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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 MA, Todd MA, Lavigne P, Vallee D, De Maria A, Picketts DJ - BMC Med. Genet. (2008)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ottawa Health Research Institute, 501 Smyth Road, Ottawa, ON K1H 8L6, Canada. maribeth_lazzaro@hc-sc.gc.ca

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

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Genomic organization and 5' transcript variants of the human SNF2L gene. A. Schematic diagram showing the 25 exons (dark boxes; exon 13 is an open box) of the human SNF2L gene (top). Below is a schematic diagram of the SNF2L transcript showing the ORF (open box) and the location of the motifs that comprise the SNF2 domain (blue boxes) and the SANT domains (red boxes). B. The 5' variants SNF2LA and SNF2LB provide alternative initiation codons and encode two forms of SNF2L with different amino-termini. They are shown aligned to the mouse Snf2l sequence. The SNF2LB transcript encodes a protein with an amino-terminus similar in length and amino acid composition to the murine Snf2l protein. C. RT-PCR analysis showing that both transcript variants are present in human cell lines and fetal brain tissue examined. The helicase I/Ia domain served as control amplification. Lane 1, 293 cells; lane 2, SH-SY5Y cells; lane 3, NT2 cells; lane 4, hNT neurons; and lane 5, human fetal brain. M, molecular weight marker.
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Figure 1: Genomic organization and 5' transcript variants of the human SNF2L gene. A. Schematic diagram showing the 25 exons (dark boxes; exon 13 is an open box) of the human SNF2L gene (top). Below is a schematic diagram of the SNF2L transcript showing the ORF (open box) and the location of the motifs that comprise the SNF2 domain (blue boxes) and the SANT domains (red boxes). B. The 5' variants SNF2LA and SNF2LB provide alternative initiation codons and encode two forms of SNF2L with different amino-termini. They are shown aligned to the mouse Snf2l sequence. The SNF2LB transcript encodes a protein with an amino-terminus similar in length and amino acid composition to the murine Snf2l protein. C. RT-PCR analysis showing that both transcript variants are present in human cell lines and fetal brain tissue examined. The helicase I/Ia domain served as control amplification. Lane 1, 293 cells; lane 2, SH-SY5Y cells; lane 3, NT2 cells; lane 4, hNT neurons; and lane 5, human fetal brain. M, molecular weight marker.

Mentions: Spatial and temporal expression studies of the Snf2l gene in mice and the purification of two human SNF2L-containing complexes have both suggested that the SNF2L protein may have an important role in neurodevelopment and that the SNF2L gene is a strong XLMR candidate gene [17-19]. In silico analysis demonstrated that the SNF2L gene (NM_003069) is highly conserved between mouse and human with a common intron/exon pattern containing 25 exons spanning ~77 kb along the X chromosome within Xq25 (Figure 1A and data not shown). However, the human cDNA and predicted protein sequences showed several significant discrepancies to the mouse Snf2l sequence (NM_053123) that required characterization, prior to mutation studies [16,17]. Indeed, we previously reported the presence of a human SNF2L variant (SNF2L+13) containing a non-conserved in-frame exon within the SNF2 catalytic domain that abolishes chromatin remodeling activity [25]. In addition, Okabe et al. reported two human cDNA clones with disparate 5' ends [16]. We will refer to these two clones as SNF2LA and SNF2LB, respectively. SNF2LB aligns with the start of the murine cDNA sequence and corresponds to a transcript that would initiate within exon 1 of the human genomic sequence (Figure 1B). The shorter SNF2LA isoform results from transcription that initiates within exon 2. The corresponding proteins differ in size at the NH2-terminus by 78 amino acids with SNF2LB corresponding to the published murine sequence (Figure 1B). The SNF2LA isoform was not present in mice suggesting that it may be unique to humans. Using RT-PCR, we detected the corresponding transcripts for both 5' variants in human fetal brain and multiple human neuronal cell lines (Figure 1C).


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 MA, Todd MA, Lavigne P, Vallee D, De Maria A, Picketts DJ - BMC Med. Genet. (2008)

Genomic organization and 5' transcript variants of the human SNF2L gene. A. Schematic diagram showing the 25 exons (dark boxes; exon 13 is an open box) of the human SNF2L gene (top). Below is a schematic diagram of the SNF2L transcript showing the ORF (open box) and the location of the motifs that comprise the SNF2 domain (blue boxes) and the SANT domains (red boxes). B. The 5' variants SNF2LA and SNF2LB provide alternative initiation codons and encode two forms of SNF2L with different amino-termini. They are shown aligned to the mouse Snf2l sequence. The SNF2LB transcript encodes a protein with an amino-terminus similar in length and amino acid composition to the murine Snf2l protein. C. RT-PCR analysis showing that both transcript variants are present in human cell lines and fetal brain tissue examined. The helicase I/Ia domain served as control amplification. Lane 1, 293 cells; lane 2, SH-SY5Y cells; lane 3, NT2 cells; lane 4, hNT neurons; and lane 5, human fetal brain. M, molecular weight marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2266716&req=5

Figure 1: Genomic organization and 5' transcript variants of the human SNF2L gene. A. Schematic diagram showing the 25 exons (dark boxes; exon 13 is an open box) of the human SNF2L gene (top). Below is a schematic diagram of the SNF2L transcript showing the ORF (open box) and the location of the motifs that comprise the SNF2 domain (blue boxes) and the SANT domains (red boxes). B. The 5' variants SNF2LA and SNF2LB provide alternative initiation codons and encode two forms of SNF2L with different amino-termini. They are shown aligned to the mouse Snf2l sequence. The SNF2LB transcript encodes a protein with an amino-terminus similar in length and amino acid composition to the murine Snf2l protein. C. RT-PCR analysis showing that both transcript variants are present in human cell lines and fetal brain tissue examined. The helicase I/Ia domain served as control amplification. Lane 1, 293 cells; lane 2, SH-SY5Y cells; lane 3, NT2 cells; lane 4, hNT neurons; and lane 5, human fetal brain. M, molecular weight marker.
Mentions: Spatial and temporal expression studies of the Snf2l gene in mice and the purification of two human SNF2L-containing complexes have both suggested that the SNF2L protein may have an important role in neurodevelopment and that the SNF2L gene is a strong XLMR candidate gene [17-19]. In silico analysis demonstrated that the SNF2L gene (NM_003069) is highly conserved between mouse and human with a common intron/exon pattern containing 25 exons spanning ~77 kb along the X chromosome within Xq25 (Figure 1A and data not shown). However, the human cDNA and predicted protein sequences showed several significant discrepancies to the mouse Snf2l sequence (NM_053123) that required characterization, prior to mutation studies [16,17]. Indeed, we previously reported the presence of a human SNF2L variant (SNF2L+13) containing a non-conserved in-frame exon within the SNF2 catalytic domain that abolishes chromatin remodeling activity [25]. In addition, Okabe et al. reported two human cDNA clones with disparate 5' ends [16]. We will refer to these two clones as SNF2LA and SNF2LB, respectively. SNF2LB aligns with the start of the murine cDNA sequence and corresponds to a transcript that would initiate within exon 1 of the human genomic sequence (Figure 1B). The shorter SNF2LA isoform results from transcription that initiates within exon 2. The corresponding proteins differ in size at the NH2-terminus by 78 amino acids with SNF2LB corresponding to the published murine sequence (Figure 1B). The SNF2LA isoform was not present in mice suggesting that it may be unique to humans. Using RT-PCR, we detected the corresponding transcripts for both 5' variants in human fetal brain and multiple human neuronal cell lines (Figure 1C).

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ottawa Health Research Institute, 501 Smyth Road, Ottawa, ON K1H 8L6, Canada. maribeth_lazzaro@hc-sc.gc.ca

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus