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Endocrinology Vol. 148, No. 6 2925-2935
Copyright © 2007 by The Endocrine Society

Lack of Gnas Epigenetic Changes and Pseudohypoparathyroidism Type Ib in Mice with Targeted Disruption of Syntaxin-16

Leopold F. Fröhlich, Murat Bastepe, Defne Ozturk, Hilal Abu-Zahra and Harald Jüppner

Endocrine Unit (L.F.F., M.B., D.O., H.A.-Z., H.J.) and Pediatric Nephrology Unit (H.J.), Department of Medicine and Massachusetts General Hospital for Children, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Harald Jüppner, Endocrine Unit, Thier 10, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: jueppner{at}helix.mgh.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pseudohypoparathyroidism type Ib (PHP-Ib) is characterized by hypocalcemia and hyperphosphatemia due to proximal renal tubular resistance to PTH but without evidence for Albright’s hereditary osteodystrophy. The disorder is paternally imprinted and affected individuals, but not unaffected carriers, show loss of GNAS exon A/B methylation, a differentially methylated region upstream of the exons encoding Gs{alpha}. Affected individuals of numerous unrelated kindreds with an autosomal dominant form of PHP-Ib (AD-PHP-Ib) have an identical 3-kb microdeletion removing exons 4–6 of syntaxin-16 (STX16) (STX16del4–6), which is thought to disrupt a cis-acting element required for exon A/B methylation. To explore the mechanisms underlying the regulation of exon A/B methylation, we generated mice genetically altered to carry the equivalent of STX16del4–6 (Stx16{Delta}4–6). Although the human GNAS locus shows a similar organization as the murine Gnas ortholog and although the human and mouse STX16/Stx16 regions show no major structural differences, no phenotypic or epigenotypic abnormalities were detected in mice with Stx16{Delta}4–6 on one or both parental alleles. Furthermore, calcium and PTH levels in Stx16{Delta}4–6 mice were indistinguishable from those in wild-type animals, indicating that ablation of the murine equivalent of human STX16del4–6 does not contribute to the development of PTH resistance. The identification of a novel intragenic transcript from within the STX16/Stx16 locus in total RNA from kidneys of Stx16{Delta}4–6 mice and lymphoblastoid cell-derived RNA of a patient with AD-PHP-Ib raises the question whether this transcript contributes, if deleted or altered, to the development of AD-PHP-Ib in humans.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE COMPLEX GNAS locus comprises at least four different promoters that generate, in humans and mice, several different sense and antisense transcripts (1, 2, 3, 4, 5, 6). Best studied is the transcript encoding the {alpha}-subunit of the stimulatory G protein (Gs{alpha}), which is encoded by exons 1–13 and is important for the cAMP-dependent actions of a large variety of hormones, neurotransmitters, and autocrine/paracrine factors (7, 8, 9). Through the use of alternative promoters, several distinct first exons are spliced onto exons 2–13 and give rise to novel mRNAs, including the possibly noncoding transcript A/B (also referred to as 1A or 1'), and transcripts that encode an extra-large Gs{alpha} variant (XL{alpha}s) and a 55-kDa neuroendocrine secretory protein (NESP55) (7, 8, 9). NESP55 is a neuroendocrine secretory protein with largely unknown biological roles (10). Ablation of 26 nucleotides of the first Nesp55 exon, including the codon for the initiation of translation, leads only to subtle behavioral changes in mice (11). The 3' end of the mRNA encoding NESP55 is also derived from GNAS exons 2–13, but this bicistronic part of the nucleic acid sequence remains untranslated because the NESP55-specific exon ends with an in-frame termination codon. Consequently, the NESP55 protein shares no amino acid sequence homology with Gs{alpha} and XL{alpha}s. In addition to these sense transcripts, the GNAS locus gives rise to at least one antisense mRNA, which is presumably noncoding (3, 6). To complicate this locus further, at least four GNAS exons and their up-stream promoter regions undergo parent-specific methylation and transcripts are derived only from the nonmethylated allele (1, 2, 3, 6).

Mutations within the GNAS locus are responsible for several human disorders (9, 12). Best studied are two disorders, the McCune-Albright syndrome, which is caused by gain-of-function mutations affecting predominantly the Gs{alpha} amino acid residue 201 (or in some cases Gln227) (13, 14), and pseudohypoparathyroidism type Ia (PHP-Ia), which is caused by a variety of different heterozygous mutations involving most of the 13 exons encoding Gs{alpha} (7, 8, 9, 15, 16, 17, 18, 19) or by larger heterozygous deletions involving the entire GNAS locus (20). PHP-Ia is characterized by features of Albright’s hereditary osteodystrophy and resistance toward several hormones, particularly toward PTH in the proximal renal tubules. Mutations in the exons encoding Gs{alpha} are also found in patients with pseudopseudohypoparathyroidism (PPHP) (7, 8, 9) and progressive osseous heteroplasia (21). PHP-Ia develops only when Gs{alpha} mutations are inherited from a female affected by either PHP-Ia or PPHP, whereas inheritance of Gs{alpha} mutations from a male leads to PPHP or progressive osseous heteroplasia, disorders without evidence of hormone resistance (7, 8, 9). Resistance toward several different hormones that mediate their actions through Gs{alpha}-coupled receptors thus occurs in a parent-specific manner, and it is related to tissue- or cell-specific silencing of Gs{alpha} transcription from the paternal allele.

Another variant of pseudohypoparathyroidism, PHP-Ib, is characterized by resistance toward PTH leading to hypocalcemia and hyperphosphatemia and is occasionally associated with mild resistance to TSH and possibly other hormones (7, 8, 9, 22). Because of these limited laboratory abnormalities and the lack of Albright’s hereditary osteodystrophy, i.e. the variable developmental defects that are observed in other forms of PHP, PHP-Ib was initially thought to be caused by mutations in the PTH receptor, i.e. the PTH/PTHrP receptor. However, mutations in this gene were excluded (23, 24, 25, 26). Instead, genetic linkage studies mapped the molecular defect for an autosomal dominant form of PHP-Ib (AD-PHP-Ib) to a region on chromosome 20q13.3 comprising portions of GNAS (27). Furthermore, it was shown that the defect is paternally imprinted and that affected individuals, but not unaffected carriers, show a loss of methylation at exon A/B (28, 29).

Recently an identical heterozygous 3-kb microdeletion was identified in numerous unrelated kindreds more than 220 kb upstream of the differentially methylated exon A/B (30, 31, 32, 33). This microdeletion removes, in-frame, syntaxin-16 (STX16) exons 4–6 (STX16del4–6), which reside between two almost perfect 391-bp direct repeats. Another heterozygous mutation, deleting STX16 exons 2–4, was identified in the affected individuals and obligate carriers of another AD-PHP-Ib kindred, in which the affected members show the same epigenetic abnormalities as those observed in the patients carrying the 3-kb deletion within STX16 (34). The region overlapping both microdeletions is thought to comprise a putative imprinting control element affecting methylation at exon A/B but not other differentially methylated regions (DMRs) within GNAS and thus appears to be directly or indirectly involved in the development of hormonal resistance.

To further explore the mechanisms underlying the differential methylation of exon A/B and the resulting development of PTH resistance in the proximal renal tubules, we generated mice with the equivalent of the 3-kb deletion identified in AD-PHP-Ib (Stx16{Delta}4–6). Although the human GNAS locus has a similar organization as its murine Gnas ortholog and although the human STX16 and the mouse Stx16 regions show no major structural differences, no phenotypic or epigenotypic abnormalities were detected in mice with disruption of Stx16 on one or both parental alleles. Instead, evidence was obtained for a novel intragenic transcript, the deletion or modification of which may contribute, in humans, to the development of AD-PHP-Ib.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of the targeting vector
To generate the gene targeting vector, pIC3, we screened a 129/SvJ mouse genomic library constructed in {lambda}-DASH II vector (a gift from Dr. T. Doetschman, University of Cincinnati, Cincinnati, OH) using a PCR-amplified genomic mouse probe comprising Stx16 exons 4–6 [hybridization of filters overnight at 65 C in 0.5 x saline sodium citrate (SSC)/0.1% sodium dodecyl sulfate (SDS); final wash 65 C in 0.5 x SSC/0.1% SDS]. The inserts of three clones, each about 15 kb in size, were subcloned into the NotI polylinker site of the pBluescript II KS (+) vector (Stratagene, La Jolla, CA) and end sequencing showed that all three inserts were identical. A 6.95-kb EcoRI fragment from one of these clones (nucleotides 44610–51565 of AL669896) was ligated into the single EcoRI site of the pACN targeting vector (35) downstream of the floxed pACN cassette and served as the long arm for homologous recombination. To generate the short arm, a 2.5-kb fragment was PCR amplified from genomic 129/SvJ ES cell DNA. After cloning into pCR4-TOPO (Invitrogen, Carlsbad, CA), the insert was excised with EcoRI, blunt ended, and inserted into a blunt-ended BglII site upstream of the pACN cassette; before this cloning step, the second BglII site of the vector downstream of the selection cassette was removed by partial endonuclease digestion of the vector followed by blunt ending with the Klenow fragment and religation.

Generation of Stx16{Delta}4–6 mice
Gene targeting was performed in J1 embryonic stem (ES) cells (129/SvJ; kindly provided by Dr. En Li, Massachusetts General Hospital, Boston, MA). The pIC3 plasmid was linearized at the unique SalI site and 60 µg of phenol-chloroform extracted DNA was electroporated into approximately 2 x 107 cells. After 24 h, selection was initiated with 350 µg/ml G418. Resistant ES cell colonies were isolated and genomic DNA after digestion with BamHI was screened by Southern blot analysis using a 5' or 3' external probe or an internal neo probe, respectively. The 5' probe was PCR amplified from intron 1 of the Stx16 gene (nucleotides 56718–57379 of AL669896), whereas the 3' probe was PCR amplified from an intergenic region between nucleotides 43262 and 44292 of AL669896; both probes were cloned into pCR4-TOPO for amplification.

Correctly targeted ES cell clones were injected into 3.5-d postcoitus C57BL/6J blastocysts, which were then transferred into the uteri of 2.5-d postcoitus pseudopregnant CD1 mice. Germ line-transmitting male chimeras were obtained from two independent cell lines. Genotyping of offspring was performed by PCR using primers mstx16ex3F1 (5'-ACAGTATGATGTTGGCCGCAT; exon 3) and mstx16in6R1 (5'-TGGTGTTCCAACAGCCGTTAC-3') for the recombinant allele and the primer pair mstx16ex4F1 (5'-CTCTTCCATAGGTGCCAGCGT-3') and mstx16ex5R1 (5'-AGTGGTACTGGGGTGTCGAAGA-3') for the wild-type allele. PCR mixtures contained 1 µM of each primer, 1.5 mM MgCl2, 200 µM of each deoxynucleoside triphosphate, 1x Q-solution, and 2.5 U of Taq DNA polymerase (Taq PCR core kit; QIAGEN, Valencia, CA). The PCR amplification program consisted of an initial 5-min denaturation step at 95 C, followed by 35 cycles of annealing (65 C, 60 sec), extension (72 C, 70 sec), and denaturation (94 C, 60 sec), with a 10-min extension after the final cycle. The mutant-specific amplification product was cloned and sequenced to confirm correct loxP-mediated Cre recombination. Stx16{Delta}4–6 heterozygous offspring from chimeras were crossed into two different genetic backgrounds (C57BL/6J and 129/Sv) to generate an inbred and outcross line.

Methylation analyses
Genomic DNA was isolated from mouse tails using standard procedures. For methylation analysis of the Gnas exon 1A DMR (1A-DMR), Southern blots were performed using genomic DNA (10 µg). After overnight digestion with BamHI/BglII, in the absence or presence of HpaII, the DNA was separated on a 0.8% agarose gel before transfer onto nitrocellulose; the blots were probed with a DNA fragment spanning the 1A-DMR that was 32P-labeled by random priming (random primed DNA labeling kit, Roche Applied Science, Mannheim, Germany); the probe was generated with the following forward primer (5'-CTGCTCCAGCAGCTTCTTCT-3') and reverse primer (5'-TATTCTAGAGCCCCGTGTGG-3') (nucleotides 183256–184972 of AL593857). Filters were incubated with probes in 50% formaldehyde hybridization solution at 42 C overnight and then washed once with 2x SSC and 0.1% SDS for 30 min at room temperature and once with 0.1x SSC and 0.1% SDS for 1 h at 42 C. Filters were exposed to Bio-Max MS films (Kodak, Rochester, NY).

To confirm these findings, differential methylation of both parental alleles was also assessed by direct restriction enzymatic analyses of PCR products amplified from bisulfite-treated genomic DNA (bisulfite PCR analysis); conditions for bisulfite treatment and primer sequences for amplification of the Gnas-DMR were as described by Liu et al. (36). The amplified fragments were purified with the QIAquick PCR purification kit (QIAGEN) and digested with the restriction enzyme AccI before electrophoresis on agarose gels.

RNA preparation and RT-PCR
Total RNA of mouse kidney (3 wk of age) and human lymphoblastoid cells was isolated by the TRIZol method (Invitrogen). An aliquot of the prepared RNA was treated with RNase-free DNaseI (Sigma, St. Louis, MO) for 20 min at 37 C to eliminate residual DNA contaminations. The reaction was phenol-chloroform (1:1) extracted once, precipitated, and resuspendend in diethylpyrocarbonate-water. RT-PCR was performed on total RNA (1 µg/sample) with or without DNaseI treatment using the SuperScript III first-strand synthesis system from Invitrogen (with oligo-dT priming). Each sample was set up in duplicate with (+) or without (–) reverse transcriptase.

For the determination of the parent of origin of exon 1A transcripts in the kidney of Stx16{Delta}4–6 mice, the following RT-PCR was performed: using the upstream primer mGnasEx1AF1 (5'-GGACACTCAGTCGCGTCGGCA-3') and the downstream primer mGnas Ex12R1 (5'-CTTAGAGCAGCTCGTATTGGC-3'), amplification was performed for 5 min at 95 C, followed by 35 cycles of annealing (65 C, 60 sec), extension (72 C, 3 min), and denaturation (94 C, 60 sec), with a final 10-min extension at 72 C. Reaction conditions were otherwise as described above. Subsequently the PCR product was digested with BanII and loaded onto an agarose gel.

The amplification of murine intragenic transcripts by RT-PCR from kidney cDNA was performed using the same conditions and primers as used for genotyping Stx16{Delta}4–6 mice (see above), with the exception of the intronic primer pair mstx16in4F1 (5’-CAACCATCTGAGACTTCTCTAAGTGGA-3’) and mstx16in4R1 (5’-CCTGCCTTTCATTGTTAACAGCTAGTT-3’) that was selected for amplifying the wild-type allele. RT-PCR of the human intragenic transcript of human lymphoblastoid cDNA was performed using the same conditions as described for amplification of the STX16del4–6 microdeletion in patients with AD-PHP-Ib (30). Additional information regarding the primers used in these studies is available upon request.

Measurement of ionized calcium and PTH
Tail vein blood was collected from individual mice (pure 129/Sv background) at 2 and 8 months of age after fasting them for 12 h. Ionized calcium concentrations were determined using a 9180 electrolyte analyzer (AVL Medical Instruments, Schaffhausen, Switzerland). Serum PTH concentrations were measured using a two-side ELISA specific for intact mouse PTH (Immutopics, San Clemente, CA). Measurements are presented as means ± SEM. Differences between groups were evaluated using Student’s t test.

Comparison of STX16/Stx16 regions/GenBank sequences
To identify regions conserved in mouse, human, and other species, the Vista Genome Browser (http://www-gsd.lbl.gov/vista/) (37), and the University of California Santa Cruz Genome browser (http://genome.ucsc.edu) (38) were used in combination with the National Center for Biotechnology Information (Bethesda, MD) BLAST program (39).

Institutional approvals
Lymphoblastoid cells were previously obtained from a patient with AD-PHP-Ib (30); this study had been approved by the Subcommittee on Human Studies of Massachusetts General Hospital, protocol 2001-P-000648/12. The animal studies were approved by the Massachusetts General Hospital Subcommittee on Research Animal Care, protocol 2001N000183.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Organization of the mouse Stx16 locus is similar to that of its human ortholog
The mouse Stx16 gene is located about 240 kb centromeric of the Gnas locus on mouse chromosome 2, which is the region syntenic to human chromosome 20q13. It was previously determined that the human STX16 gene is not imprinted, although it harbors a CpG island in its 5' end and exhibits stretches of CpG dinucleotides in exon 4 (30, 34). Inspection of the mouse genomic region comprising Stx16 revealed a gene organization that showed marked similarity to that of the human locus (Fig. 1Go, A and B). Nucleotide sequence identity between the mouse and human exons ranged from 85 to 98%. Furthermore, the exon sizes were similar except for the first exon, which was found to be smaller in the mouse (132 vs. 290 bp). Introns were smaller in the mouse than in the human gene, except for introns 1 and 2, which were significantly larger (13,432 vs. 2,135 bp for intron 1; 754 vs. 390 bp for intron 2).


Figure 1
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  		FIG. 1. Comparison of human GNAS (A) and mouse Gnas (B) and the upstream STX16/Stx16 regions. Depicted are the paternal and maternal alleles on chromosome 20q13.3 (human) and distal chromosome 2 (mouse) with individual exons (black, gray, hatched, or white boxes). The alternative first exons of GNAS/Gnas, which splice onto GNAS exons 2 to 13 (human) or Gnas exons 2–12 (mouse; note that exons 9 and 10 are fused in the mouse) (hatched box) are designated with individual characters. N, Exon NESP55/Nesp55 (human/mouse); XL, exon XL{alpha}s/Gnasxl; A/B or 1A, exon A/B or exon 1A; AS, exon antisense/Nespas. In contrast to the human antisense mRNA, which is transcribed from six exons, the murine antisense mRNA is derived only from five antisense exons; the third of these Nespas exons is located within the intron between Nesp55 exons 1 and 2 (note that human NESP55 is encoded by a single exon). Arrows indicate the orientation of individual monoallelically derived Gnas transcripts; p, paternal allele; m, maternal allele; the dashed arrow for exon 1 on the paternal allele indicates that this promoter is active in some tissues and silenced in others. The methylation status of each DMR of the GNAS/Gnas locus is shown by the presence or absence of crosses (+, methylated). Triangles mark the position of retroviral elements representing the 3-kb microdeletion identified in most patients with AD-PHP-Ib. The human deletion is flanked by two almost identical repeats of 391 bp that are composed of two different retroviral elements (MER46A; MIRb), whereas the mouse locus comprises only one simple MIR element. The human STX16 gene harbors additional small exons located between exon 1 and exon 2 (not shown). The approximate locations and the sizes of the two different STX16 deletions that were identified in humans affected by AD-PHP-Ib, STX16del4–6, and deletion of STX16 exons 2–4 are displayed below the scheme of the human gene. The dashed line indicates that both STX16 microdeletions eliminate exon A/B methylation on the maternal allele.

 
The human STX16 gene comprises two almost identical 391-bp repeats located upstream of exon 4 and downstream of exon 6, respectively (30), whereas the mouse gene was found to lack regions that are homologous to these repeats. Instead the mouse Stx16 locus revealed only one simple repeat element in intron 3 without homology to the 391-bp repeat in humans but no evidence for a similar repeat downstream of exon 6. Furthermore, the two almost identical direct 391-bp repeats within the human STX16 gene comprise MIRb (mammalian-wide interspersed repeat) and MER46A (medium reiterated repeat) retroviral elements, whereas the mouse Stx16 gene comprises only a single MIR element that is only 173 bp in size. Comparison of the mouse introns within the region deleted in humans affected by AD-PHP-Ib revealed small stretches of sequence conservation with more than 50% homology. These conserved regions included a 98-bp segment within intron 4, which showed 80% nucleotide sequence identity between the mouse and human genes, as described previously (30).

In comparison with the human genome, the organization of the mouse regions comprising Gnas and Stx16 are largely conserved. Furthermore, there is an identical epigenetic pattern of parent-specific methylation at the conserved Gnas-DMRs, and several mouse intronic regions share significant nucleotide sequence homology with the equivalent region of the human gene (see Fig. 1Go, A and B). We predicted the presence of a region within STX16 that regulates, directly or indirectly, the methylation of exon A/B and its promoter. Consistent with this hypothesis, a novel 4.4-kb STX16 microdeletion was recently identified in the affected members and unaffected carriers of another AD-PHP-Ib kindred (34). This novel microdeletion overlaps with the previously identified 3-kb deletion by about 1200 bp, and it is therefore conceivable that the region critical for exon A/B methylation resides within this DNA fragment.

Targeted ablation of Stx16 in the mouse
Through homologous recombination in ES cells, we ablated a 2.3-kb region extending from Stx16 intron 3 to intron 6 (Fig. 2AGo), which is roughly equivalent to the 3-kb deletion identified in patients with AD-PHP-Ib (30). The targeting vector (Fig. 2BGo) included a floxed pACN selection cassette (35), which is designed to self-excise the testis-specific angiotensin-converting enzyme (tACE) promoter driven Cre recombinase and the neomycin resistance gene (neo) upon passage through the male germline (Fig. 2Go, C and D). Recombinant clones were screened by Southern blot analysis (Fig. 2Go, E–G), and mice carrying the deletion (Stx16{Delta}4–6) were generated from two independent correctly targeted clones. Offspring of female and male Stx16{Delta}4–6 mice were analyzed for obvious phenotypic abnormalities, allele-specific changes in exon 1A methylation, and allelic expression of exon 1A transcripts as well as blood serum parameters.


Figure 2
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  		FIG. 2. Targeting strategy and Southern blot analysis to document deletion of Stx16 exons 4–6. A, Map of the Stx16 region with a magnified depiction of the deleted area within the wild-type allele. The introduced deletion encompasses a 2.3-kb sequence extending from intron 3 to intron 6 of Stx16, which comprises exons 4–6 and a MIR element (hatched triangle) within intron 3 and two simple AT-rich repeats within introns 5 and 6 (gray bars). Bold black lines indicate regions that are targeted for homologous recombination. Black rectangles show the positions of the 5' probe and the 3' probe that were used for Southern blot analysis. Arrowheads mark the positions of primers a and b that were used for genotyping the wild-type Stx16 allele. E, EcoRI; B, BamHI. B, The targeting construct contains a self-splicing floxed ACN cassette (35 ) comprising a neomycin resistance gene (neo) and a Cre-recombinase gene (Cre), which replaces the 2.3-kb region corresponding approximately to the 3-kb deletion identified in most patients with AD-PHP-Ib. Crosses represent homologous recombination events. The SalI site (S) for vector linearization is marked by an angular line. C, Restriction map of the correctly targeted locus. The BamHI (B) restriction fragments used for Southern blot screening are indicated. The positions of the 5' probe, the 3' probe, and the neo probe are indicated. D, The targeted locus after self-splicing of the pACN cassette by Cre-mediated recombination during passage through the germ line of male chimeric mice. Arrowheads and lowercase letters mark the position of the primers used to genotype Stx16{Delta}4–6 mice. E, Screening of genomic DNA derived from ES cell for integration of the 3' end of the targeting construct by Southern blot analysis. Digestion of genomic wild-type DNA with BamHI gave rise to a hybridizing 16.7-kb wild-type band, whereas the targeted allele with deletion leads to a hybridizing recombinant 10.4-kb band when probed with the 3' probe. Lane 1, Wild-type DNA; lanes 2 and 3, DNA from two independent ES cells clones heterozygous for Stx16{Delta}4–6. F, Screening for integration of the 5' end of the targeting construct by Southern blot analysis. Genomic ES cell DNA was digested with BamHI and probed with the 5' probe. Digestion of wild-type DNA gives rise to a 16.7-kb hybridizing band, whereas the targeted allele gives rise to a 6.0-kb hybridizing recombinant band, indicating that it had undergone homologous recombination. Lane 1, Wild-type DNA; lanes 2 and 3, DNA from two independent ES cell clones heterozygous for Stx16{Delta}4–6. G, Screening for integration of the targeting construct with an internal neo probe by Southern blot analysis. The BamHI-digested genomic DNA shows the expected single 10.4-kb hybridizing band.

 
Stx16{Delta}4–6 mice are phenotypically indistinguishable from their wild-type littermates
Matings of chimeras with wild-type C57BL/6 animals yielded several mice heterozygous for Stx16{Delta}4–6, which were then bred further with C57BL/6 animals or among each other. PCR-based genotyping of the offspring resulting from these crosses (Fig. 3AGo) confirmed the presence of the different possible genotypes with the expected Mendelian frequency (Table 1Go). PCR amplification of genomic DNA from mice heterozygous or homozygous for Stx16{Delta}4–6 revealed a 1060-bp fragment, indicating that the pACN cassette of 3676 bp had been correctly self-excised in the mutant allele; this was also confirmed through nucleotide sequence analysis of the amplified PCR products across the deleted region (data not shown). To determine whether a BanII polymorphism, present within Gnas exon 10 of 129Sv-derived DNA, can be used to determine the parental origin of mRNA transcripts (see below), we PCR amplified genomic DNA from wild-type, and heterozygous or homozygous Stx16{Delta}4–6 mice in the 129Sv or the C57BL/6 genetic background (Fig. 3Go, B and C). To determine whether Stx16 exons 4–6 were indeed removed, we further amplified Stx16 transcripts by RT-PCR using total RNA from kidneys of wild-type mice as well as animals heterozygous and homozygous for Stx16{Delta}4–6 (Fig. 3Go, D and E). The reduction in transcript size by 405 bp confirmed that deletion of exons 4–6 had occurred in the mutant animals. Because deletion of these exons occurs in-frame, i.e. like in humans with AD-PHP-Ib (30), these findings further revealed that the mutant mRNA is sufficiently stable and not particularly prone to nonsense mediated decay. Mice that inherited the Stx16{Delta}4–6 allele from either parent as well as homozygous animals were viable, showed no gross phenotype, no evidence for reduced fertility, and appeared healthy until at least 8 months of age.


Figure 3
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  		FIG. 3. Germline transmission and genotyping of Stx16{Delta}4–6 mice. A, Genotyping of Stx16{Delta}4–6 mice by PCR. Genomic mouse tail DNA was subjected to separate PCRs using primer pairs specific for either wild-type Stx16 (lower panel; primer pair a/b; see also Fig. 2AGo) or the mutant alleles (upper panel; primer pair c/d; see also Fig. 2DGo). A 615-bp band was obtained for wild-type DNA; the mutant Stx16{Delta}4–6 allele revealed a 1060-bp PCR product. M, Marker lane; +/+, wild-type; +/{Delta}, heterozygous; {Delta}/{Delta}, homozygous. B, BanII-polymorphism in Gnas exon 10. Genomic tail DNA was subjected to PCR to amplify the region surrounding Gnas exon 10 (primers g and h). The resulting PCR product was subjected to BanII digestion before agarose gel electrophoresis. Due to an additional site for BanII digestion in exon 10 of Gnas in the 129Sv background (C, asterisk), the restriction pattern is different for the allele derived from C57BL/6 mice. Sites for BanII digestion fragments (primer pair g/h) are indicated above each strain-specific allele; the 460-bp band is specific for the C57BL/6 strain (paternal), whereas the 264- and 196-bp bands are specific for the 129Sv strain (maternal)). +, Wild-type allele; {Delta}, mutant allele; s, 129Sv allele; c, C57BL/6 allele. D, RT-PCR to amplify the Stx16 transcript from kidney of wild-type and Stx16{Delta}4–6 mice. Primers in exon 1 (primer e) and exon 8 (primer f) were used to PCR amplify cDNA (see E). Amplification of wild-type cDNA revealed the expected size of 870 bp, whereas the band derived from the Stx16{Delta}4–6 cDNA revealed a PCR product of 465 bp. +/+, Wild-type; +/{Delta}, heterozygous; {Delta}/{Delta}, homozygous; M, marker lane.

 

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  		TABLE 1. Genotypes of offspring from matings between mice with one mutant Stx16{Delta}4–6 allele1

 
Stx16{Delta}4–6 is associated with unchanged differential methylation at Gnas exon 1A
After maternal transmission of the Stx16{Delta}4–6 allele, methylation of genomic tail DNA at the Gnas exon 1A DMR was analyzed in newborn mice. For Southern blot analysis, DNA was digested either with BamHI/BglII alone or in combination with the CpG methylation-sensitive enzyme HpaII (Fig. 4Go, A and B). DNA from mice carrying Stx16{Delta}4–6 on either the maternal or paternal allele, or on both parental alleles, all revealed the same pattern of DNA fragments. When the methylation-sensitive endonuclease HpaII was used to further digest the 2000-bp fragment obtained with BamHI and BglII, a 449-bp fragment was obtained with 129Sv-derived DNA, whereas a 397-bp fragment was obtained with C57BL/6-derived DNA (note that an additional HpaII site is present in the C57BL/6 background; see Fig. 4AGo); DNA from both mouse strains led to the generation of an approximately 900-bp fragment. This experiment thus provided no evidence for Stx16{Delta}4–6-dependent methylation changes at exon 1A.


Figure 4
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  		FIG. 4. Methylation analysis of the exon 1A DMR after maternal, paternal, and biallelic transmission of the Stx16{Delta}4–6 deletion. A, All HpaII sites within a BamHI/BglII fragment comprising the exon 1A region (black box) are indicated as vertical lines on the paternal allele; these are not methylated and Southern analysis therefore revealed DNA fragments of approximately 900 and 449 bp in the C75BL/6 background and approximately 900 and 397 bp in the 129Sv background (several smaller DNA fragments are not shown); the polymorphic HpaII site is marked by an asterisk. The recognition sites for HpaII on the maternal allele are all methylated; therefore, the approximately 2000 bp BamHI/BglII fragment cannot be digested further with HpaII. B, BamHI; Bg, BglII; p, paternal; m, maternal. B, Methylation of the exon 1A DMR was analyzed using tail DNA from mutant Stx16{Delta}4–6 mice and their wild-type littermates by Southern blot analysis. Genomic DNA samples were digested with BamHI and BglII, in the presence or absence of the methylation-sensitive enzyme HpaII. The blot was hybridized with an approximately 1.7-kb PCR fragment spanning the entire BamHI/BglII fragment shown in the restriction map in A); note that the 397-bp fragment was obtained only in offspring that received one allele from an 129Sv male. C, A fragment comprising the exon 1A-DMR was amplified by nested PCR using initially the primer pair i/j. The PCR products were then digested with the methylation-sensitive restriction enzyme AccI; note that AccI digestion products derived from the maternal allele (108 and 115 bp bands), but not from the unmethylated paternal allele (223 bp band). +, Wild-type allele; {Delta}, mutant allele; {Delta}P, paternal Stx16{Delta}4–6; {Delta}M, maternal Stx16{Delta}4–6; M, marker lane. D, Bisulfite PCR analysis of the exon 1A-DMR. Differential methylation of both parental alleles was assessed by direct restriction enzymatic analyses of PCR products amplified from bisulfite-treated genomic DNA.

 
These findings were confirmed by digesting PCR-amplified bisulfite-treated DNA with AccI, an endonuclease that digests only products derived from the methylated maternal allele at a site within exon 1A (Fig. 4Go, C and D). Irrespective as to whether Stx16{Delta}4–6 was derived from the maternal or the paternal allele or both parental alleles, only the PCR product from one parental allele was digested with AccI. Two independent methods thus revealed no evidence for abnormal methylation at the exon 1A DMR after maternal or paternal transmission of the Stx16{Delta}4–6 mutation.

Stx16{Delta}4–6 mice, like their wild-type littermates, display paternal, monoallelic expression of 1A transcripts and normal mineral ion homeostasis
We next tested whether maternal transmission of the deletion can de-repress maternal exon 1A transcription, which is normally silenced on the maternal allele and is expressed only paternally in all examined tissues (36). For these experiments, heterozygous Stx16{Delta}4–6 females (F1 generation; 129Sv/C57BL/6) were mated to either wild-type C57BL/6 or heterozygous Stx16{Delta}4–6 129Sv x C57BL/6 males, and total RNA was isolated from the kidneys once the animals were 3 wk old. Using the strain-specific BanII polymorphism in Gnas exon 10 (see above), we were able to distinguish between 1A transcripts derived from the maternal or the paternal allele (Fig. 5AGo; see also Fig. 3Go, B and C). Allelic expression was unchanged in that both wild-type and Stx16{Delta}4–6 mutant animals expressed the 1A transcript only from the paternal chromosome, a finding consistent with the lack of methylation changes at exon 1A (Fig. 5BGo).


Figure 5
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  		FIG. 5. Analysis of allele-specific expression of 1A transcripts and lack of imprinting after maternal transmission of the Stx16{Delta}4–6 deletion. Allele-specific expression of the exon 1A transcript was analyzed by RT-PCR using total RNA of kidneys from 3-wk-old wild-type, heterozygous, or homozygous mice. A, Primers annealing in exon 1A (Ex 1A) (primer k) and Gnas exon 11 (Ex 11) (primer l) were used to specifically amplify the cDNA derived from the exon 1A promoter. A 129Sv-specific BanII polymorphism in Gnas exon 10 (Ex 10) was used to distinguish between both parental alleles (see Fig. 3BGo). B, The PCR product of 1195 bp was separated on an agarose gel and stained with ethidium bromide (upper panel). Restriction enzymatic digestion of the PCR product with BanII reduces the 1195-bp PCR product derived from the 129Sv allele by 203 bp (lower panel). Amplification of cDNA from Stx16{Delta}4–6 mice revealed only a paternally derived 1A transcript but no evidence for biallelic expression from the exon 1A promoter, which would have provided a 1195-bp band derived from C57BL/6 and 992-bp band derived from 129Sv. +, Wild-type allele; {Delta}, mutant allele; {Delta}P, paternal allele; {Delta}M, maternal allele; M, marker lane.

 
To determine whether the deleted Stx16 region could be involved in the regulation of the Gs{alpha} expression and thereby PTH signaling through means that do not involve a loss of exon 1A DMR methylation, we measured blood ionized calcium and serum PTH. Consistent with the lack of methylation changes at exon 1A, male and female mice with Stx16{Delta}4–6 on the maternal or paternal allele had, at the ages of 2 and 8 months, ionized calcium and PTH levels that were indistinguishable from those of wild-type littermates (Table 2Go).


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  		TABLE 2. Ionized calcium and PTH concentrations of Stx16{Delta}4–6 mice and wild-type littermates

 
A novel intragenic transcript can be amplified from the Stx16 locus
There are no obvious imprinting control elements within the Stx16 region. Furthermore, our Stx16{Delta}4–6 mice lacked epigenetic changes, and it was therefore not possible to develop a mouse model of AD-PHP-Ib. However, in other imprinted disorders, noncoding sense or antisense RNA transcripts were shown to be involved in regulating gene expression (40, 41, 42). To determine whether our Stx16{Delta}4–6 mice could be used to reveal the presence of such antisense transcripts, we first screened several databases for evidence of Stx16-derived RNAs. The recently released database of fully sequenced mouse cDNAs (based on the FANTOM2 clone set), which predicts as many as 2500 antisense transcripts (43), revealed one entry derived from the Stx16 locus that predicted the presence of an antisense transcript extending from Stx16 intron 5 to intron 1 (no. D730019D15; National Center for Biotechnology Information AK052807, http://genome.gsc.riken.go.jp/m/antisense/). To verify the existence of this putative transcript, RT-PCR was performed using DNase-treated total RNA from kidney of mutant Stx16{Delta}4–6 mice and wild-type littermates. When using the same primers as those used for genotyping the Stx16{Delta}4–6 mice, amplicons of 615 bp for the Stx16{Delta}4–6 allele and 544 bp for the wild-type allele were observed (Fig. 6Go, A and B). Although at least one primer annealed to an intronic sequence, each RT-PCR yielded intronic nonspliced sequences. Only very little amplification was obtained from wild-type cDNA when using primers c and d. However, in the presence of high amounts of genomic DNA, primers c and d were able to amplify a product of the expected size (2944 bp), suggesting that reverse transcription (RT) of the novel mRNA was not sufficiently efficient or that the transcript exists only in low abundance.


Figure 6
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  		FIG. 6. RT-PCR to amplify an unspliced transcript from the Stx16/STX16 locus. A, Diagram depicting the location of primers a–d in the Stx16 gene. Only primer c anneals to an exonic region (Stx16 exon 3), whereas the other primers anneal to intronic sequences. B, RT-PCR using kidney RNA of Stx16{Delta}4–6 mice and the same primer pairs as for genotyping Stx16{Delta}4–6 mice (see Fig. 2Go) to amplify transcripts derived either from the wild-type (lower panel) or the mutant allele (upper panel). A 615-bp band (upper panel; primers c/d) could be amplified from cDNA of mice that are heterozygous or homozygous for Stx16{Delta}4–6, whereas the expected wild-type band of 2944 bp is barely detectable due to insufficient RT and/or low abundance of the mRNA yet clearly visible when amplified from wild-type genomic DNA (positive control). Primers located within the deleted intron 4 of Stx16 (lower panel; primers a/b) amplified a 544-bp band from cDNA derived from wild-type and heterozygous Stx16{Delta}4–6 mice but not in cDNA from homozygous Stx16{Delta}4–6 animals. Wild-type genomic DNA served as positive control. Treatment of the samples with or without DNaseI and RT is indicated. +, Wild-type allele; {Delta}, mutant allele; {Delta}P, paternal allele; {Delta}M, maternal allele; M, marker lane. C, RT-PCR for STX16 amplification using RNA of a patient with AD-PHP-Ib. DNase-treated total RNA was isolated from lymphoblastoid cells of patient F-III/37 (30 ). After RT, the cDNA was subjected to PCR using intronic primers (n/m) flanking the 3-kb microdeletion (see diagram in D). A 1367-bp PCR product was amplified, which could not be amplified when RT was omitted (neg Ctr.) or when cDNA obtained from lymphoblastoid cells of a healthy individual was used (normal cDNA).

 
To confirm the presence of the presumed novel transcript in humans, a similar experiment was performed using DNase-treated total RNA isolated from lymphoblastoid cells of an AD-PHP-Ib patient with the 3-kb microdeletion (patient F-III/37; described in Ref. 30). These studies revealed the presence of a 1367 bp cDNA containing only intronic sequences, when the RNA was amplified by RT-PCR across the deletion using primers located just downstream of exon 3 and just upstream of exon 7 (Fig. 6Go, C and D). No amplicons were obtained when RT was omitted. When total RNA from lymphoblastoid cells of a healthy individual was used for RT, no amplification was obtained either. This may be related, just as observed with total RNA from wild-type mice (see Fig. 6BGo), to the large size and/or the low abundance of the putative novel transcript. Control experiments attempting the amplification of intronic regions of the ß-globin gene, revealed no PCR products indicating that DNase treatment had efficiently removed all genomic DNA (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A unique identical 3-kb microdeletion within STX16 gene is associated with the loss of exon A/B methylation on the maternal allele and thus PTH-resistance in numerous patients with AD-PHP-Ib (30, 31, 32, 33). Another microdeletion, which removes 4.4-kb of STX16, was more recently identified in an unrelated AD-PHP-Ib kindred; it leads to the same epigenetic change that was observed in patients with the 3-kb deletion, i.e. a loss of methylation limited to exon A/B (34). The two deletions overlap by about 1200 bp, suggesting that this region in humans is involved in maintaining or establishing methylation of a single GNAS DMR, namely the exon A/B DMR.

To determine whether ablation of the equivalent of the 3-kb STX16 microdeletion in humans leads to the Gnas methylation changes and to the development of PHP-Ib, we generated mice with a 2.3-kb targeted ablation of the Stx16 region that corresponds to human STX16del4–6. However, when this microdeletion was present on the maternal allele, no loss of exon 1A methylation was observed, and the mutant mice failed to develop PTH resistance. Likewise, ablation of Stx16 exons 4–6 on the paternal allele or on both parental alleles did not result in any obvious phenotype. All investigated patients with an isolated loss of exon A/B methylation carry deletions on the maternal allele that disrupt STX16, and it was therefore surprising that the ablated mouse genomic region does not appear to contain an imprinting control element of GNAS. Although loss of one copy of STX16 could have been involved in the pathogenesis of AD-PHP-Ib, our previous efforts had failed to document that human STX16 is imprinted, thus making this hypothesis unlikely. However, a role for syntaxin-16 or its transcript in establishing exon A/B methylation, especially through yet undefined mechanisms during oogenesis, has remained hitherto possible. The present study now clearly rules out this latter possibility. Hence, it appears more likely that the deleted region in humans contains a cis-acting element that happens to overlap with the STX16 locus. Similar to the findings in fascioscapulohumeral muscular dystrophy (44), this element may physically interact with exon A/B, thereby facilitating or maintaining methylation of its promoter. Taken together, our findings demonstrate that disruption of STX16, or haploinsufficiency thereof, is not responsible for the PTH resistance in PHP-Ib and that the translated gene product itself is not involved in the control of GNAS imprinting.

Organization of the Stx16 locus and the overall structure and regulation of the Gnas cluster are well conserved in mouse and human (and other mammals, e.g. dog). Furthermore, the four DMRs affecting the activity of the promoters for exons Nesp55, Gnasxl-Nespas, and 1A undergo the same parent-specific methylation and thus silencing in both species (1, 2, 3, 4, 5, 6). It was therefore surprising that ablation of the 3-kb Stx16 region in mice (Stx16{Delta}4–6) did not lead to the same phenotype and epigenotype as in humans after maternal inheritance of the deletion. These findings suggest that the presumed regulatory element necessary for establishing or maintaining exon 1A methylation resides in mice at a location different from that in humans, or it is not present at all; similarly discordant observations have been made for a growing number of imprinted genes in human and mouse (45). Interestingly, a database entry for a spliced, chimeric mRNA that comprises exons of both STX16 and its telomeric neighbor NPEPL1, is present in the human (GenBank accession no. AB209296); a similar transcript is not present in the mouse databases. Considering that the STX16 microdeletions in patients with AD-PHP-Ib are predicted to disrupt this putative transcript, it appears possible that it plays a role in the control of imprinting at the GNAS locus. Alternatively, it is conceivable that the forms of AD-PHP-Ib caused by these microdeletions are disorders specific for humans (or primates) or that two independent regions need to be disrupted or mutated in cis to prevent normal exon 1A (or exon A/B) methylation on the maternal allele. However, all AD-PHP-Ib patients investigated thus far have failed to show evidence for the presence of such a second mutation within the STX16/Stx16-GNAS region.

Because Stx16{Delta}4–6 mice had failed to provide insights into the mechanisms leading to loss of exon 1A methylation and PTH resistance, we searched for other clues as to how the 3-kb STX16 region that is missing in a large number of AD-PHP-Ib patients might be involved in regulating methylation at the exon 1A (or exon A/B) DMR located about 220 kb downstream. These efforts led to the identification of an antisense transcript in a public database (43), and the use of intronic primers confirmed the presence of this putative transcript in kidney-derived cDNA from wild-type mice and animals that are heterozygous or homozygous for Stx16{Delta}4–6 (see Fig. 6Go). The presence of the novel transcript was furthermore confirmed in human by using lymphoblastoid-derived total RNA from a patient with STX16del4–6 for RT-PCR amplification with intronic primers located downstream of exon 3 and upstream of exon 7. Additional findings with RNA from mouse kidney and lymphoblastoid cells furthermore suggest that the STX16/Stx16-derived antisense transcript is large, but it is currently unknown whether it undergoes splicing and whether it is involved in the pathogenesis of AD-PHP-Ib. If it contributes to the development of the disease, the lack of an epigenetic or biochemical phenotype in the Stx16{Delta}4–6 mouse would suggest that important elements of the human antisense RNA transcript are either located elsewhere in the mouse genome or that these elements are not well conserved.

In recent years, naturally occurring antisense transcripts have been implicated in many aspects of eukaryotic gene expression, including genomic imprinting, RNA interference, translational regulation, alternative splicing, X-inactivation, RNA editing, gene silencing, and methylation (for reviews see Refs. 46, 47, 48, 49, 50). Similar mechanisms might therefore contribute to the abnormal methylation in AD-PHP-Ib and the subsequent development of PTH-resistance. In fact, an antisense transcript has already been described for the human GNAS and the mouse Gnas locus (3, 6), and this transcript appears to have an important role for regulating, in cis, the expression of other transcripts derived from the GNAS locus, particularly the silencing of Nesp55 transcripts (51).

Antisense transcripts similar to that in patients with AD-PHP-Ib have been identified within the large SNURF-SNRPNsense/UBE3A locus, and these have been implicated in reducing UBE3A expression in patients with the Prader-Willi or Angelman syndrome (40). Furthermore, abnormal imprinting of LIT1, the antisense transcript encoded on the opposite strand of the potassium voltage-gated channel Kcnq1, is associated with the Beckwith-Wiedemann syndrome (52), and microdeletions within or telomeric of LIT1 have been reported in patients affected by this disease (42). If inherited maternally, these deletions silence expression of p57KIP2, an imprinted cyclin-dependent kinase inhibitor located several hundred kilobases centromeric of the deletion. Lastly, an inherited form of ß-thalassemia appears to be associated with silencing of hemoglobin ß-2 (HBA2) gene expression through a cis-acting antisense RNA, which causes de novo methylation of a CpG island (53). Silencing of gene expression through mechanisms that involve epigenetic modifications through antisense transcripts has also been proposed for Igf2r (41, 54) and Xist (55, 56). However, despite a clear correlation between antisense transcription, CpG-island methylation and the localized modifications of chromatin structure, the molecular mechanisms underlying this method of gene silencing remain to be explored.

It is plausible that expression from the Gnas locus involves a completely different mechanism of action, particularly because the STX16/Stx16 locus shows no features associated with imprinting control elements, such as a DMR. The only obvious sequence elements present in the human region are two direct repeats, which are flanking the deleted sequence (30) (see Fig. 1Go, A and B). These repeat elements are likely to facilitate the mutation process by unequal homologous crossing over because exactly the same deletion was thus far found in 22 unrelated families (30, 31, 32, 33) and probably have no direct functional role in genomic imprinting. In support of this latter view was the identification of a different 4.4-kb microdeletion within STX16, which results in AD-PHP-Ib with an indistinguishable epigenotype and phenotype (34). This mutation overlaps partially with the 3-kb deletion, and it also eliminates one of the repeats, whereas the second retroviral element is left intact. In addition, both repeat elements are not conserved across other species, such as dog or cow, and they do not represent tandem repeats similar to those described for the Rasgrf1 DMR that, when deleted, facilitate aberrant DNA methylation and imprinting (57).

In conclusion, further analysis of the STX16 region in human and mouse is required to unravel the role of specific sequences that might be involved in silencing transcription from the Gs{alpha} promoter through mechanisms that involve DNA methylation. Whether the newly discovered putative STX16/Stx16 antisense transcript could have a regulatory role in these processes remains uncertain. It may be necessary to search for trans-acting factors that normally interact with the portion of the STX16 region, which is deleted in patients with AD-PHP-Ib. Such factors may help explore the complex mechanisms, which appear to be involved in the long-range regulation of human exon A/B (and mouse exon 1A) methylation and thus Gs{alpha} expression.


   Acknowledgments
 
We thank Siglinde Hirmer for excellent technical assistance.


   Footnotes
 
This work was supported by Grant RO1 46718-10 from the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (to H.J.).

Present address for L.F.F.: Institute of Pathophysiology, University of Veterinary Medicine, Vienna, Austria.

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 22, 2007

Abbreviations: DMR, Differentially methylated region; 1A-DMR, exon 1A DMR; ES cell, embryonic stem cell; GNAS/Gnas, gene encoding the guanine nucleotide-binding {alpha}-subunit and splice variants thereof; Gs{alpha}, {alpha}-subunit of the stimulatory G protein; NESP55/Nesp55, neuroendocrine secretory protein of Mr 55,000; PHP-Ib, pseudohypoparathyroidism type Ib; PPHP, pseudopseudohypoparathyroidism; RT, reverse transcription; SDS, sodium dodecyl sulfate; SSC, saline sodium citrate; STX16/Stx16, syntaxin 16; STX16del4–6, deleting exons 4–6 of STX16; XL{alpha}s, extra-large Gs{alpha} variant.

Received September 25, 2006.

Accepted for publication February 14, 2007.


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 Introduction
 Materials and Methods
 Results
 Discussion
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