SHORT COMMUNICATION A Heterozygous 4-bp Deletion Mutation in the G,Q Gene (GNASI) in a Patient with Albright Hereditary Osteodystrophy LEE 5. WEINSTEIN, PABLO V. GEJMAN, PHILIPPE DE MAZANCOURT, Molecular
Pathophysiology National
NADINE AMERICAN, AND ALLEN M. SPIEGEL
Branch, National Institute of Diabetes, Digestive and Kidney Disease, and Clinical Neurogenetics Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892 Received
February
Several heterozygous mutations within the gene encoding the a-subunit of G, (GNASl), the G protein that stimulates adenylyl cyclase, have been previously identified in patients with Albright hereditary osteodystrophy (AHO). We have now identified a fourth GNASl mutation from an AH0 patient. Amplification by the polymerase chain reaction (PCR) of a genomic fragment encompassing GNAS 1 exons 7 and 8 from one patient resulted in a product with aberrant migration on nondenaturing polyacrylamide and agarose gels. Direct DNA sequencing identified a 4-bp deletion in one allele of exon 7 encoding a frameshift with a premature stop codon. Analysis of lymphocyte RNA by reverse transcription-PCR and direct sequencing showed that the GNASl allele bearing the mutation is not expressed as mRNA. Consistent with this, Northern analysis revealed an approximate 50% deficiency in steady-state levels of GNASl mRNA. These findings further illustrate the heterogeneity of GNASl gene defects in o 1992 Academic Press, ~nc. AHO.
18, 1992
tumors (11) and are present in a mosaic distribution in patients with the McCune-Albright syndrome (17). We obtained lymphoblast cultures from five unrelated AH0 patients from the NIGMS Human Genetic Mutant Cell Repository (Accession Nos. GM04411A, GM04425-GM04428). Three of the five reportedly had G protein deficiency. GNASl exons 2 through 13 were screened for mutations as previously described using the polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) (16). Lymphoblast RNA was isolated by previously described methods (3). For reverse transcription-PCR (RT-PCR), a 19-pl mixture containing 5 pg of total RNA, 1~1 of RNasin (Promega), 10 pmol of downstream oligonucleotide primer, 50 mMTris-HCl (pH 8.91), 6 mMMgCl,, 10 mMdithiothreitol, and 100 mM NaCl was heated to 70°C and slowly cooled to 42°C. AMV reverse transcriptase (ProAcrylamide A
The Albright hereditary osteodystrophy (AHO) phenotype is characterized by short stature, obesity, rounded facies, and skeletal defects. AH0 may be expressed alone [pseudopseudohypoparathyroidism (PPHP)] or in association with resistance to multiple hormones that are coupled to stimulation of adenylyl cyclase [pseudohypoparathyroidism (PHP)]. Most affected patients (either PHP or PPHP) have an approximate 50% deficiency of functional G,, the G protein that stimulates adenylyl cyclase (9, 10). G proteins involved in signal transduction consist of a-subunits, which bind guanine nucleotides and confer specificity, as well as /3and y-subunits. Steady-state levels of G,ol mRNA are reduced in the majority of AH0 patients (1, 10). The human G,a gene (GNASl) is located on the distal long arm of chromosome 20 (4) and contains 13 exons spanning 20 kb (7). Recently, three unique heterozygous GNASl mutations that disrupt the expression of a normal G,cu protein have been identified in affected members from three unrelated families (12,16). Somatic mutations of this gene encoding a constitutively activated G,a protein are also present in sporadic endocrine 1319
Branch,
rd
603-
310-
Agarose A ::
1.1 -603
-310
FIG. 1. Migration of the exon 7-8 genomic DNA fragment in nondenaturing acrylamide and agarose gels. The oligonucleotides used for PCR TGTG 3’ (an
were
5’ AAACTGCAGCAAATTGATGTGAGCGC-
upstream primer containing the underlined intron sequence)and 5’ CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGC-
6
CCCCGCCCCAGAAACCATGATCTCTGTTATA 3’ (a downstream primer containing the underlined complementary intron 9 sequence with an attached 5’ GC clamp). Samples were electrophoresed in a nondenaturing 5% acrylamide gel, shown on the left, and a 3% agarose gel (2% NuSieve, 1% standard agarose), shown on the right. The positions of molecular weight markers (in bp) are on the side of each gel. DNA from patient GM04426 (AHO) shows two additional slowly migrating bands not seen in other control or patient samples (N) in polyacrylamide. The same sample in agarose shows one slower migrating band just above the wildtype band. GENOMICS
13,
1319-1321 088%X43/92
(19%) $5.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
1320
SHORT
COMMUNICATIONS
mega), 8 units, was added and the reactions were incubated at 42°C for 1 h. The RT mixture (10 ~1) was used as template in a 100-/J PCR reaction under previously described conditions (16). Direct DNA sequencing was performed by a modification of the method of Kadowaki et al. (6). Northern analysis was performed on total RNA (5 pg/lane) as previously described (8) except that denatured salmon sperm DNA (500 pg/ml) was included in the prehybridization and hybridization. The signals were quantified with a phosphorimager (Molecular Dynamics). In one individual (GM04426) with PHP, the amplified fragments encompassing GNASl exons 7 and 8 displayed an atypical pattern on nondenaturing polyacrylamide and agarose gels (Fig. 1). In a 5% polyacrylamide gel (5O:l acrylamide to bisacrylamide in Tris-borateEDTA), two slower migrating bands were present in addition to the wildtype band. These abnormally migrating bands were not present in multiple other patient and control samples. The pattern in agarose [2% NuSieve (FMC), 1% standard agarose in Tris-borate-EDTA] showed one slower migrating band close to the wildtype band. Direct sequencing of the GNASl exon 7-8 containing genomic fragments revealed a heterozygous 4-bp deletion in exon 7, resulting in a frameshift encoding 13 incorrect amino acid residues followed by a premature stop codon (Fig. 2). PCR products heterozygous for a common 4-bp insertion mutation in the HEX A gene in Tay-Sachs disease show similar patterns in polyacrylamide and agarose gels (13, 14). Heteroduplexes containing 4-bp insertions or deletions presumably have an altered secondary structure and therefore a retarded mobility.
GENOMIC
A region of GNASl including exon 7 was amplified by RT-PCR from lymphoblast RNA of patient GM04426. In contrast to the PCR product from genomic DNA, the RT-PCR product did not have abnormally migrating bands on a nondenaturing acrylamide gel (data not shown). Direct sequencing of this product revealed only the wildtype sequence (Fig. 2), suggesting minimal or no expression of the mutant allele as mRNA. Northern analysis was performed using a full-length rat GNASl cDNA probe (5) and a 650-bp fi-actin cDNA probe (provided by B. Patterson, National Institutes of Health, Bethesda, MD). Consistent with the results of RT-PCR and direct sequencing, the expression of GNASl mRNA corrected for &actin mRNA in patient GM04426 was approximately half of that in an individual with normal functional G, (data not shown). We have not as yet identified GNASl mutations in the two other lymphoblast cell lines from patients with deficient G,. In these patients, it is possible that GNASl mutations may be located in regions of the gene that we have not analyzed (introns, exon 1, and the 5’ flanking region) or that additional mutations were undetectable by parallel DGGE alone. The diminished levels of GNASl mRNA in this patient are consistent with the findings in patients from other AH0 kindreds (1,lO). Mutations encoding premature terminations in other genes (e.g., the genes coding for triose phosphate isomerase and dihydrofolate reductase) have been associated with diminished levels of mRNA, and it has been proposed that translation to the distal end of the penultimate exon may be required for normal RNA processing and transport to the cytoplasm (2,15). This newly identified mutation in a patient with
DNA
AH0
RNA AH0
Control
GATC
GATC
GATC
2: EL
Normal
Allele
5’CAG Gin
+mb Ala
Mutant
Allele
5’CAG Gin
GCT Ala
200 GTG Val
CCG Pro
AGC Ser
GAT Asp
CAG Gin
GAC Asp
CTG Leu
CTT Leu
CGC Arg
TGC Cys
CGT Arg
GTC
Asp
190 TAT Tyr
ATG Met
TGC Cys
CGA Arg
GCG Ala
ATC Ile
AGG Arg
ACC Thr
TGC Cys
TTC Phe
GCT Ala
GCC Ala
GTG Val
TCC Ser
TGA 3’ STOP
FIG. 2. Direct DNA sequencing of genomic DNA and RT-PCR products. Sequencing of the heterozygous 4bp deletion mutation present in the patient’s genomic DNA (AHO) that is absent sequences are shown below with the codon number (7). The deleted bases in the coding strand are termination at codon 202. On the right are the results of RT-PCR and direct sequencing of the detected. The primers for RT-PCR were 5’ TACGAACGCTCCAACGAGTACC 3’ (upstream CACG 3’ (downstream primer).
3’
Val
exon 7-8 genomic DNA fragments identified a in a normal control. Normal and mutant allele boxed. The mutant allele encodes a premature patient’s RNA. Only the wildtype sequence is primer) and 5’ TTCATCGCGCTGGCCACC-
SHORT
AH0 is further evidence that this disease is associated with heterogeneous molecular defects of the GNASl gene.
mediated motility of highly ht. J. Cancer 48: 113-120.
3. Chomczynski, RNA form
4.
5. 6.
7.
8.
isolation extraction.
P., and Sacchi, N. (1987). Single-step method by acid guanidinium thiocyanate-phenol-chloroAnal. Biochem. 162: 156-159.
621. 11.
Lyons, J., Landis, C. A., Harsh, G., Vallar, L., Griinewald, K., Feichtinger, H., Duh, Q., Clark, 0. H., Kawasaki, E., Bourne, H. R., and McCormick, F. M. (1990). Two G protein oncogenes in human endocrine tumors. Science 249: 655-659.
12.
Patten, J. L.. Johns, D. R., Valle, D., Eil, C., Gruppuso, P. A., Steele, G., Smallwood, P. M., and Levine, M. A. (1990). Mutation in the gene encoding the stimulatory G protein of adenylate cyclase in Albright’s hereditary osteodystrophy. N. Engl. J. Med. 322: 1412-1419. Shore, S., and Myerowitz, R. (1990). A gel electrophoretic assay for detecting the insertion defect in Ashkenazi Jewish carriers of Tay-Sachs disease. Anal. Biochem. 186: 179-181.
of
Gejman, P. V., Weinstein, L. S., Martinez, M., Spiegel, A. M., Cao, Q., Hsieh, W., Hoehe, M. R., and Gershon, E. S. (1991). Genetic mapping of the Gs-(Y subunit gene (GNASl) to the distal long arm of chromosome 20 using a polymorphism detected by denaturing gradient gel electrophoresis. Genomics 9: 782-783. Jones, D. T., and Reed, R. R. (1987). Molecular cloning of five GTP-binding protein cDNA species from rat olfactory neuroepithelium. J. Biol. Chem. 262: 14241-14249. Kadowaki, T., Kadowaki, H., and Taylor, S. I. (1990). A nonsense mutation causing decreased levels of insulin receptor mRNA: Detection by a simplified technique for direct sequencing of genomic DNA amplified by the polymerase chain reaction. Proc. Natl. Acad. Sci. USA 87: 658-662. Kozasa, T., Itoh, H., Tsukamoto, T., and Kaziro, Y. (1988). Isolation and characterization of the human G,a gene. Proc. N&l. Acad. Sci. USA 85: 2081-2085. Lester, B. R., Weinstein, L. S., McCarthy, R. S., and Furcht, L. T. (1991). The role
J. B., Sun, Z., Smith, of G-protein in matrix-
cells.
Levine, M. A., Ahn, T. G., Klupt, S. F., Kaufman, K. D., Smallwood, P. M., Bourne, H. R., Sullivan, K. A., and Van Dop, C. (1988). Genetic deficiency of the N subunit of the guanine nucleotide-binding protein Gs as the molecular basis for Albright hereditary osteodystrophy. Proc. Natl. Acad. Sci. USA 85: 617-
2. Cheng,
J., Fogel-Petrovic, M., and Maquat, L. E. (1990). Translation to near the distal end of the penultimate exon is required for normal levels of spliced triosephosphate isomerase mRNA. Mol. Cell. Biol. 10: 5215-5225.
melanoma
10.
Collins for maintaining the lymphoblast cultures. was supported by the University of Paris V and
Carter, A., Bardin, C., Collins, R., Simons, C., Bray, P., and Spiegel, A. (1987). Reduced expression of multiple forms of the cy subunit of the stimulatory GTP-binding protein in pseudohypoparathyroidism type Ia. Proc. Natl. Acad. Sci. USA 84: 72667269.
invasive
Levine, M. A., Jap, T-J., Mauseth, R. S., Downs, R. W., and Spiegel, A. M. (1986). Activity of the stimulatory guanine nucleotide-binding protein is reduced in erythrocytes from patients withpseudohypoparathyroidismandpseudopseudohypoparathyroidism: Biochemical, endocrine, and genetic analysis of Albright’s hereditary osteodystrophy in six kindreds. J. Clin. Endocrinol. Metab. 62: 497-502.
REFERENCES 1.
and poorly
9.
ACKNOWLEDGMENTS We thank Regina Dr. P. de Mazancourt CNRS.
1321
COMMUNICATIONS
13. 14.
15.
Triggs-Raine, B. L., and Gravel, R. A. (1990). Diagnostic heteroduplexes: Simple detection of carriers of a 4-bp insertion mutation in Tay-Sachs disease. Am. J. Hum. Genet. 46: 183-184. Urlaub, G., Mitchell. P. J., Ciudad, C. J., and Chasin, L. A. (1989). Nonsense mutations in the dihydrofolate reductase gene affect RNA processing. Mol. Cell. Biol. 9: 2868-2880.
16.
Weinstein, L. S., Gejman, P. V., Friedman, E., Kadowaki, T., Collins, R. M., Gershon, E. S., and Spiegel, A. M. (1990). Mutations of the Gs a-subunit gene in Albright hereditary osteodystrophy detected by denaturing gradient electrophoresis. Proc. Natl. Acad. Sci. USA 87: 8287-8290.
17.
Weinstein, L. S., Shenker, A., Gejman, P. V., Friedman, E., and Spiegel, A. M. (1991). Activating the stimulatory G protein in the McCune-Albright Engl. J. Med. 325: 1688-1695.
Merino, M. J., mutations of syndrome. N.
Pathophysiology National
NADINE AMERICAN, AND ALLEN M. SPIEGEL
Branch, National Institute of Diabetes, Digestive and Kidney Disease, and Clinical Neurogenetics Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892 Received
February
Several heterozygous mutations within the gene encoding the a-subunit of G, (GNASl), the G protein that stimulates adenylyl cyclase, have been previously identified in patients with Albright hereditary osteodystrophy (AHO). We have now identified a fourth GNASl mutation from an AH0 patient. Amplification by the polymerase chain reaction (PCR) of a genomic fragment encompassing GNAS 1 exons 7 and 8 from one patient resulted in a product with aberrant migration on nondenaturing polyacrylamide and agarose gels. Direct DNA sequencing identified a 4-bp deletion in one allele of exon 7 encoding a frameshift with a premature stop codon. Analysis of lymphocyte RNA by reverse transcription-PCR and direct sequencing showed that the GNASl allele bearing the mutation is not expressed as mRNA. Consistent with this, Northern analysis revealed an approximate 50% deficiency in steady-state levels of GNASl mRNA. These findings further illustrate the heterogeneity of GNASl gene defects in o 1992 Academic Press, ~nc. AHO.
18, 1992
tumors (11) and are present in a mosaic distribution in patients with the McCune-Albright syndrome (17). We obtained lymphoblast cultures from five unrelated AH0 patients from the NIGMS Human Genetic Mutant Cell Repository (Accession Nos. GM04411A, GM04425-GM04428). Three of the five reportedly had G protein deficiency. GNASl exons 2 through 13 were screened for mutations as previously described using the polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) (16). Lymphoblast RNA was isolated by previously described methods (3). For reverse transcription-PCR (RT-PCR), a 19-pl mixture containing 5 pg of total RNA, 1~1 of RNasin (Promega), 10 pmol of downstream oligonucleotide primer, 50 mMTris-HCl (pH 8.91), 6 mMMgCl,, 10 mMdithiothreitol, and 100 mM NaCl was heated to 70°C and slowly cooled to 42°C. AMV reverse transcriptase (ProAcrylamide A
The Albright hereditary osteodystrophy (AHO) phenotype is characterized by short stature, obesity, rounded facies, and skeletal defects. AH0 may be expressed alone [pseudopseudohypoparathyroidism (PPHP)] or in association with resistance to multiple hormones that are coupled to stimulation of adenylyl cyclase [pseudohypoparathyroidism (PHP)]. Most affected patients (either PHP or PPHP) have an approximate 50% deficiency of functional G,, the G protein that stimulates adenylyl cyclase (9, 10). G proteins involved in signal transduction consist of a-subunits, which bind guanine nucleotides and confer specificity, as well as /3and y-subunits. Steady-state levels of G,ol mRNA are reduced in the majority of AH0 patients (1, 10). The human G,a gene (GNASl) is located on the distal long arm of chromosome 20 (4) and contains 13 exons spanning 20 kb (7). Recently, three unique heterozygous GNASl mutations that disrupt the expression of a normal G,cu protein have been identified in affected members from three unrelated families (12,16). Somatic mutations of this gene encoding a constitutively activated G,a protein are also present in sporadic endocrine 1319
Branch,
rd
603-
310-
Agarose A ::
1.1 -603
-310
FIG. 1. Migration of the exon 7-8 genomic DNA fragment in nondenaturing acrylamide and agarose gels. The oligonucleotides used for PCR TGTG 3’ (an
were
5’ AAACTGCAGCAAATTGATGTGAGCGC-
upstream primer containing the underlined intron sequence)and 5’ CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGC-
6
CCCCGCCCCAGAAACCATGATCTCTGTTATA 3’ (a downstream primer containing the underlined complementary intron 9 sequence with an attached 5’ GC clamp). Samples were electrophoresed in a nondenaturing 5% acrylamide gel, shown on the left, and a 3% agarose gel (2% NuSieve, 1% standard agarose), shown on the right. The positions of molecular weight markers (in bp) are on the side of each gel. DNA from patient GM04426 (AHO) shows two additional slowly migrating bands not seen in other control or patient samples (N) in polyacrylamide. The same sample in agarose shows one slower migrating band just above the wildtype band. GENOMICS
13,
1319-1321 088%X43/92
(19%) $5.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
1320
SHORT
COMMUNICATIONS
mega), 8 units, was added and the reactions were incubated at 42°C for 1 h. The RT mixture (10 ~1) was used as template in a 100-/J PCR reaction under previously described conditions (16). Direct DNA sequencing was performed by a modification of the method of Kadowaki et al. (6). Northern analysis was performed on total RNA (5 pg/lane) as previously described (8) except that denatured salmon sperm DNA (500 pg/ml) was included in the prehybridization and hybridization. The signals were quantified with a phosphorimager (Molecular Dynamics). In one individual (GM04426) with PHP, the amplified fragments encompassing GNASl exons 7 and 8 displayed an atypical pattern on nondenaturing polyacrylamide and agarose gels (Fig. 1). In a 5% polyacrylamide gel (5O:l acrylamide to bisacrylamide in Tris-borateEDTA), two slower migrating bands were present in addition to the wildtype band. These abnormally migrating bands were not present in multiple other patient and control samples. The pattern in agarose [2% NuSieve (FMC), 1% standard agarose in Tris-borate-EDTA] showed one slower migrating band close to the wildtype band. Direct sequencing of the GNASl exon 7-8 containing genomic fragments revealed a heterozygous 4-bp deletion in exon 7, resulting in a frameshift encoding 13 incorrect amino acid residues followed by a premature stop codon (Fig. 2). PCR products heterozygous for a common 4-bp insertion mutation in the HEX A gene in Tay-Sachs disease show similar patterns in polyacrylamide and agarose gels (13, 14). Heteroduplexes containing 4-bp insertions or deletions presumably have an altered secondary structure and therefore a retarded mobility.
GENOMIC
A region of GNASl including exon 7 was amplified by RT-PCR from lymphoblast RNA of patient GM04426. In contrast to the PCR product from genomic DNA, the RT-PCR product did not have abnormally migrating bands on a nondenaturing acrylamide gel (data not shown). Direct sequencing of this product revealed only the wildtype sequence (Fig. 2), suggesting minimal or no expression of the mutant allele as mRNA. Northern analysis was performed using a full-length rat GNASl cDNA probe (5) and a 650-bp fi-actin cDNA probe (provided by B. Patterson, National Institutes of Health, Bethesda, MD). Consistent with the results of RT-PCR and direct sequencing, the expression of GNASl mRNA corrected for &actin mRNA in patient GM04426 was approximately half of that in an individual with normal functional G, (data not shown). We have not as yet identified GNASl mutations in the two other lymphoblast cell lines from patients with deficient G,. In these patients, it is possible that GNASl mutations may be located in regions of the gene that we have not analyzed (introns, exon 1, and the 5’ flanking region) or that additional mutations were undetectable by parallel DGGE alone. The diminished levels of GNASl mRNA in this patient are consistent with the findings in patients from other AH0 kindreds (1,lO). Mutations encoding premature terminations in other genes (e.g., the genes coding for triose phosphate isomerase and dihydrofolate reductase) have been associated with diminished levels of mRNA, and it has been proposed that translation to the distal end of the penultimate exon may be required for normal RNA processing and transport to the cytoplasm (2,15). This newly identified mutation in a patient with
DNA
AH0
RNA AH0
Control
GATC
GATC
GATC
2: EL
Normal
Allele
5’CAG Gin
+mb Ala
Mutant
Allele
5’CAG Gin
GCT Ala
200 GTG Val
CCG Pro
AGC Ser
GAT Asp
CAG Gin
GAC Asp
CTG Leu
CTT Leu
CGC Arg
TGC Cys
CGT Arg
GTC
Asp
190 TAT Tyr
ATG Met
TGC Cys
CGA Arg
GCG Ala
ATC Ile
AGG Arg
ACC Thr
TGC Cys
TTC Phe
GCT Ala
GCC Ala
GTG Val
TCC Ser
TGA 3’ STOP
FIG. 2. Direct DNA sequencing of genomic DNA and RT-PCR products. Sequencing of the heterozygous 4bp deletion mutation present in the patient’s genomic DNA (AHO) that is absent sequences are shown below with the codon number (7). The deleted bases in the coding strand are termination at codon 202. On the right are the results of RT-PCR and direct sequencing of the detected. The primers for RT-PCR were 5’ TACGAACGCTCCAACGAGTACC 3’ (upstream CACG 3’ (downstream primer).
3’
Val
exon 7-8 genomic DNA fragments identified a in a normal control. Normal and mutant allele boxed. The mutant allele encodes a premature patient’s RNA. Only the wildtype sequence is primer) and 5’ TTCATCGCGCTGGCCACC-
SHORT
AH0 is further evidence that this disease is associated with heterogeneous molecular defects of the GNASl gene.
mediated motility of highly ht. J. Cancer 48: 113-120.
3. Chomczynski, RNA form
4.
5. 6.
7.
8.
isolation extraction.
P., and Sacchi, N. (1987). Single-step method by acid guanidinium thiocyanate-phenol-chloroAnal. Biochem. 162: 156-159.
621. 11.
Lyons, J., Landis, C. A., Harsh, G., Vallar, L., Griinewald, K., Feichtinger, H., Duh, Q., Clark, 0. H., Kawasaki, E., Bourne, H. R., and McCormick, F. M. (1990). Two G protein oncogenes in human endocrine tumors. Science 249: 655-659.
12.
Patten, J. L.. Johns, D. R., Valle, D., Eil, C., Gruppuso, P. A., Steele, G., Smallwood, P. M., and Levine, M. A. (1990). Mutation in the gene encoding the stimulatory G protein of adenylate cyclase in Albright’s hereditary osteodystrophy. N. Engl. J. Med. 322: 1412-1419. Shore, S., and Myerowitz, R. (1990). A gel electrophoretic assay for detecting the insertion defect in Ashkenazi Jewish carriers of Tay-Sachs disease. Anal. Biochem. 186: 179-181.
of
Gejman, P. V., Weinstein, L. S., Martinez, M., Spiegel, A. M., Cao, Q., Hsieh, W., Hoehe, M. R., and Gershon, E. S. (1991). Genetic mapping of the Gs-(Y subunit gene (GNASl) to the distal long arm of chromosome 20 using a polymorphism detected by denaturing gradient gel electrophoresis. Genomics 9: 782-783. Jones, D. T., and Reed, R. R. (1987). Molecular cloning of five GTP-binding protein cDNA species from rat olfactory neuroepithelium. J. Biol. Chem. 262: 14241-14249. Kadowaki, T., Kadowaki, H., and Taylor, S. I. (1990). A nonsense mutation causing decreased levels of insulin receptor mRNA: Detection by a simplified technique for direct sequencing of genomic DNA amplified by the polymerase chain reaction. Proc. Natl. Acad. Sci. USA 87: 658-662. Kozasa, T., Itoh, H., Tsukamoto, T., and Kaziro, Y. (1988). Isolation and characterization of the human G,a gene. Proc. N&l. Acad. Sci. USA 85: 2081-2085. Lester, B. R., Weinstein, L. S., McCarthy, R. S., and Furcht, L. T. (1991). The role
J. B., Sun, Z., Smith, of G-protein in matrix-
cells.
Levine, M. A., Ahn, T. G., Klupt, S. F., Kaufman, K. D., Smallwood, P. M., Bourne, H. R., Sullivan, K. A., and Van Dop, C. (1988). Genetic deficiency of the N subunit of the guanine nucleotide-binding protein Gs as the molecular basis for Albright hereditary osteodystrophy. Proc. Natl. Acad. Sci. USA 85: 617-
2. Cheng,
J., Fogel-Petrovic, M., and Maquat, L. E. (1990). Translation to near the distal end of the penultimate exon is required for normal levels of spliced triosephosphate isomerase mRNA. Mol. Cell. Biol. 10: 5215-5225.
melanoma
10.
Collins for maintaining the lymphoblast cultures. was supported by the University of Paris V and
Carter, A., Bardin, C., Collins, R., Simons, C., Bray, P., and Spiegel, A. (1987). Reduced expression of multiple forms of the cy subunit of the stimulatory GTP-binding protein in pseudohypoparathyroidism type Ia. Proc. Natl. Acad. Sci. USA 84: 72667269.
invasive
Levine, M. A., Jap, T-J., Mauseth, R. S., Downs, R. W., and Spiegel, A. M. (1986). Activity of the stimulatory guanine nucleotide-binding protein is reduced in erythrocytes from patients withpseudohypoparathyroidismandpseudopseudohypoparathyroidism: Biochemical, endocrine, and genetic analysis of Albright’s hereditary osteodystrophy in six kindreds. J. Clin. Endocrinol. Metab. 62: 497-502.
REFERENCES 1.
and poorly
9.
ACKNOWLEDGMENTS We thank Regina Dr. P. de Mazancourt CNRS.
1321
COMMUNICATIONS
13. 14.
15.
Triggs-Raine, B. L., and Gravel, R. A. (1990). Diagnostic heteroduplexes: Simple detection of carriers of a 4-bp insertion mutation in Tay-Sachs disease. Am. J. Hum. Genet. 46: 183-184. Urlaub, G., Mitchell. P. J., Ciudad, C. J., and Chasin, L. A. (1989). Nonsense mutations in the dihydrofolate reductase gene affect RNA processing. Mol. Cell. Biol. 9: 2868-2880.
16.
Weinstein, L. S., Gejman, P. V., Friedman, E., Kadowaki, T., Collins, R. M., Gershon, E. S., and Spiegel, A. M. (1990). Mutations of the Gs a-subunit gene in Albright hereditary osteodystrophy detected by denaturing gradient electrophoresis. Proc. Natl. Acad. Sci. USA 87: 8287-8290.
17.
Weinstein, L. S., Shenker, A., Gejman, P. V., Friedman, E., and Spiegel, A. M. (1991). Activating the stimulatory G protein in the McCune-Albright Engl. J. Med. 325: 1688-1695.
Merino, M. J., mutations of syndrome. N.
