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The Journal of Clinical Endocrinology & Metabolism Vol. 86, No. 10 4627-4629
Copyright © 2001 by The Endocrine Society

Special Features

G{alpha}s Transcripts Are Biallelically Expressed in the Human Kidney Cortex: Implications for Pseudohypoparathyroidism Type 1b

Hong Zheng, Genia Radeva, Jennifer A. McCann, Geoffrey N. Hendy and Cynthia G. Goodyer

Departments of Pediatrics (H.Z., G.N.H., C.G.G.), Medicine (J.A.M., G.N.H., C.G.G.), Human Genetics (G.N.H.), and Physiology (G.R., G.N.H.), McGill University, Montréal, Québec, Canada H3Z 2Z3

Address all correspondence and requests for reprints to: Cynthia G. Goodyer, Ph.D., Endocrine Research Laboratory, 4th Floor, Place Toulon, Room 415/1, McGill University–Montreal Children’s Hospital Research Institute, 4060 Ste. Catherine Street W., Westmount, Québec, Canada H3Z 2Z3. E-mail: cindy.goodyer{at}muhc.mcgill.ca

Abstract

Pseudohypoparathyroid type 1b patients are characterized by renal resistance to PTH in the absence of Albright’s hereditary osteodystrophy or other endocrine abnormalities. Kindred studies have suggested that the cause of this resistance is a specific decrease in G{alpha}s activity in renal proximal tubules due to paternal imprinting of G{alpha}s. To test this, allelic expression of G{alpha}s was analyzed in human fetal kidney cortex samples by RT-PCR assays. The results showed that, in contrast to the parent-specific expression of exon 1A and XL{alpha}s (paternal) or NESP (maternal) mRNAs, G{alpha}s transcripts are biallelically expressed in human kidney cortex. These data implicate abnormal imprinting of alternative regions within the GNAS1 locus as a more likely cause of pseudohypoparathyroid type 1b.

GNAS1 [GUANINE NUCLEOTIDE (binding) {alpha}-subunit 1] encodes four products due to alternative splicing of four different first exons into a common site in exon 2 (Fig. 1aGo) (1). mRNA expression from these alternative first exons is under complex imprinting controls (1). Exon 1 mRNA is biallelically expressed in all human tissues examined to date and encodes G{alpha}s, the {alpha}-subunit of the heterotrimeric guanine nucleotide-binding protein that couples hormone receptors to stimulation of adenylate cyclase (2, 3, 4, 5). Exon 1A transcripts are expressed only from the paternal allele in peripheral blood cells (6) and encode a G{alpha}s isoform that lacks the GTP-binding domain (7, 8). The third alternatively spliced mRNA is transcribed only from the paternal allele (5, 9) and encodes XL{alpha}s, a large isoform of G{alpha}s that lacks the ability to be activated by G protein-coupled receptors, although it binds GTP and ß{gamma}-subunits and activates adenylate cyclase (10, 11). mRNA transcribed from the most upstream exon (NESP) is expressed only from the maternal allele and encodes the 55kD NESP chromogranin-like protein (4, 12). In addition, one antisense transcript, derived solely from the NESP region of the paternal allele, has been described (13, 14, 15).



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  		Figure 1. GNAS1 imprinting in human fetal tissues. a, Genomic structure of GNAS1. RT-PCR primers are indicated by arrows. The arrowhead marks the polymorphic FokI site in exon 5. b, GNAS1 mRNA expression patterns in kidney cortex from two informative fetuses. Each pair of lanes contains undigested (left) or FokI-digested (right) RT-PCR products. The two bands in most undigested samples result from alternative splicing of exon 3. In kidney cortex #46 (FokI m+/p-), all NESP-specific but none of the XL{alpha}s- or exon 1A-specific products are cut, whereas exon 1 and exons 4–6 products are partially cleaved. Opposite results were obtained for kidney cortex #17 (FokI m-/p+). Thus, in kidney cortex, the G{alpha}s-encoding exon 1-derived transcripts are biallelically expressed. c, GNAS1 expression in multiple tissues from fetus #42 (FokI m-/p+). The two bands in most undigested samples result from alternative splicing of exon 3; in the central nervous system (CNS) only the full-length transcript was detected (n = 11). Maternally expressed NESP-derived RT-PCR products are FokI resistant, but not paternally expressed XL{alpha}s and exon 1A or the biallelically expressed exon 1 and exons 4–6 products. These data parallel the imprinting expression patterns observed in human fetal kidney cortex. Thus, no tissue- or individual-specific imprinting patterns were found in any of the tissues examined. d, Schematic of maternal vs. paternal expressed mRNAs encoding NESP55, XL{alpha}s, a protein of unknown function (exon 1A), and G{alpha}s (exon1) in human tissues. The horizontal arrows designate transcription start sites.

 
Genetic defects in GNAS1 contribute to pseudohypoparathyroidism type 1b (PHP-1b), although the precise mechanism is not known (1). PHP-1b is characterized by G{alpha}s resistance to PTH primarily in the renal proximal tubule (1, 16), but no mutations in PTH or its receptor have been reported, and the G{alpha}s level in erythrocytes of PHP1b patients is normal (16, 17, 18, 19, 20). However, kindred studies have mapped the PHP-1b locus to the GNAS1 region of chromosome 20q and determined that maternal inheritance of genetic defects at this locus is the major cause of PHP-1b (21). Based on these findings, it has been proposed, but never demonstrated, that G{alpha}s expression is paternally imprinted in a tissue (renal cortex)-specific manner in normal humans, as has been shown in mice: G{alpha}s is maternally expressed in murine renal proximal tubules and adipose tissues and biallelically expressed in other tissues (22).

We, therefore, compared the imprinted status of GNAS1-derived transcripts in 11 different human fetal tissues, including kidney cortex, and determined that in all the tissue samples G{alpha}s is biallelically expressed.

Materials and Methods

Tissues

Human fetal tissues were collected at the time of therapeutic abortion. Informed consent was obtained in all cases, and prior approval for the study was received from the local institutional review board. The outer layer (cortex) of the human fetal kidneys was removed using scalpel blades. Histological analysis of the kidney cortex samples revealed an enrichment of cortex-related cells (proximal tubules, glomeruli, condensing mesenchyme, developing nephron structures) with less than 15% medullary tubular structures (data not shown).

RNA and DNA extractions and assay methods

DNA and RNA were extracted as described previously (23). A transcribed, silent T to C polymorphism in exon 5, creating a FokI site, was used to distinguish two copies of the gene (24). PCR assays were carried out using 125 ng genomic DNA in a 25-µl reaction mixture containing 0.25 µM of primers (exon 4 sense, 5'-TGAGAAGGCAACCAAAGTGC-3'; and intron E antisense, 5'-GGGCTAAGGCCACACAAGT-3'), 2 mM MgCl2, 500 µM dNTPs, 7% DMSO, and 1.25 U Taq DNA polymerase. Amplification occurred over 35 cycles of 94/58/72 C for 45/30/90 sec. PCR products were digested with 1 U FokI for 1 h at 37 C. Of the 45 fetuses genotyped, 17 (13–18 wk fetal age) were heterozygous. To examine allele-specific gene expression, 5 µg total RNA were reverse transcribed using Superscript II (Life Technologies, Inc., Burlington, Ontario, Canada) and an antisense primer in exon 6 (no. 1, 5'-ACTCCTTCATCCCACAGA-3'). Reverse transcribed products were amplified using exon-specific sense primers [NESP (5), XL{alpha}s (5), exon 1 (5), exon 1A (5'-AGCGAGCCCCTGTTCCCGGCG-3'), exon 4 (see above)] and a common antisense primer in exon 6 (no. 2) (5) (Fig. 1aGo) under the same conditions as for the genomic DNA. RT-PCR products were digested with FokI and analyzed on agarose gels.

Results and Discussion

Kidney cortex samples (n = 12) uniformly expressed all four GNAS1 sense mRNAs (Fig. 1Go, b and c, and Table 1Go). NESP-derived transcripts showed opposite imprinting (maternal expression) to XL{alpha}s and exon 1A-derived mRNAs (paternal). In contrast, exon 1 transcripts were always biallelically expressed (Fig. 1Go, b and c, and Table 1Go). Finally, these mRNAs had the same imprinting pattern in all fetal tissues examined (Fig. 1Go, c and d, and Table 1Go). Thus, no individual- or tissue-specific patterns of expression were observed.


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  		Table 1. Summary of the GNAS1 imprinting patterns observed in human fetal tissues

 
Our findings demonstrate that PHP-1b cannot be due to paternal imprinting of G{alpha}s within the renal proximal tubule. Recently, Liu et al. (6) showed that, in peripheral blood cells of PHP-1b patients, there were abnormal imprinting patterns of GNAS1-derived mRNAs: biallelic expression of exon 1A (n = 13 of 13 patients) and XL{alpha}s (n = 2 of 13), and loss of NESP mRNA expression (n = 5 of 13). Similar abnormal imprinting patterns have been reported in an additional nine PHP1b kindreds (25). Furthermore, Bastepe et al. (26) have described a PHP-1b patient with paternal uniparental isodisomy/maternal deletion of chromosome 20q. Thus, our data, in the context of these three reports, strongly suggest that maternal inheritance of abnormal imprinting of the three upstream GNAS1 exons, resulting in a double dose of paternally expressed (XL{alpha}s- and exon 1A-derived) transcripts, might be responsible for PHP1b. Enhanced expression of these protein products, which have a similar carboxy-terminal region as G{alpha}s but significantly different NH2-terminal sequences, may act as specific inhibitors of G{alpha}s activity in renal proximal tubule cells.

Acknowledgments

We thank Drs. Constantin Polychronakos and Aimée Ryan for helpful discussions.

Footnotes

This work was supported by the Canadian Institutes for Health Research (to C.G.G. and G.N.H.).

Abbreviations: PHP-1b, Pseudohypoparathyroidism type 1b.

Received April 3, 2001.

Accepted June 25, 2001.

References

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