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Original Studies |
Ospedale Maggiore IRCCS, Institute of Endocrine Sciences (G.M., R.R., P.B.-P., A.S.), and Pediatric Department, Endocrine Unit, Scientific Ospedale S. Raffaele (G.W., S.B.), University of Milan, and Pediatric Department, Ospedale Buzzi (V.B.), 20122 Milan; and First Divisione Medica, Ospedale Regionale Veneto (E.D.M.), 31100 Treviso, Italy
Address all correspondence and requests for reprints to: Dr. Anna Spada, Istituto di Scienze Endocrine, Padiglione Granelli, Ospedale Maggiore IRCCS, Via Francesco Sforza 35, 20122 Milan, Italy. E-mail: endosci{at}mailserver.unimi.it
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Abstract |
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gene (GNAS1). The present study reports clinical,
biochemical, and molecular data of 8 unrelated families with PHP Ia and
PPHP. The 13 exons of GNAS1 were screened for mutations by PCR and
direct sequencing of the amplified products. We detected heterozygous
mutations in the affected members of the 4 families in which PHP Ia was
present. In 2 families 2 previously reported deletions in exons 5 and 7
were found, whereas in the other 2 families, 2 novel frameshift
deletions were identified in exons 1 and 11, causing a premature stop
codon in the mutant allele. No mutation was detected in the families in
which PPHP was the only clinical manifestation. In conclusion, we report the first mutational analysis of Italian patients with PHP Ia and PPHP, and we describe two novel deletions in GNAS1. Furthermore, we confirm that these mutations cannot be detected in families with isolated PPHP, suggesting that these forms of AHO are genetically distinct from PHP Ia.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-subunit (Gs)
messenger ribonucleic acid (mRNA) and protein. The human
Gs gene (GNAS1) contains 13 exons encoding the
Gs and is located at 20q13.11. Heterozygous
loss of function mutations in GNAS1 have been identified in the
majority of patients with PHP Ia and in their relatives affected with
PPHP (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19), whereas there is a general agreement that
PHP Ib is not associated with this defect. GNAS1 mutations have been
localized in the entire coding sequence of the gene; only a mutational
hot spot has been identified to date, and it maps within exon 7
(7, 19, 20, 21), involving 35% of all mutations described.
However, not all patients with PHP Ia and PPHP were found to carry
detectable GNAS1 mutations (19, 22). Many features of AHO are quite nonspecific or are present in other disorders, some of which are ascribed to specific chromosomal defects, such as the small terminal deletions on chromosome 2 in the AHO-like syndrome (23, 24). This makes the diagnosis of PPHP in families in whom PHP Ia is not present particularly difficult. Therefore, it has been proposed that the coexistence of multiple AHO manifestations probably increases the likelihood of a correct diagnosis of PPHP (22).
Here we report clinical, biochemical, and molecular analysis of eight unrelated families affected with AHO features associated or not with PHP Ia. In the four families in whom PHP Ia was present, heterozygous GNAS1 mutations were detected; in two of them we identified two novel frameshift deletions. No mutations were found in any of the families in whom PPHP was the only clinical manifestation.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The study includes the families of eight probands: four affected
with PHP Ia, three with PPHP, and one in whom the diagnosis of PHP Ia
vs. PPHP is still uncertain. In PHP Ia patients the
diagnosis was based upon the occurrence of PTH resistance
(e.g. hypocalcemia, hyperphosphatemia, and raised serum PTH
levels) together with AHO manifestations. In all PHP Ia patients
hypothyroidism due to resistance to TSH (documented by raised serum TSH
levels with an exaggerated response to TRH in the absence of
antithyroid antibodies and in the presence of a normal thyroid scan)
and requiring thyroid hormone replacement therapy was present.
Hypogonadism was present in only 1 PHP Ia patient (A II 1), but it
should be considered that two of six PHP Ia patients were of prepuberal
age. All PPHP patients had several AHO features in association,
including short stature, obesity, round face, brachydactyly, and sc
calcifications without evidence of PTH resistance (Table 1
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) the diagnosis of PHP Ia vs.
PPHP is still uncertain; she was first referred to our hospital at the
age of 11 yr for growth retardation. At that time she presented short
stature, round face, obesity, brachydactyly, sc calcifications, and
only partial PTH resistance (high PTH levels with normal calcium and
phosphate levels) with normal thyroid and gonadal function. The
Ellsworth-Howard test, which would have been helpful in confirming the
diagnosis, was not performed because of the unavailability of the human
PTH-(1–34) synthetic peptide analog. Considering the families of PHP
Ia probands (families A–D), PHP Ia was the only clinical manifestation
in two families (A and B); in one (C) both PHP Ia and PPHP were
present; and one case (D) was sporadic. Probands with PPHP belonged to
families in whom familial (F) or sporadic (G and H) PPHP was the only
clinical manifestation. In proband F III 1, who has inherited the
disease from the mother, the diagnosis of PHP Ia cannot be ruled out,
as she may manifest the hormonal resistance at a later age; this seems
unlikely, however, as resistance, even in its mild forms, usually
manifests within the first decade of life. Clinical and biochemical
details for patients with PHP Ia and PPHP and their relatives are given
in Table 1. Informed consent was obtained from all subjects involved in
the study. The study was approved by the local ethical committee. Methods
Genomic DNA was extracted by the phenol-chloroform method from
peripheral blood leukocytes (Nucleon, Amersham Pharmacia Biotech, Aylesbury, UK). The Gs gene
(exons 1–13) (25) was then amplified by the PCR using the
specific primers pairs shown in Table 2.
Amplification of exons 2–12 included each bordering intron region,
whereas for exon 1, because of the abundance of guanine and cytosine in
the bordering regions, a DNA fragment from 20 bp downstream of the
initiation codon to the donor site of intron 1 was amplified
(22). A 50-µL reaction mixture [0.5–1 µg DNA sample,
50 nmol/L KCl, 50 nmol/L Tris-KCl (pH 8.3), 1–2 nmol/L
MgCl2, 40 pmol of each primer, and 2.5 U
Taq DNA polymerase-AmpliTaq (Perkin-Elmer Corp., Foster City, CA)] was subjected to denaturation at 94 C
for 3 min, followed by 34 cycles of 94 C for 45 s, at specific
annealing temperature (Table 2) for 45 s, and 72 C for 45 s.
A final cycle at 72 C for 10 min was carried out to allow complete
extension of the amplified fragments. The amplified products were then
visualized on a 3% agarose gel stained with ethidium bromide. Both
strands of each exon were finally sequenced for each patient;
sequencing of the PCR products using both sense and antisense primers
was performed using the AmpliTaq BigDye Terminator kit and 310 Genetic
Analyzer (Perkin-Elmer Corp., PE Applied Biosystems). Whenever a mutation was detected, it was confirmed
by performing a new genomic DNA extraction and subsequent sequencing
analysis.
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RNA transcripts were
evaluated by semiquantitative RT-PCR using an appropriately selected
primer pair (5'-AGCACCATTGTGAAGCAGATG-3' and
5'-TGCTTGTTGAGGAAACAGGAT-3', spanning exons 2–11, with an annealing
temperature of 58 C) and compared with those from three normal
subjects; the hypoxanthine-guanine phosphoribosyltransferase gene was
used as an internal standard. For each complementary DNA, preliminary
experiments were conducted to determine the PCR cycles corresponding to
the exponential phase, as previously described (26). PCR
products were visualized on a 2% Nusieve-1% agarose gel, and the
bands were evaluated by an imaging densitometer (GS-700, Bio-Rad Laboratories, Inc. Richmond, CA). Standard chromosomal analysis on peripheral lymphocytes from patient H II 1 was performed.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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). The same deletion
was found in the patient’s mother, who presented the same clinical and
biochemical features. No other family member was available for study.
The other novel mutation was a heterozygous 1-bp deletion within exon 1
(codon 38), which results in a premature stop codon in position 57
(Fig. 2). This mutation was found in
patient C II 1, who was affected with PPH Ia, and in the patient’s
mother, who was affected with PPHP. No mutation was detected in the
unaffected father and brother.
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No mutations could be found in any of the PPHP patients without
relatives with PHP Ia (families F, G, and H) or in the patient with
uncertain diagnosis (E II 1). It is worth noting that all of these
patients showed the typical constellation of physical features,
including short stature, obesity, round face, brachydactyly, and sc
calcifications, that were superimposable to those observed in PPHP
patients with GNAS1 mutations (Table 1). In the familial case (F),
these features were present in the proband, her mother, her maternal
aunt, and her grandfather (Table 1). Semiquantitative RT-PCR from
peripheral lymphocytes of patient H II 1 showed levels of
Gs RNA comparable to those found in controls
(OD target/standard ratio, 0.63 ± 0.09 in controls vs.
0.70 in the patient).
In patient H II 1 chromosomal analysis on peripheral lymphocytes was also performed. The chromosomal study revealed a normal female karyotype (46,XX), with no visible deletions on the long arm of chromosome 2.
A T
C transition in exon 5 within the codon corresponding to
isoleucine at position 131 was detected in four of eight patients (C II
1, D II 1, F III 1, and G II 1), thus confirming the high prevalence of
this polymorphism (47%) in the population (8).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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By direct DNA sequencing we identified two novel frameshift mutations
within exons 1 and 11, thus expanding the spectrum of GNAS1 mutations
associated with PHP1 and PPHP. The mutation in exon 1 (
R38) was a
1-bp deletion affecting codon 38 and resulting in a premature stop at
codon 57, thus determining a truncated protein in the guanosine
triphosphatase domain. This heterozygous alteration is predicted to be
responsible for a reduced Gs activity as
demonstrated for the other previously reported deletions in exon 1
(5, 18). This novel mutation was associated with PHP Ia in
the proband and with PPHP in the mother, providing further evidence
that the same genetic defect may lead to two distinct presentations in
PHP families. A normal GNAS1 sequence was detected in the father and
brother, who were both unaffected. As frequently observed in PHP Ia
patients, the two affected subjects showed resistance to other hormones
acting through Gs-coupled receptors, in
particular TSH. This mutation is the third mutation affecting exon 1,
confirming that this exon is subject to undergoing genetic alterations
(5, 18) and should therefore be included in the analysis
of GNAS1 mutations.
The second novel mutation was a 2-bp deletion identified in exon 11,
where no mutations have been reported to date. This frameshift
(V287) encodes a premature stop 11 codons downstream from the
deletion and results in a severe alteration of the C-terminal region,
lacking the G protein-coupled receptor interaction domain (encoded by
exons 12 and 13) (25, 27). This mutant protein is thus
likely to be functionally inactive. This mutation was detected in the
proband and her mother, who were both affected by PHP Ia; the other
family members were not available for biochemical and molecular
analysis.
The pattern of transmission of these two novel heterozygous mutations is consistent with the general model proposed for AHO. Indeed, in our series all familial PHP Ia cases inherited the disease from the mother, in agreement with a possible role of paternal imprinting in the genesis of AHO. In fact, it has been proposed that when the mutated paternal allele is inherited, offspring will show a PPHP phenotype, whereas when the mutated maternal allele is inherited, clinical and biochemical characteristics of PHP Ia will be present (28, 29).
Of the two previously reported mutations, one was a heterozygous
deletion in exon 5 affecting proline 116 (P116) and introducing a
stop codon 16 amino acids downstream. Also, this mutation has been
shown to prevent the generation of a normal full-length
Gs protein, resulting in a partial deficiency
(50%) of Gs activity (14). This
alteration, which is close to another known substitution
(Pro115Ser) (19), is predicted to
disrupt the highly conserved domain of Gs that
interacts with adenylate cyclase (27). Thus, it seems that
the particular region located in exon 5 and involving prolines 115 and
116 is subject to undergoing mutational changes with a significant
frequency, and it could represent a new mutational hot spot in GNAS1.
The last mutation described in this study (patient D II 1) is the well
known deletion in exon 7 that in this family represented a de
novo mutation, as it was detected in neither the mother nor the
father. To date, this 4-bp deletion in exon 7 represents the only
defined deletional hot spot in GNAS1, involving a defined consensus
sequence for the arrest of DNA polymerase (30).
In the other families included in our analysis, in whom sporadic or
familial PPHP was the only clinical manifestation, no alterations in
the GNAS1-coding sequence were found, in agreement with previous
reports (19, 22). Our results support the view that PHP Ia
and isolated PPHP may represent two genetically distinct entities.
Admittedly, the possibility that a defect might exist in the promoter
region or in other regulatory intronic sequences of GNAS1 cannot be
completely excluded. However, the finding of normal levels of
Gs mRNA in patient H II 1 does not support
this hypothesis; semiquantitative RT-PCR was, in fact, performed using
a primer pair that spanned exons 2–11, thus including almost the
entire Gs mRNA; functional regulatory regions
are supposed to be necessary for the transcription of such product.
Furthermore, it should be considered that isolated PPHP is often difficult to distinguish from other heterogeneous disorders, such as the AHO-like syndrome or other skeletal defects, for which other chromosomal locations have been identified (23, 24, 31). However, in the PPHP patient (H II 1) in whom it was possible to perform chromosomal analysis on peripheral lymphocytes, no visible deletions on the long arm of chromosome 2, which have been reported to occur in a subset of patients with AHO-like disorders (23, 24), were present. Moreover, despite the difficulties in correctly defining subjects with AHO, the patients included in our study were diagnosed with PPHP on the basis of strict criteria, i.e. coexistence of specific and aspecific features, including short stature, obesity, round face, brachydactyly, and sc calcifications, all actually indistinguishable from those present in patients with PHP Ia and PPHP carrying GNAS1 mutations.
In conclusion, we report here the first mutational analysis of Italian patients with PHP Ia and PPHP. By analyzing the entire GNAS1-coding sequence we detected heterozygous mutations in the totality of patients affected with PHP Ia and their relatives with PPHP and identified two novel frameshift deletions affecting exons 1 and 11. No genetic alterations were found in families with PPHP as the only clinical manifestation. Further studies will be necessary to characterize the molecular defects responsible for PPHP and to understand the relationship between this disorder and PHP Ia.
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Footnotes |
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Received April 19, 2000.
Revised July 20, 2000.
Accepted July 27, 2000.
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References |
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J. Liu, B. Erlichman, and L. S. Weinstein The Stimulatory G Protein {alpha}-Subunit Gs{alpha} Is Imprinted in Human Thyroid Glands: Implications for Thyroid Function in Pseudohypoparathyroidism Types 1A and 1B J. Clin. Endocrinol. Metab., September 1, 2003; 88(9): 4336 - 4341. [Abstract] [Full Text] [PDF]
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