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The Journal of Clinical Endocrinology & Metabolism 86(10):4630 – 4634
Copyright © 2001 by The Endocrine Society
Analysis of the GNAS1 Gene in Albright’s Hereditary
Osteodystrophy
W. AHRENS, O. HIORT, P. STAEDT, T. KIRSCHNER, C. MARSCHKE,
AND
K. KRUSE
Department of Pediatrics, Medical University of Lübeck, Lübeck, Germany
Albright’s hereditary osteodystrophy (AHO) is characterized
by phenotypic signs that typically include brachydactyly and
sc calcifications occurring with or without hormone resistance toward PTH or other hormones such as thyroid hormone or gonadotropins. Different inactivating mutations of
the gene GNAS1 encoding Gs␣ lead to a reduced Gs␣ protein
activity in patients with AHO and pseudohypoparathyroidism
type Ia or without resistance to PTH (pseudopseudohypoparathyroidism).
We investigated 29 unrelated patients with AHO and
pseudohypoparathyroidism type Ia or pseudopseudohypoparathyroidism and their affected family members performing functional and molecular genetic analysis of Gs␣.
In vitro determination of Gs␣ protein activity in erythrocyte
membranes was followed by the investigation of the whole
coding region of the GNAS1 gene using PCR, nonisotopic
A
LBRIGHT’S HEREDITARY OSTEODYSTROPHY
(AHO) is a genetic disorder characterized by phenotypic signs of brachydactyly, sc calcifications, short stature,
obesity, and mental retardation (1). The disease is often
associated with pseudohypoparathyroidism (PHP) and
other endocrinopathies such as hypothyroidism and hypogonadism. It is caused by a reduced activity of the adenylyl
cyclase stimulating protein Gs␣. These patients with AHO
and PHP (PHP Ia) can be distinguished from patients with
signs of AHO and Gs␣ deficiency but without biochemical
evidence of hormone resistance [pseudopseudohypoparathyroidism (PPHP)]. Both autosomal dominant inherited
disorders can occur in the same family. It has been recognized that in the case of maternal transmission the children
develop PHP Ia; on the other hand, if the mutation is derived from the father, the children develop PPHP. This
parental origin effect has led to the suggestion of Gs␣ imprinting (2, 3). In addition, an AHO-like syndrome associated with a deletion of chromosome 2q37 but normal Gs␣
protein activity is known (4, 5).
The human Gs␣ gene GNAS1 is located at chromosome
20q13 spanning 20 kb. The coding region is divided into 13
exons (6); however, additional exons and alternative splicing
products have been described (7). During the last years about
35 different inactivating mutations in the GNAS1 gene have
been identified in patients with AHO and PHP or PPHP (3,
8 –26). Except for a 4-bp deletion in exon 7 (10, 18, 22, 27, 28),
no mutational hot spot has been described. Due to the clinical
Abbreviations: AHO, Albright’s hereditary osteodystrophy; d, deoxy;
P, patient; PHP, pseudohypoparathyroidism; PPHP, pseudopseudohypoparathyroidism; SSCA, single strand conformation analysis.
single strand conformation analysis, and direct sequencing
of the PCR products.
All patients showed a reduced Gs␣ protein activity (mean
59% compared with healthy controls). In 21/29 (72%) patients,
15 different mutations in GNAS1 including 11 novel mutations
were detected. In addition we add five unrelated patients with a
previously described 4 bp deletion in exon 7 (⌬ GACT, codon 189/
190), confirming the presence of a hot spot for loss of function
mutations in GNAS1. In eight patients, no molecular abnormality
was found in the GNAS1 gene despite a functional defect of Gs␣.
We conclude that biochemical and molecular analysis of
Gs␣ and its gene GNAS1 can be valuable tools to confirm the
diagnosis of AHO. However, in some patients with reduced
activity of Gs␣, the molecular defect cannot be detected in the
exons encoding the common form of Gs␣. (J Clin Endocrinol
Metab 86: 4630 – 4634, 2001)
and genetic heterogeneity, a clear-cut phenotype/genotype
correlation seems not to be possible.
We investigated a cohort of patients with AHO and in
some cases affected family members to verify the diagnosis
employing biochemical analysis of Gs␣ protein activity. In
addition, we aimed to characterize the underlying molecular
defect by analyzing the GNAS1 gene.
Materials and Methods
Patients
Twenty-eight children (15 girls and 13 boys, age between 4 months
and 15 yr) and one female adult (age 31 yr) from unrelated families were
investigated. The patients showed an AHO phenotype with clinical
symptoms as described above including brachydactyly in the case of 16
patients and sc calcifications noted in 9 children. Moreover, for 25 patients the diagnosis of hypothyroidism was made. In addition, hypogonadism was diagnosed in case of the affected adult woman. Depending on the analysis of calcium metabolism, which included the
measurement of serum calcium, phosphate and intact PTH, 26 patients
were classified as PHP Ia. Three young children showed no resistance
to PTH at the time of investigation (Table 1).
Furthermore, we investigated 16 affected family members with AHO
[13 mothers (M), two brothers (B), and one sister (S)]. The siblings and
one mother were classified as PHP Ia, 12 mothers showed no alteration
of calcium metabolism (PPHP).
All patients or parental guardians consented to the determination of
Gs␣ protein activity and molecular genetic analysis of the GNAS1 gene.
Gs␣ protein activity
In heparinized blood samples the activity of Gs␣ protein from erythrocyte membranes of the patients was analyzed in vitro adapted to the
method of Levine et al. (29) as previously described (30). Briefly, after
solubilization and activation of the Gs protein with GTP␥S the generation of cAMP using adenylyl cyclase from turkey red cell membranes
in presence of ATP was measured by RIA (Immuno Biological Labo-
4630
Ahrens et al. • Analysis of GNAS1 Gene
J Clin Endocrinol Metab, October 2001, 86(10):4630 – 4634 4631
ratories, Hamburg, Germany). Results obtained in triplicate were expressed as per cent of the mean of healthy controls. The normal range
was between 85–115%.
quencer (ALF express II, Amersham Pharmacia Biotech, Freiburg, Germany) using Biozym sequencing kit (Biozym, Hessisch Oldendorf, Germany), according to the directions provided by the manufacturer.
Molecular genetic analysis
Results
Genomic DNA was isolated from peripheral leukocytes by standard
procedures. Exons 1–13 of the GNAS1 gene were individually amplified
including intron/exon boundaries (except for exon 1) in 10 fragments by
PCR using the oligonucleotide primers listed in Table 2.
The amplification reactions contained 100 ng of DNA in a final volume of 50 ␮l using 20 pmol of each primer, 200 ␮mol or 2 mmol deoxy
(d)NTP (dATP, dCTP, dGTP, dTTP), 0.5, or 1 U Taq polymerase (Ampli
Taq, Perkin-Elmer Corp., Norwalk, CT), 20 mmol Tris (pH 8.4), 1.0 –2.5
mmol magnesium chloride. An initial denaturation at 94 C for 5 min was
followed by 34 cycles of annealing at 52⫺72 C for 1 min 30 sec, elongation
at 72 C for 2 min, denaturation at 94 C for 1 min 15 sec, and final
elongation at 72 C for 5 min. Amplification products were between 141
and 381 bp long (Table 2). To generate shorter fragments for further
analysis, fragment containing exon 4/5 was digested with the restriction
enzyme HaeII to yield two fragments of 150 bp and 230 bp.
For mutation screening, a nonisotopic single strand conformation
analysis (SSCA) on 5–10% polyacrylamid gels was used as previously
described (31). Electrophoretic band shifts were visualized by silver
staining, and DNA samples with an aberrant migration pattern were
reamplified from genomic DNA and directly sequenced.
Direct sequence analysis of DNA was performed with CY5-labeled
primers in sense and antisense direction analyzed in an automatic se-
All 29 unrelated patients and the 16 investigated relatives
with AHO showed a reduced Gs␣ protein activity compared
with healthy controls (range between 41 and 75%, mean 59%;
Table 1).
For 21 patients and 14 relatives, SSCA analysis revealed
the presence of an aberrant migration pattern in one of the
fragments of GNAS1 leading to direct DNA sequence analysis. In eight families, SSCA molecular genetic abnormalities
were not detected.
Performing DNA sequence analysis
Fifteen different mutations including 11 novel mutations
were found consisting of nine missense, four frameshift, one
splice site mutation and one in-frame deletion (Table 1).
Patient (P) 1 showed an insertion of adenine in codon 51 of
exon 2 of GNAS1. For P 2–5, mutations were revealed in exon
4 of GNAS1: P2 had a cytosine to thymine transversion
TABLE 1. Diagnosis and results of the determination of Gs␣ protein activity and the molecular genetic analysis of GNAS1 of 29 patients
(P) and their relatives [mother (M), sister (S), brother (B)] with AHO
Patient
Diagnosis
P1/M1
P2/M2
P3/M3
P4/M4
P5/M5
P6
P7/M7/S7
P8 –13
Ia/PPHP
Ia/PPHP
Ia/PPHP
Ia/PPHP
Ia/PPHP
Ia
Ia/PPHP/Ia
Ia
M8/11/12
B13
P14
P15–16
P17
P18/M18
P19/M19
P20
P21
P22/M22/B22
P23/M23
P24 –29
PPHP/Ia (M12)
Ia
PPHP
Ia/Ia
Ia
Ia/PPHP
Ia
PPHP
Ia
Ia/PPHP/Ia
Ia/PPHP
Ia (24 –28)
PPHP (29)
Gs␣ protein activity (%)
69/59
67/66
54/55
54/58
57/68
59
64/73/58
44/71/56
56/49/73
60/75/58
60
58
48/49
56
55/61
47/70
68
44
56/48/50
68/48
56/41/59
54/61/56
Exon
Codon
2
4
4
5
5
6
I6/E7
7
51
102
102
115
140
165
9
11
11
11
12
13
13
231
298
299
313
336
357
377
189/190
Nucleotide sequence
Amino acid change
insertion A (GAA TCT3 GAAATC)
GCG3 GTG
GCG3 GAG
CCC3 CTC
insertion T (TTT GAC3 TTTTGA)
CGC3 TGC
splice site mutation (ag C TTC3gg C TTC)
4 bp deletion
(⌬ GACT)
stop codon 53
Ala3 Val
Ala3 Gln
Pro3 Leu
stop codon 141
Arg3 Cys
CGC3 TGC
GCT3 CCT
insertion A (GAG3 GAAG)
CCA3 CTA
CGG3 TGG
CAC3 CTC
3 bp deletion (⌬ AAC)
no mutation
no mutation
no mutation
Arg3 Cys
Ala3 Pro
stop codon 309
Pro3 Leu
Arg3 Trp
His3 Leu
⌬ Asn
stop codon 202
TABLE 2. Oligonucleotide primers used to amplify the 13 exons of GNAS1
Exon
Fragment
size (bp)
Annealing
temperature (C)
Upstream primer
Downstream primer
1
2
3
4/5
6
7/8
9/10
11
12
13
141
176
238
381
217
345
347
212
293
322
70
61
54
54
57
61
72
62
53
52
5⬘-CCG-CCA-TGG-GCT-GCC-TCG-GGA-A-3⬘
5⬘-CAG-ACC-TCC-CTG-CCC-AAA-GTG-3⬘
5⬘-GGC-GCG-CGA-ATT-GTT-GCT-TT-3⬘
5⬘-TAC-TCC-TAA-CTG-ACA-TGG-TG-3⬘
5⬘-CAT-AGG-GAA-CTC-TGG-TCT-CA-3⬘
5⬘-GCG-CTG-TGA-ACA-CCC-CAC-GTG-TCT-3⬘
5⬘-CTC-TGG-AAT-AAC-CAG-CTG-TCC-TC-3⬘
5⬘-CCT-GGC-CGA-AAG-CGC-GCT-TCT-3⬘
5⬘-GGA-GCT-ACA-GAG-ATG-CTA-GC-3⬘
5⬘-TGG-ATT-TGA-GCT-CTT-TGC-GC-3⬘
5⬘-CCC-GCC-CGC-CCT-TAC-CCA-GCA-3⬘
5⬘-TTA-CTT-GGT-GCC-CCC-CTG-CA-3⬘
5⬘-CAG-TTT-CCC-AGT-ATG-ATC-TT-3⬘
5⬘-ATA-TGG-ACA-CTG-TGC-TCA-GG-3⬘
5⬘-GAT-GGG-TTG-GGT-GGC-GGT-TA-3⬘
5⬘-GAA-ACC-ATG-ATC-TCT-GTT-AT-3⬘
5⬘-CGC-AGG-GGG-TGG-GCG-GTC-ACT-CCA-3⬘
5⬘-AGC-CAG-CAA-GAG-TGG-AAG-CC-3⬘
5⬘-AGT-AAA-TTT-ACA-TCC-ATG-AGA-3⬘
5⬘-TAA-TTA-AAT-TTG-GGG-GTT-CC-3⬘
4632
J Clin Endocrinol Metab, October 2001, 86(10):4630 – 4634
mutation in codon 102 resulting in a substitution of alanine
by valine. In the same position of exon 4P3 showed a different
missense mutation leading to the substitution of alanine by
glutamine. For P 4 and 5, mutations in exon 5 were detected
(P 4: Pro3 Leu 115, described before by de Sanctis et al. (23);
P 5: insertion of thymine in codon 140 initiating a stop in
codon 141). P 6 showed a single base substitution from cytosine to thymine in codon 165 of exon 6, which has been
previously described for another kindred by Miric et al. (11),
resulting in an arginine to cysteine change. A splice site
mutation (intron 6/exon 7) occurred in P 7 (Fig. 1). The
previously known 4 bp deletion (10, 18, 22, 27, 28) involving
codons 189 and 190 of exon 7 followed by a frameshift with
a premature stop at codon 202 was observed in P 8 –13. P 14
showed a missense mutation in the GTP-dependent conformational change domain encoded by exon 9 (Arg3 Cys 231).
Three different novel mutations were detected in exon 11
(Ala3 Pro 298 identified in 2 unrelated patients (P 15 and 16);
additional adenine inserted in codon 299 initiating a stop in
codon 309 in P 17; Pro3 Leu 313 in P 18). P 19 had a cytosine
to thymine transversion mutation resulting in a substitution
of arginine to tryptophane in codon 336 of exon 12, identified
for another unrelated patient by de Sanctis et al. (21). Finally,
two different mutations were found in exon 13 (His3 Leu
357 in P 20; 3-bp deletion in codon 377 (Asn) causing an
in-frame mutation in P 21).
The affected family members of the 21 patients showed the
same mutations as their relatives (Table 1).
Discussion
In the present study, we identified 15 different mutations
in the GNAS1 gene (Fig. 2) investigating a collective of 29
patients with AHO from different families and 16 affected
FIG. 1. Family investigation of patient 7. Beside the patient her
mother and one sister show physical features of AHO, the father and
two other siblings are not affected. Determination of Gs␣ protein
activity revealed a reduced activity for all three family members with
AHO. PCR-SSCA showed an aberrant migration pattern compared
with the healthy relatives. Performing direct sequence analysis of
DNA a splice site mutation (intron 6/exon 7) with a single base substitution from adenine to guanine was detected for patient 7 and her
affected family members. Filled symbols, PHP Ia; hatched symbol,
PPHP; open symbols, unaffected.
Ahrens et al. • Analysis of GNAS1 Gene
family members. To our knowledge 11 of these mutations
including six missense, three frameshift, one splice site mutation and one in-frame deletion have not been described up
to now. These results underscore the genetic heterogeneity of
AHO.
The investigated affected family members included 12 unrelated mothers with AHO and PPHP and one mother with
PHP Ia. Interestingly, in every case the children developed
a PHP Ia, if they inherited the mutation from their mother.
This result supports the hypothesis of a genomic imprinting
for this disorder. It is believed that Gs␣ is expressed in certain
tissues only from the maternal allele (32). Therefore, mutations of the active maternal allele lead to a decreased Gs␣
expression and to hormone resistance whereas mutations on
the inactive paternal allele have no effect.
All patients and their affected relatives showed a reduced
Gs␣ protein activity. In eight families GNAS1 mutations
were not detected. We believe in accordance with a previous
study (18) that in these cases the genetic defect may be located outside the coding region rather in the promoter region
of GNAS1 or in other regulatory regions leading to Gs␣
deficiency. In the other 21 investigated patients from different families with novel or known mutations in GNAS1, no
second mutation was identified in the gene. Therefore, we
conclude following Miric et al. (11) that the described mutations are responsible for the reduced Gs␣ activity.
In five unrelated children with AHO and PHP Ia and three
mothers with either PPHP or PHP Ia, the previously described 4-bp deletion in exon 7 was detected. This result was
confirmed by the repetition of the molecular analysis of
GNAS1 of two brothers (P 13, B 13) who had initially been
described by Nakamoto et al. (24) to carry the same deletion.
Except for this hot spot for loss of function mutations in
GNAS1, only three other mutations located in exon 1
(Met3 Val 1; 8, 25), exon 5 (Pro3 Ser 115; 18, 25) and exon
13 (Ala3 Ser 366; 12) have been identified so far in more than
one kindred. This study adds now four different single base
substitution mutations occurring in more than one unrelated
patient. The missense mutation in exon 5 resulting in a proline to leucine change in codon 115 has been described before
by de Sanctis et al. (23). A different missense mutation
(Pro3 Ser 115) has been detected in the same location in exon
5 in unrelated patients by Ahmed et al. (18) and Aldred and
Trembath (25). The substitution of arginine by cysteine in
codon 165 of exon 6 found in one girl with AHO and PHP
Ia has been noticed by Miric et al. (11) for a mother and her
daughter with PHP Ia. Moreover, we report two unrelated
patients with the identical mutation in exon 11 leading to a
change of alanine to proline in codon 298. The substitution
of proline in other proteins is known to be often followed by
the synthesis of unstable proteins (11). The fourth missense
mutation in codon 336 of exon 12 resulting in the substitution
of arginine by tryptophane was found in a patient with AHO
and PHP Ia and his mother with PPHP. It has been identically
described as a de novo mutation in an Italian patient with PHP
Ia (21). We conclude that molecular genetic analysis of a large
number of patients from different kindreds with AHO may
lead to the identification of additional mutational hot spots
for inactivating mutations in GNAS1.
Several of the novel mutations are located in exons known
Ahrens et al. • Analysis of GNAS1 Gene
J Clin Endocrinol Metab, October 2001, 86(10):4630 – 4634 4633
FIG. 2. Schematic representation of GNAS1 with the location of 15 mutations detected in patients with AHO by the present study. Reference
numbers of the mutations described previously are set in parentheses.
to encode for different activity domains of GNAS1. Farfel et
al. (15) and Iiri et al. (26) described a mutation found in codon
231 of exon 9 that substitutes arginine by histidine leading to
a disturbance of the interaction between the switch 2 and 3
regions of Gs␣, which are necessary to stabilize the active
conformation. We discovered in the same location in exon 9 a
different missense mutation resulting in an arginine to cysteine change.
Interestingly, we were able to characterize the molecular
defect in only 21 of 29 patients with reduced Gs␣ activity.
This may be due to technical reasons. SSCA is a widely used
screening technique for the identification of unknown single
nucleotide variations in short DNA fragments. In the approach we took, the analyzed fragments were shorter than
350 bp, in the majority even shorter than 300 bp. It has been
demonstrated that the sensitivity of SSCA to identify a single
nucleotide variation should be more than 80% in fragments
of this size (33). Thus, if all patients with reduced Gs␣ activity
really have a molecular defect in one of the exons investigated, our yield should have been higher to detect these
abnormalities. So it has to be postulated that in some cases
of AHO with reduced Gs␣ activity, the molecular defect may
be outside of the coding region covering exons 1 to 13 of the
GNAS1 gene.
The diagnosis of AHO during childhood is often difficult
because phenotypic signs as brachydactyly, short stature,
and mental retardation and hormone resistance develop over
the first years of life and show a variety in their expression.
Gs␣ protein activity and the molecular genetic analysis of
GNAS1 proved to be important parameters for an early diagnosis of AHO in affected individuals and also make an
investigation of family members possible even before symptoms may occur. Despite the genetic heterogeneity of AHO
a phenotype/genotype correlation may be possible for single
mutations if molecular analysis is performed in a large collective of patients. In addition a follow up of these patients
during childhood is necessary concerning the clinical features until the end of the development of phenotypic signs
of AHO.
Acknowledgments
We are grateful to the Developmental Biology Unit of the Faculty of
Medicine, Rouen, France, especially to E. Mallet, J. P. Basuyau, and M.
Leroy for introducing the determination of Gs␣ protein activity to us.
Furthermore, we thank all corresponding physicians of the investigated
families for their cooperation.
Received January 10, 2001. Accepted July 5, 2001.
Address all correspondence and requests for reprints to: Wiebke
Ahrens, M.D., Klinik für Kinder- und Jugendmedizin, Medizinische
Universität zu Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany.
E-mail: [email protected].
This work was presented in part at the 39th Annual Meeting of the
European Society for Pediatric Endocrinology in Brussels, Belgium, September 2000.
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