- Research article
- Open Access
- Open Peer Review
Characterization of novel isoforms and evaluation of SNF2L/SMARCA1 as a candidate gene for X-linked mental retardation in 12 families linked to Xq25-26
© Lazzaro et al; licensee BioMed Central Ltd. 2008
- Received: 01 November 2007
- Accepted: 26 February 2008
- Published: 26 February 2008
Mutations in genes whose products modify chromatin structure have been recognized as a cause of X-linked mental retardation (XLMR). These genes encode proteins that regulate DNA methylation (MeCP2), modify histones (RSK2 and JARID1C), and remodel nucleosomes through ATP hydrolysis (ATRX). Thus, genes encoding other chromatin modifying proteins should also be considered as disease candidate genes. In this work, we have characterized the SNF2L gene, encoding an ATP-dependent chromatin remodeling protein of the ISWI family, and sequenced the gene in patients from 12 XLMR families linked to Xq25-26.
We used an in silico and RT-PCR approach to fully characterize specific SNF2L isoforms. Mutation screening was performed in 12 patients from individual families with syndromic or non-syndromic XLMR. We sequenced each of the 25 exons encompassing the entire coding region, complete 5' and 3' untranslated regions, and consensus splice-sites.
The SNF2L gene spans 77 kb and is encoded by 25 exons that undergo alternate splicing to generate several distinct transcripts. Specific isoforms are generated through the alternate use of exons 1 and 13, and by the use of alternate donor splice sites within exon 24. Alternate splicing within exon 24 removes a NLS sequence and alters the subcellular distribution of the SNF2L protein. We identified 3 single nucleotide polymorphisms but no mutations in our 12 patients.
Our results demonstrate that there are numerous splice variants of SNF2L that are expressed in multiple cell types and which alter subcellular localization and function. SNF2L mutations are not a cause of XLMR in our cohort of patients, although we cannot exclude the possibility that regulatory mutations might exist. Nonetheless, SNF2L remains a candidate for XLMR localized to Xq25-26, including the Shashi XLMR syndrome.
- Chromatin Remodel
- Rett Syndrome
- Flag Epitope
- Flank Intron Sequence
- Chromatin Remodel Protein
The isolation of genes underlying X-linked mental retardation (XLMR) disorders has been hampered, in part, by the broad phenotypic variability observed in patients that restricts linkage analysis to large single families or instances where a specific trait facilitates phenotypic splitting. More recently, the use of large scale genomic methods including comparative genome hybridization (CGH) arrays and patient sequencing projects has increased the identification rate of XLMR disease genes. Surprisingly, each gene identified accounts for a small proportion of cases and there remain many conditions for which a gene has not been identified. Nonetheless, several trends have emerged. These include the identification of XLMR genes encoding proteins that modulate chromatin structure . The cloning of the ATRX gene as the cause of the α-thalassemia mental retardation (ATR-X) syndrome established the paradigm for chromatin remodeling proteins in neurodevelopmental disorders . This gene, encoding a SWI/SNF-like protein, is also mutated in other severe XLMR syndromes lacking α-thalassemia and in patients with mild-to-moderate XLMR . Subsequently, the RSK2 gene encoding a histone kinase was identified as the causative gene for Coffin-Lowry syndrome and non-specific XLMR [4, 5], and the methyl-CpG-binding protein 2 (MeCP2) gene was identified as the causative gene for Rett syndrome  and other non-specific male MR [7–9]. More recently, the PHF6 (Borjeson-Forssman-Lehmann syndrome;), ZNF41 , ZNF81 , and JARID1C  genes have also been implicated in XLMR and have roles in transcriptional regulation and/or chromatin remodeling. Taken together, these studies suggest that additional chromatin interacting proteins whose genes reside on the X chromosome should be considered as disease candidates for both syndromal and non-specific XLMR disorders.
The Drosophila ISWI gene was identified as a distinct SWI/SNF subclass named the Imitation SWI (ISWI) family . Two human orthologs of Drosophila ISWI (dISWI) have been described, SNF2H (SMARCA5) which maps to 4q31.1 and SNF2L (SMARCA1) which maps to Xq25-26 [15, 16]. Moreover, analysis of the murine Snf2h and Snf2l genes demonstrated that Snf2h was expressed in proliferating neuroblast layers whereas Snf2l expression was enhanced in differentiating neuronal populations . Indeed, purification of the SNF2L-containing human NURF complex demonstrated that it regulated expression of the engrailed genes, which are important in mid/hind-brain development . In addition, the latter study also demonstrated that SNF2L could promote neuronal differentiation when expressed ectopically in neuroblasts . SNF2L was also found to be a component of a second chromatin remodeling complex, called CERF that contains the CECR2 protein, a transcription factor involved in neurulation and a cause of exencephaly in mice when mutated . These studies suggest that SNF2L is an excellent candidate gene for the cause of XLMR. In this study, we have characterized multiple splice forms and examined 12 families with XLMR for mutations in SNF2L.
For cell lines, total RNA was prepared from cell lines by acid phenol extraction of cell lysates . Poly A+ RNA for reverse transcription was purified from total RNA using the PolyATtract mRNA Isolation System (Promega, Nepean, Ontario). Total RNA from human tissues and specific brain regions were obtained commercially (Applied Biosystems Canada, Streetsville, Ontario). Total RNA (2 ug) or Poly A+ RNA (100 ng) was reverse transcribed using Superscript RT (Invitrogen) and a combination of oligo dT and random hexamers. PCR reactions for analysis of SNF2L splice variants were at 94°C for 30 seconds, 53°C for 30 seconds and 72°C for 2 minutes for 35 cycles, followed by a final extension of 15 minutes at 72°C. For the 5' splice variants, the following primers were used: 5'UTR SNF2L1 Fwd, 5' CAAACTTGCTGCTAAAGCGCC 3'; 5'UTR SNF2L2 Fwd, 5' GGAATTCATGGAGCAGGACACTGC 3'; SNF2L5'splice variants Rev, 5'CACCAAGACAATTTTTAGTG 3'. For the NLS splice variants: SNF2L NLS Fwd, 5' GGAGGTCATGGAGTATTC 3' and SNF2L NLS Rev, 5'CAGTAGCTGACTCTGCTTTTCTTTTCTGTG 3'.
DNA samples from 12 individuals affected with XLMR and previously mapped to a region encompassing the SNF2L gene were used for direct sequencing analysis. Samples from families F85-19 , F93-04, and F91-02 were provided by Drs. Ben Hamel and Hans van Bokhoven (University Hospital Nijmegen, Nijmegen, Netherlands). Samples K8045 , K8135 , K8320, K8395, K8725, K8895 , and K8923 were generously provided by Dr. Charles Schwartz (JC Self Research Institute, Greenwood Genetic Center, Greenwood South Carolina). The non-syndromic XLMR sample 24981 was provided by Dr. Judith Allanson (Children's Hospital of Eastern Ontario, Ottawa Ontario). The Pettigrew syndrome DNA sample was prepared from a commercially available EBV transformed lymphoblast cell line (GM12523; Coriell Cell Repository, Camden, NJ).
The SNF2L NLS splice variants were cloned into the pcDNA3 expression plasmid (Invitrogen Canada Inc. Burlington, ON), each with an amino terminal tag encoding the FLAG epitope. Annealed primers encoding the FLAG epitope were inserted in the KpnI and EcoRI sites of the pcDNA3 plasmid. The 5' region encoding the amino terminus of the SNF2L2 splice variant (SNF2LB) was engineered with an EcoRI restriction site for cloning in frame with the FLAG epitope. The primers used were: FLAG-sense EcoRI, 5'-CCCACCATGGATTACAAGGATGACGACGATAAGG-3' and FLAG-antisense EcoRI, 5'-AATTCCTTATCGTCGTCATCCTTGTAATCCATGGTGGGGTAC-3'. The remainder of each cDNA (+ NLS or -NLS) was inserted in two additional cloning steps to construct plasmids encoding full length SNF2L proteins with and without a nuclear localization signal.
Cell transfections and immunofluorescence
293HEK cells were cultured in EMEM (Eagle's Minimal Essential Medium, Wisent, St Bruno, Quebec) supplemented with 10% fetal bovine serum (FBS) in a humidified 95% air with 5% CO2 incubator at 37°C. One day prior to transfection, cells were plated on glass coverslips coated with poly D-lysine (100 μg/ml, Sigma, Oakville, Ontario) in 6 well tissue culture plates at a density of approximately 3 × 105 cells/well. Cells were transfected with 5 μg DNA [pcDNA3 control, pcDNA3 FLAG-SNF2LΔNLS, or pcDNA3 FLAG SNF2L] and the Lipofectamine 2000 reagent (Life Technologies, Burlington, Ontario) according to the manufacturer's protocol. After 48 hours, cells were fixed in cold ethanol/methanol (3:1) prior to blocking with 10% FBS diluted in PBS. Coverslips were incubated with diluted primary antibody (murine anti-FLAG monoclonal clone M2, 2 μg/ml, Sigma, Oakville, Ontario) for 1 hour at room temperature, washed and then incubated for 1 hour at room temperature with FITC-conjugated donkey-anti-murine IgG secondary antibody (Sigma) diluted 1:100 in PBS. Cell nuclei were counterstained with 1 μg/ml DAPI (Sigma) and images were captured using a Zeiss Axioplan 2 microcope outfitted with an AxioCam camera and AxioVision software.
SNF2L Mutation Analysis
Genomic DNA from individuals affected with XLMR in each of the 12 families analyzed was used to amplify each exon of the SNF2L gene with forward and reverse primers that annealed to flanking intron sequences, approximately 100 bp from each exon/intron boundary (primer sequences available upon request). Genomic DNA from an individual unaffected by XLMR was the control for all PCR reactions. For each reaction, 100–200 ng DNA was combined with 1 μM each of forward and reverse PCR primers, 200 μM dNTPs, 2.5 mM MgCl2, 20 mM Tris-HCl pH 8.4, 50 mM KCl, and 1 unit Taq DNA polymerase in a volume of 100 μl. PCR reactions were incubated at 95°C for 40 seconds, 57°C for 40 seconds and 72°C for 3 minutes for 30 cycles, followed by a final extension of 15 minutes at 72°C. DNA obtained from PCR reactions was purified using the Qiaquick PCR Purification kit (Qiagen, Mississauga, Onatario) and sequenced using Perkin Elmer dye terminator and ABI automated sequencing. Sequences were aligned and compared to sequences obtained from the control DNA and sequence available in public databases to identify mutations.
The SNF2L gene undergoes alternative splicing at multiple sites to generate a wide number of different isoforms. Some of these isoforms will affect the activity of the protein. For example, the inclusion of exon 13 abolishes the chromatin remodeling activity , and the altered splicing within exons 24/25 removes the NLS resulting in cytoplasmic localization. In the latter case, the SNF2LΔNLS variant was detected in most tissues examined, suggesting that the cytoplasmic isoform of SNF2L may fulfill an alternative role to the chromatin remodeling function of the nuclear isoforms. Alternatively, it could represent a SNF2L dominant negative protein that sequesters other components of the chromatin remodeling complex in the cytoplasm. As such, it will be important to further explore the function of these novel SNF2L isoforms to determine their specific roles, similar to work done with the SNF2L+13 variant. Indeed, with splicing occurring at the 5'-end of the gene (isoforms SNF2LA and B), the 3'-end of the gene (NLS or ΔNLS) and encompassing exon 13 (+exon 13 or -exon 13) there are 8 possible isoforms of the SNF2L protein that can impinge on the function/activity of the protein.
Mutation analysis of 12 families linked to Xq25 revealed 3 SNPs
Location of SNP
% of control sample with SNP (n = 100)
SNP Heterozygosity (SNP Database No.)
Exon 1 – 5'UTR 29 bp 5' of AUG
Intron 13 48 bp 5' of exon14
Intron 18 74 bp 3' of exon18
Despite the absence of mutations in these families, SNF2L remains a logical candidate for XLMR localized to Xq25-26 because of (a) its similarity to ATRX, a SNF2-domain containing protein mutated in several XLMR disorders; (b) its high expression in the brain including areas important for learning and development; and (c) its ability to induce neuroblastoma cells to undergo differentiation when ectopically expressed [17, 18]. In addition, SNF2L has been shown to regulate the engrailed genes, En-1 and En-2, both of which are important for mid/hind-brain development [27, 28], the latter of which has been associated with autism in genetic linkage studies [29–31]. Given these criteria and the small sample size available for mutation analysis, other syndromes should be considered for screening, including the Shashi XLMR syndrome [32, 33] and a family with syndromal XLMR and late-onset testicular failure (Cillier syndrome)  that both map to Xq26. In addition, there are 3 other syndromes (Wilson/MRXS12, Gustavson, and CMTX4/Cowchock-Fishbeck) and 8 MRX families (MRX 27, 35, 42, 62, 70, 71, 75, and 82) that map to this region that should also be considered in future screening endeavors . The generation of a transgenic mouse ablated for the Snf2l gene should provide valuable phenotypic insight for its potential involvement in specific XLMR disorders. Together, the analysis of additional samples and the characterization of transgenic mice should define whether the SNF2L gene is a cause of mental retardation.
We have shown that there are multiple SNF2L isoforms that result from alternative splicing of the gene. How these different isoforms are spatially and temporally regulated and defining their specific role during neural development remains to be established. In our collection of 12 patients with XLMR linked to Xq25-26, we did not identify any mutations within the coding region. However, we cannot exclude intronic mutations (outside of the consensus splice sites) that would affect alternative splicing and hence the expression levels of the different isoforms. Similarly, we cannot exclude mutations in regulatory regions in the 12 families screened for mutations in this gene. Alternatively, our failure to identify mutations may arise from (1) the small sample size which may have prevented us from ascertaining a family with a mutation in this gene, or (2) the possibility that mutations in SNF2L could cause a more severe phenotype that may be lethal in males. Indeed, SNF2L remains a candidate XLMR gene for Xq25-26 linked XLMR families including the Shashi syndrome as well as in sporadic mental retardation cases.
The authors would like to thank Drs. Allanson, Hamel, Schwartz, and Van Bokhoven for their generosity in providing patient DNA samples. This work was supported by grants from the Ontario Mental Health Foundation and the Neuromuscular Research Partnership (JNM-78384), an initiative of the ALS Society of Canada, Muscular Dystrophy Canada, and the Canadian Institutes of Health Research.
- Chelly J, Mandel JL: Monogenic causes of X-linked mental retardation. Nat Rev Genet. 2001, 2 (9): 669-680. 10.1038/35088558.View ArticlePubMedGoogle Scholar
- Gibbons RJ, Picketts DJ, Higgs DR: Syndromal mental retardation due to mutations in a regulator of gene expression. Hum Mol Genet. 1995, 4 (Spec No): 1705-1709.PubMedGoogle Scholar
- Gibbons RJ, Higgs DR: Molecular-clinical spectrum of the ATR-X syndrome. Am J Med Genet. 2000, 97 (3): 204-212. 10.1002/1096-8628(200023)97:3<204::AID-AJMG1038>3.0.CO;2-X.View ArticlePubMedGoogle Scholar
- Merienne K, Pannetier S, Harel-Bellan A, Sassone-Corsi P: Mitogen-regulated RSK2-CBP interaction controls their kinase and acetylase activities. Mol Cell Biol. 2001, 21 (20): 7089-7096. 10.1128/MCB.21.20.7089-7096.2001.View ArticlePubMedPubMed CentralGoogle Scholar
- Trivier E, De Cesare D, Jacquot S, Pannetier S, Zackai E, Young I, Mandel JL, Sassone-Corsi P, Hanauer A: Mutations in the kinase Rsk-2 associated with Coffin-Lowry syndrome. Nature. 1996, 384 (6609): 567-570. 10.1038/384567a0.View ArticlePubMedGoogle Scholar
- Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY: Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl- CpG-binding protein 2. Nat Genet. 1999, 23 (2): 185-188. 10.1038/13810.View ArticlePubMedGoogle Scholar
- Meloni I, Bruttini M, Longo I, Mari F, Rizzolio F, D'Adamo P, Denvriendt K, Fryns JP, Toniolo D, Renieri A: A mutation in the Rett syndrome gene, MECP2, causes X-linked mental retardation and progressive spasticity in males. Am J Hum Genet. 2000, 67 (4): 982-985. 10.1086/303078.View ArticlePubMedPubMed CentralGoogle Scholar
- Orrico A, Lam C, Galli L, Dotti MT, Hayek G, Tong SF, Poon PM, Zappella M, Federico A, Sorrentino V: MECP2 mutation in male patients with non-specific X-linked mental retardation. FEBS Lett. 2000, 481 (3): 285-288. 10.1016/S0014-5793(00)01994-3.View ArticlePubMedGoogle Scholar
- Couvert P, Bienvenu T, Aquaviva C, Poirier K, Moraine C, Gendrot C, Verloes A, Andres C, Le Fevre AC, Souville I, Steffann J, des Portes V, Ropers HH, Yntema HG, Fryns JP, Briault S, Chelly J, Cherif B: MECP2 is highly mutated in X-linked mental retardation. Hum Mol Genet. 2001, 10 (9): 941-946. 10.1093/hmg/10.9.941.View ArticlePubMedGoogle Scholar
- Lower KM, Turner G, Kerr BA, Mathews KD, Shaw MA, Gedeon AK, Schelley S, Hoyme HE, White SM, Delatycki MB, Lampe AK, Clayton-Smith J, Stewart H, van Ravenswaay CM, de Vries BB, Cox B, Grompe M, Ross S, Thomas P, Mulley JC, Gecz J: Mutations in PHF6 are associated with Borjeson-Forssman-Lehmann syndrome. Nat Genet. 2002, 32 (4): 661-665. 10.1038/ng1040.View ArticlePubMedGoogle Scholar
- Shoichet SA, Hoffmann K, Menzel C, Trautmann U, Moser B, Hoeltzenbein M, Echenne B, Partington M, Van Bokhoven H, Moraine C, Fryns JP, Chelly J, Rott HD, Ropers HH, Kalscheuer VM: Mutations in the ZNF41 gene are associated with cognitive deficits: identification of a new candidate for X-linked mental retardation. Am J Hum Genet. 2003, 73 (6): 1341-1354. 10.1086/380309.View ArticlePubMedPubMed CentralGoogle Scholar
- Kleefstra T, Yntema HG, Oudakker AR, Banning MJ, Kalscheuer VM, Chelly J, Moraine C, Ropers HH, Fryns JP, Janssen IM, Sistermans EA, Nillesen WN, de Vries LB, Hamel BC, van Bokhoven H: Zinc finger 81 (ZNF81) mutations associated with X-linked mental retardation. J Med Genet. 2004, 41 (5): 394-399. 10.1136/jmg.2003.016972.View ArticlePubMedPubMed CentralGoogle Scholar
- Jensen LR, Amende M, Gurok U, Moser B, Gimmel V, Tzschach A, Janecke AR, Tariverdian G, Chelly J, Fryns JP, Van Esch H, Kleefstra T, Hamel B, Moraine C, Gecz J, Turner G, Reinhardt R, Kalscheuer VM, Ropers HH, Lenzner S: Mutations in the JARID1C Gene, Which Is Involved in Transcriptional Regulation and Chromatin Remodeling, Cause X-Linked Mental Retardation. Am J Hum Genet. 2005, 76 (2): 227-236. 10.1086/427563.View ArticlePubMedGoogle Scholar
- Elfring LK, Deuring R, McCallum CM, Peterson CL, Tamkun JW: Identification and characterization of Drosophila relatives of the yeast transcriptional activator SNF2/SWI2. Mol Cell Biol. 1994, 14 (4): 2225-2234.View ArticlePubMedPubMed CentralGoogle Scholar
- Aihara T, Miyoshi Y, Koyama K, Suzuki M, Takahashi E, Monden M, Nakamura Y: Cloning and mapping of SMARCA5 encoding hSNF2H, a novel human homologue of Drosophila ISWI. Cytogenet Cell Genet. 1998, 81 (3-4): 191-193. 10.1159/000015027.View ArticlePubMedGoogle Scholar
- Okabe I, Bailey LC, Attree O, Srinivasan S, Perkel JM, Laurent BC, Carlson M, Nelson DL, Nussbaum RL: Cloning of human and bovine homologs of SNF2/SWI2: a global activator of transcription in yeast S. cerevisiae. Nucleic Acids Res. 1992, 20 (17): 4649-4655. 10.1093/nar/20.17.4649.View ArticlePubMedPubMed CentralGoogle Scholar
- Lazzaro MA, Picketts DJ: Cloning and characterization of the murine Imitation Switch (ISWI) genes: differential expression patterns suggest distinct developmental roles for Snf2h and Snf2l. J Neurochem. 2001, 77 (4): 1145-1156. 10.1046/j.1471-4159.2001.00324.x.View ArticlePubMedGoogle Scholar
- Barak O, Lazzaro MA, Lane WS, Speicher DW, Picketts DJ, Shiekhattar R: Isolation of human NURF: a regulator of Engrailed gene expression. Embo J. 2003, 22 (22): 6089-6100. 10.1093/emboj/cdg582.View ArticlePubMedPubMed CentralGoogle Scholar
- Banting GS, Barak O, Ames TM, Burnham AC, Kardel MD, Cooch NS, Davidson CE, Godbout R, McDermid HE, Shiekhattar R: CECR2, a protein involved in neurulation, forms a novel chromatin remodeling complex with SNF2L. Hum Mol Genet. 2005, 14 (4): 513-524. 10.1093/hmg/ddi048.View ArticlePubMedGoogle Scholar
- Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate- phenol-chloroform extraction. Anal Biochem. 1987, 162 (1): 156-159. 10.1016/0003-2697(87)90021-2.View ArticlePubMedGoogle Scholar
- Hamel BC, Smits AP, Otten BJ, van den Helm B, Ropers HH, Mariman EC: Familial X-linked mental retardation and isolated growth hormone deficiency: clinical and molecular findings. Am J Med Genet. 1996, 64 (1): 35-41. 10.1002/(SICI)1096-8628(19960712)64:1<35::AID-AJMG5>3.0.CO;2-Q.View ArticlePubMedGoogle Scholar
- Arena JF, Schwartz C, Stevenson R, Lawrence L, Carpenter A, Duara R, Ledbetter D, Huang T, Lehner T, Ott J, et al: Spastic paraplegia with iron deposits in the basal ganglia: a new X-linked mental retardation syndrome. Am J Med Genet. 1992, 43 (1-2): 479-490. 10.1002/ajmg.1320430172.View ArticlePubMedGoogle Scholar
- Cabezas DA, Slaugh R, Abidi F, Arena JF, Stevenson RE, Schwartz CE, Lubs HA: A new X linked mental retardation (XLMR) syndrome with short stature, small testes, muscle wasting, and tremor localises to Xq24-q25. J Med Genet. 2000, 37 (9): 663-668. 10.1136/jmg.37.9.663.View ArticlePubMedPubMed CentralGoogle Scholar
- Christianson AL, Stevenson RE, van der Meyden CH, Pelser J, Theron FW, van Rensburg PL, Chandler M, Schwartz CE: X linked severe mental retardation, craniofacial dysmorphology, epilepsy, ophthalmoplegia, and cerebellar atrophy in a large South African kindred is localised to Xq24-q27. J Med Genet. 1999, 36 (10): 759-766.View ArticlePubMedPubMed CentralGoogle Scholar
- Barak O, Lazzaro MA, Cooch NS, Picketts DJ, Shiekhattar R: A tissue-specific, naturally occurring human SNF2L variant inactivates chromatin remodeling. J Biol Chem. 2004, 279 (43): 45130-45138. 10.1074/jbc.M406212200.View ArticlePubMedGoogle Scholar
- Predict_NLS_program: [http://cubic.bioc.columbia.edu/predictNLS/]
- Joyner AL, Herrup K, Auerbach BA, Davis CA, Rossant J: Subtle cerebellar phenotype in mice homozygous for a targeted deletion of the En-2 homeobox. Science. 1991, 251 (4998): 1239-1243. 10.1126/science.1672471.View ArticlePubMedGoogle Scholar
- Wurst W, Auerbach AB, Joyner AL: Multiple developmental defects in Engrailed-1 mutant mice: an early mid-hindbrain deletion and patterning defects in forelimbs and sternum. Development. 1994, 120 (7): 2065-2075.PubMedGoogle Scholar
- Benayed R, Gharani N, Rossman I, Mancuso V, Lazar G, Kamdar S, Bruse SE, Tischfield S, Smith BJ, Zimmerman RA, Dicicco-Bloom E, Brzustowicz LM, Millonig JH: Support for the homeobox transcription factor gene ENGRAILED 2 as an autism spectrum disorder susceptibility locus. Am J Hum Genet. 2005, 77 (5): 851-868. 10.1086/497705.View ArticlePubMedPubMed CentralGoogle Scholar
- Gharani N, Benayed R, Mancuso V, Brzustowicz LM, Millonig JH: Association of the homeobox transcription factor, ENGRAILED 2, 3, with autism spectrum disorder. Mol Psychiatry. 2004, 9 (5): 474-484. 10.1038/sj.mp.4001498.View ArticlePubMedGoogle Scholar
- Kuemerle B, Gulden F, Cherosky N, Williams E, Herrup K: The mouse Engrailed genes: a window into autism. Behav Brain Res. 2007, 176 (1): 121-132. 10.1016/j.bbr.2006.09.009.View ArticlePubMedGoogle Scholar
- Castro NH, dos Santos RC, Nelson R, Becak W, Hane B, Lindsey CJ, Lubs HA, Stevenson RE, Schwartz CE: Shashi XLMR syndrome: report of a second family. Am J Med Genet A. 2003, 118 (1): 49-51. 10.1002/ajmg.a.10888.View ArticleGoogle Scholar
- Shashi V, Berry MN, Shoaf S, Sciote JJ, Goldstein D, Hart TC: A unique form of mental retardation with a distinctive phenotype maps to Xq26-q27. Am J Hum Genet. 2000, 66 (2): 469-479. 10.1086/302772.View ArticlePubMedPubMed CentralGoogle Scholar
- Cilliers DD, Parveen R, Clayton P, Cairns SA, Clarke S, Shalet SM, Black GC, Newman WG, Clayton-Smith J: A new X-linked mental retardation (XLMR) syndrome with late-onset primary testicular failure, short stature and microcephaly maps to Xq25-q26. Eur J Med Genet. 2007, 50 (3): 216-223. 10.1016/j.ejmg.2007.01.003.View ArticlePubMedGoogle Scholar
- Chiurazzi P, Schwartz CE, Gecz J, Neri G: XLMR genes: update 2007. Eur J Hum Genet. 2008Google Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/9/11/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.