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Department of Pediatrics, Medical University of Lübeck, Lübeck, Germany
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: ahrens{at}paedia.ukl.mu-luebeck.de
Abstract
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 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.
ALBRIGHT’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, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 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 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
).
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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 Laboratories,
Hamburg, Germany). Results obtained in triplicate were expressed as per
cent of the mean of healthy controls. The normal range was between
85–115%.
Molecular genetic analysis
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.
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). 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 sequencer (ALF express II, Amersham Pharmacia Biotech, Freiburg, Germany) using Biozym sequencing kit (Biozym, Hessisch Oldendorf, Germany), according to the directions provided by the manufacturer.
Results
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 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:
Pro
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 (ArgCys
231). Three different novel mutations were detected in exon 11
(AlaPro 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; ProLeu 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 (HisLeu 357 in P 20; 3-bp
deletion in codon 377 (Asn) causing an in-frame mutation in P 21).
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). 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 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.
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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 (MetVal 1; 8, 25), exon 5 (ProSer 115; 18, 25)
and exon 13 (AlaSer 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 (ProSer
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 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.
Footnotes
This work was presented in part at the 39th Annual Meeting of the European Society for Pediatric Endocrinology in Brussels, Belgium, September 2000.
Abbreviations: AHO, Albright’s hereditary osteodystrophy; d, deoxy; P, patient; PHP, pseudohypoparathyroidism; PPHP, pseudopseudohypoparathyroidism; SSCA, single strand conformation analysis.
Received January 10, 2001.
Accepted July 5, 2001.
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