguineous parents. At the age of 1, she suffered from hyperthermic convulsions. Apart from mental retardation (IQ<45, WISC-R scale), she suffers from scoliosis and spotty hypopigmentation of the skin. She also presents with slight facial asymmetry and clinodactily (MRX39) (15 ,17 ).
FISH procedures used were essentially as described elsewhere (41 -43 ). The X chromosome centromere specific alphoid sequence probe pBAMX5 was labelled with Fluorolink Cy3-dCTP (BDS Inc., Pittsburg) while the YAC clones were labelled with biotin-14-dATP using a nick-translation kit (Gibco, Life Technologies). 200 ng labelled YAC probe was dissolved in 6 [mu]l of a hybridization solution containing 50% v/v deionized formamide, 10% w/v dextran sulphate, 2* SSC and 1% v/v Tween-20 at pH 7.0 as well as 10 mg Cot-1 DNA (Gibco, Life Technologies). Prior to hybridization, the probe was denatured at 80oC for 10 min, chilled on ice and incubated for 30 min at 37oC allowing preannealing. For the pBAMX5 probe, 20 ng of DNA was used in 6 [mu]l of the hybridization solution without competitor DNA. Metaphase spreads were prepared using standard procedures. After denaturation of the slides, probe incubations were carried out under an 18*18 mm coverslip in a moist chamber for 45 h.
Immunocytochemical detection of the hybridizing probes was achieved as described elsewhere (41 -43 ).
YAC yWXD940 is a 350 kb YAC containing the CCG1 gene (20 ). Yeast cell culturing and DNA isolation were done as described elsewhere (44 ). A partial cosmid contig from YAC yWXD940 was generated essentially as described elsewhere (45 ).
Methods employed for the isolation of high-molecular-weight DNA, restriction-endonuclease digestion, as well as separation and blotting of DNA fragments have been described elsewhere (45 ). Hybridization of the cosmid inserts in the presence of excess human competitor DNA was done essentially as described by Blonden et al. (46 ). Hybridized filters were washed at 65oC with 40 mM Na2HPO4 (pH 7.2)/0.5% (w/v) SDS for 3*5 min and 1*30 min. Autoradiography took 4-16 h at -70oC using two intensifying screens.
Fetal and adult multiple tissue northern blots (Clontech) were hybridized in a solution containing 5* SSPE, 10* Denhardt's solution, 100 mg/ml herring sperm DNA, 50% formamide and 2% SDS for 18 h at 42oC after prehybridization for 3 h under the same condidtion. Hybridized filters were washed 30 min in 2* SSC, 0.05% SDS at room temperature and 30 min at 50oC in 0.1* SSC, 0.1% SDS. Autoradiography took 16-40 h at -70oC using two intensifying screens.
A [lambda]ZapII human fetal brain cDNA library (Stratagene) was screened by standard methods (47 ) and Bluescript KS(+) plasmids containing cDNA inserts were isolated by in vivo excision according to the manufacturer's instructions.
Double-stranded sequence analysis was performed on DNA isolated from the cDNA clones and on PCR amplified DNA products using T3 and T7 oligonucleotides as well as gene-specific oligonucleotides (Isogen Biosciences, the Netherlands). Plasmid DNA was isolated using a Qiaprep 8 kit (Qiagen) while PCR products were first separated on an agarose gel and isolated with a gel extraction kit (Qiagen). Sequence analysis was performed radioactively with the T7 sequencing Kit (Pharmacia Biotech) and, in addition, with the Taq DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems) on an Applied Biosystems 373A DNA sequencer according to the manufacturer's instructions.
Total RNA was isolated from EBV-transformed cell lines, fetal brain, adult muscle and liver with RNAzol according to the manufacturer's instructions (Cinna Biotecx). For synthesis of cDNAs, 200 ng of total RNA was reversed transcribed with MMLV reverse transcriptase essentially as described elsewhere (48 ). The cDNA products were directly used as template for PCR amplification in a buffer containing 10 mM Tris, 50 mM KCl, 0.01% gelatine, 2 mM MgCl2 (DXS6673E) or 3 mM MgCl2 (SMCX), 10 mM dNTPs, 250 ng of each oligonucleotide 2,5 Taq DNA polymerase (Perkin Elmer). Amplification was done in 30 (DXS6673E) or 35 (SMCX) cycles of 1 min 94oC, 1 min 60oC and 1.5 min 72oC. Inactivation studies of the DXS6673E gene were done with primers 973 (5'-TGCTCGTCATCCTGCCTCAC-3') and 974 (5'-ACACTGGTCACAACAGTTGG-3') amplifying 380 bp from exons 5 and 6. For expression studies of exon 1A the following primer combinations located on both sides of the breakpoint were used: primers 736 (5'-AGACAAGGACAGAAAGGGGG-3') and 968 (5'-CCAGCAACTCAGTGGCTCCA-3'), followed by a nested PCR with primers 900 (5'-TTACTGCACAGGCGTTGCCA-3') and 969 (5'-GGGTCCATGAGTATGGCTGG-3') amplifying 490 bp of exon 1A and exon 2. Additionally, exon 1A sequences located proximally from the breakpoint were amplified with the primers 1195 (5'-CTGCATGGCGTAGGTAGGGA-3') and 968 followed by a nested amplification with the primers 379 (5'-GGTGAACACGAGTGGGAAGC-3') and 969 (184 bp). Expression of exon 1B was studied with the primers 1196 (5'-CTGTCCCGGCTGCTAGGGAG-3') and 968, amplifying a product of 356 bp. Amplification of SMCX cDNA was done with the oligonucleotides S40HX (5'-GAAGGTGGAGTCAACATCGCCT-3') and S41HX (5'-AGCCATCACACAGCAGGAGC-3') (26 ).
The authors are indebted to Mrs S. D. van der Velde-Visser and E. Boender-van Rossum for expert technical assistance. We thank D. Röhme, Mrs. Y. J. M. de Kok and A. Kataki for their contributions to this study, and J. Leunisse for his contribution to homology searches. The research of F.P.M.C. has been made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences. This work was supported by the Netherlands Organization of Scientific Research (NWO grant 960-10-809) and the Dutch Preaventiefonds (grant 28-2447).
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*To whom correspondence should be addressed at: Max-Planck-Institut für Molekulare Genetik, Ihnestraße 73, D-14195 Berlin-Dahlem, Germany+Present address: Department of Medical Genetics, University of Helsinki, Helsinki, Finland
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