This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Lietman, S. A. Articles by Levine, M. A. Search for Related Content PubMed PubMed Citation Articles by Lietman, S. A. Articles by Levine, M. A. Related Collections Pediatric Endocrinology Calcium and Bone Metabolism

Preimplantation Genetic Diagnosis for Severe Albright Hereditary Osteodystrophy

Department of Orthopedic Surgery (S.A.L.), Cleveland Clinic Foundation; Department of Biomedical Engineering (S.A.L., M.A.L.), Cleveland Clinic Lerner Research Institute; Department of Obstetrics and Gynecology (J.G., N.D.), Cleveland Clinic; and Section of Pediatric Endocrinology (M.A.L.), Cleveland Clinic Children’s Hospital, Cleveland, Ohio 44195

Address all correspondence and requests for reprints to: Steven Lietman, M.D., Cleveland Clinic Lerner Research Institute, 9500 Euclid Avenue, Mailstop ND20, Cleveland, Ohio 44195. E-mail: lietmas{at}ccf.org.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context: Preimplantation genetic diagnosis (PGD) enables the selection of embryos without mutations for implantation and has not been described to our knowledge for mutations in GNAS. Phocomelia in a patient with Albright hereditary osteodystrophy (AHO) has also not been previously described.

Objective: The aim of this study was to identify a GNAS mutation in a patient with a severe form of AHO and pseudohypoparathyroidism type 1a with phocomelia and to perform PGD on embryos derived by in vitro fertilization to deliver an unaffected infant.

Design: A proband and his family are described clinically, the GNAS gene was sequenced to identify a novel mutation in the proband, and PGD was performed on embryos.

Setting: The setting was in a tertiary-care hospital.

Patients: The patients were from a single family in which the proband has a severe form of AHO.

Interventions: Interventions were PGD and in vitro fertilization.

Main Outcome Measures: The main outcome measures were the clinical phenotypes and GNAS gene sequences of the proband, embryos, and family members.

Results: After PGD, three genotypically normal embryos were transferred back to the mother. Pregnancy ensued, and a healthy male infant was delivered at 36.5 wk gestation. The GNAS genes in the baby were confirmed as wild-type, and the infant is free of any signs of AHO.

Conclusions: We describe herein a proband with AHO and severe skeletal deformities (including phocomelia) related to a novel GNAS mutation and the delivery of a male infant with homozygous normal GNAS genotype after PGD.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Albright hereditary osteodystrophy (AHO) is characterized by a distinctive constellation of developmental and skeletal defects that include a round face, a short stocky physique, brachydactyly, heterotopic ossification, and mental retardation (1). The molecular basis for AHO is heterozygous mutation of the GNAS gene located at 20q13.2 that leads to loss of expression or function of the -chain of Gs, the heterotrimeric G protein that couples heptahelical receptors to activation of adenylyl cyclase.

AHO patients with GNAS mutations on maternally inherited alleles also manifest resistance to multiple hormones such as PTH, TSH, gonadotropins, GHRH, and glucagon (2, 3, 4, 5), a condition referred to as pseudohypoparathyroidism (PHP) type 1a. By contrast, AHO patients with GNAS mutations on paternally inherited alleles have only the phenotypic features of AHO without hormonal resistance, a condition termed pseudo-pseudohypoparathyroidism (pseudo-PHP) (6). This unusual inheritance pattern was first observed clinically by inspection of published pedigrees and was subsequently ascribed to genomic imprinting of the GNAS gene that leads to preferential expression of G_s from the maternal GNAS allele in some tissues (3, 7) Imprinting of GNAS also accounts for the development of PTH resistance in subjects with PHP type 1b, who lack features of AHO and have normal G_s activity in accessible tissues (8). The coding region of GNAS is normal, but an epigenetic defect switches the maternal GNAS allele to a paternal pattern of methylation (i.e. paternal epigenotype) (9, 10, 11).

Preimplantation genetic diagnosis (PGD) is a powerful adjunct to assisted reproductive technology and offers the advantage over conventional prenatal diagnosis of preselecting unaffected embryos before a pregnancy is established. This report describes, for the first time, the use of PGD to select embryos that do not have GNAS mutations. Our report also describes the first patient with AHO with phocomelia as a severe manifestation of the skeletal disorder.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Clinical description

The proband is a 32-yr-old French male with PHP 1a manifested as PTH resistance (hypocalcemia and hyperphosphatemia), TSH resistance (hypothyroidism), and mild hypogonadism plus severe AHO with short stature, mild mental retardation, and skeletal defects including congenital phocomelia of the left upper extremity. X-ray of the left upper extremity demonstrated absence of the humerus with an intact radius and absence of the proximal three fourths of the ulnar bone. The right hand showed severe shortening of the fourth metacarpal with moderate shortening of the third metacarpal and milder shortening of other metacarpals and of many of the phalanges. There was no history of fetal exposure to thalidomide or other drugs.

When he was seen for reproductive evaluation, the patient was euthyroid and normocalcemic and was taking T₄ and calcitriol. Semen analysis showed a volume of 1.3 ml of normal color and viscosity. Sperm concentration was 2 million/ml with motility after 1 h showing only 10% normal, 10% diminished, and 80% without any motility. There was 70% sperm vitality. Round cells were approximately 1 million/ml. The spermatocytogram showed 48% typical forms and 52% atypical forms with an overall diagnosis of significant oligospermia.

Family history

There is no history of AHO, PHP, or any endocrine defect in the family. His parents, a 35-yr-old brother, and a 37-yr-old sister are normal. Informed consent was obtained from all members of the family for the DNA studies.

Molecular studies

Peripheral blood samples were obtained from the proband, his parents, and siblings. DNA was extracted from leukocytes by standard methods, and exons 1–13, the flanking intron sequences, and the exon 1 promoter region of the human GNAS gene were amplified by PCR as described previously (12) and analyzed by direct nucleotide sequencing.

Genomic DNA was obtained from the infant by firmly scraping the inside of one of his cheeks about 10 times with a sterile cotton swab. The DNA was then extracted and purified using the QIAamp DNA Blood Mini Kit (QIAGEN, Valencia, CA) following the buccal swab spin protocol without modification.

Eight of the nine mature oocytes were successfully fertilized by intracytoplasmic sperm injection. Embryos were highly fragmented, and only six were suitable for blastomere biopsy. For each biopsied cell, a blank control was prepared from the final wash drop. PCR control tubes containing a single lymphoblast from a control cell line and paternal lymphocytes were added to the overall analysis. PCR for the GNAS mutation was performed by nested PCR; the first reaction was performed in a 100-µl volume with K⁺-free buffer, 1 mM dNTPs, 1 µM outer PCR primers (forward 5'-TTCAGCTACCTCCAATCTTTGC-3', reverse 5'-GCACGTTCATCACACTCAGGA-3') and AmpliTaq Gold Taq DNA polymerase. The PCR conditions were 95 C for 5 min; 97 C for 30 sec, 55 C for 30 sec, and 72 C for 55 sec for 10 cycles; 94 C for 30 sec, 55 C for 30 sec, and 72 C for 55 sec for 25 cycles; and 72 C for 5 min, with a 4 C hold. For the second PCR, 4 µl of the PCR product was used as template in a 50-µl final volume that contained 2.5 mM MgCl₂, 0.8 mM dNTPs, 0.8 µM primers (forward 5'-ACAGATCCGAACCCACAACT-3'; reverse 5'-CATTTTCAGACCATTGTGGC-3', to which sequences corresponding to the -21M13For and 28M13Rev were added to the 5'-ends of the forward and reverse PCR primers, respectively, to facilitate subsequent sequencing) and AmpliTaq Gold Taq DNA polymerase. The inner cycling conditions were 95 C for 5 min; 95 C for 30 sec, 53 C for 30 sec,and 72 C for 55 sec for 35 cycles; and 72 C for 5 min, with a 4 C hold. The resulting product was sequenced directly (ABI Prism BigDye Terminator version 3.1 cycle sequencing kit; Applied Biosystems, Foster City, CA).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The proband was found to be heterozygous for a splice site mutation in intron 4, IVS4 + 5GA mutation, that was present on the maternally inherited GNAS allele (Fig. 1). The IVS4 + 5S sequence change likely represents a pathogenic de novo mutation for several reasons: 1) it was present only in the single affected proband in the family and was not present in either of his parents or his two unaffected siblings, 2) it was not found in 50 randomly selected control subjects, and 3) it involved the consensus splice donor site. Splice site mutations are a common inactivating event for several human genes and are estimated to account for up to 15% of all point mutations causing human genetic disease (13). In the GNAS gene, they represent about 10% of the identified mutations. Most frequent targets of splice site mutations are the consensus GT and AG donor and acceptor dinucleotides, respectively. To assess the potential pathogenicity of the mutation IVS4 + 5A, we performed splice site analysis (https://splice.cmh.edu/index.html); the genomic sequence was examined using software that scans genomic sequences with weight matrices for sites with positive R_i (individual information content) values, and the results were displayed using the Sequence Walker program (14). The IVS4 + 5A nucleotide substitution causes an 11.5-fold reduction in the strength of the natural donor site. This change is predicted to result in aberrant splicing with removal of exon 4. Immunoblot analysis showed an approximately 50% reduction in the level of erythrocyte G_s protein compared with normal control (data not shown), suggesting that the mutant allele did not produce normal G_s protein.

View larger version (28K): [in this window] [in a new window]   FIG. 1. Electrochromatograms demonstrating the heterozygous GA GNAS mutation in the proband (CnT) (above left) and the abnormal embryo (above right) and the wild-type alleles in the normal embryo (mother, CGT) (below left) and baby (below right).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
PGD can be used to identify genetic defects in embryos created through in vitro fertilization before transferring them into the uterus. Because only unaffected embryos are transferred to the uterus for implantation, PGD provides an attractive alternative to current postconception diagnostic procedures. PGD has been successfully applied to the diagnosis of about 200 diseases and disorders that are due to single gene defects. In this particular family, an assay was developed specifically to follow the AHO mutation. The location of the heterozygous GNAS donor splice site mutation (IVS4 + 5GA) in the proband is the most common nucleotide change (GA) in the second most common location (the fifth intron nucleotide, +5) for donor splice site mutations in human disease genes (15).

The development of similar skeletal defects in patients with PHP type 1a and pseudo-PHP (who are heterozygous for inactivating GNAS mutations) is consistent with growing evidence that G_s pathways are important regulators of growth plate development and that G_s is biallelically expressed in human bone (16) and in murine chondrocytes (17). Although inheritance of a defective paternal GNAS allele is not predicted to cause endocrine defects, a 50% deficiency in G_s expression is sufficient to cause brachydactyly and other skeletal defects. Although phocomelia has not previously been reported in patients with AHO, several lines of evidence support the premise that this skeletal defect is also a result of the GNAS mutation. First, the patient had no other features to suggest a second genetic disorder such as Fuhrmann syndrome or Al-Awadi/Raas-Rothschild/Schinzel phocomelia syndromes (18). Second, sporadic unilateral upper limb phocomelia is very rare, occurring in fewer than five in 4 million individuals (19). Thus, the coincidental occurrence of both AHO and unilateral phocomelia seems highly unlikely. Third, a wide variety of reduced limb segments or elements (phocomelia, ectromelia, ectrodactyly, and brachydactyly) result from a common molecular mechanism in which expression of Shh (sonic hedgehog) is reduced, leading to down-regulation of Ihh (Indian hedgehog). Similarly, the process of chondrocyte proliferation and differentiation within the growth plate is regulated by an intricate interplay of Ihh and PTHrP (20). PTHrP acts predominantly through the G_s/cAMP pathway in chondrocytes, and G_s signaling plays the critical role in regulating chondrocyte proliferation and differentiation. Thus, loss of G_s leads to PTHrP resistance with premature differentiation of proliferating chondrocytes into hypertrophic chondrocytes, early closure of the growth plate, and limb reduction defects.

In conclusion, we have described a novel mutation leading to a severe form of AHO in the proband. Furthermore, we described the first successful application of PGD for AHO caused by the same mutation in the GNAS gene, with the subsequent delivery of a homozygous normal baby. In this case, we believe that the risk of severe skeletal dysplasia in a child with a GNAS mutation was averted by selection of a normal embryo. Our results demonstrate that PGD for the detection of GNAS mutations is a powerful diagnostic tool for an affected couple who desire a healthy child.

	Footnotes

 
This work was supported in part by U.S. Public Health Service Research Grants K08-AR47661 (S.A.L.) and DK34281 and DK56178 (M.A.L.) and General Clinical Research Center Grant RR0035. S.A.L. is the recipient of and this work is supported by a Career Development Award from the Orthopaedic Research and Education Foundation.

Disclosure Statement: The authors have nothing to disclose.

First Published Online December 18, 2007

Abbreviations: AHO, Albright hereditary osteodystrophy; PHP, pseudohypoparathyroidism; PGD, preimplantation genetic diagnosis.

Received September 11, 2007.

Accepted December 10, 2007.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

1. Mann JB, Alterman S, Hills AG 1962 Albright’s hereditary osteodystrophy comprising pseudohypoparathyroidism and pseudo-pseudohypoparathyroidism. With a report of two cases representing the complete syndrome occurring in successive generations. Ann Intern Med 56:315–342[Abstract/Free Full Text]
2. Juppner H, Bastepe M 2006 Different mutations within or upstream of the GNAS locus cause distinct forms of pseudohypoparathyroidism. J Pediatr Endocrinol Metab 19(Suppl 2):641–646
3. Germain-Lee EL, Ding CL, Deng Z, Crane JL, Saji M, Ringel MD, Levine MA 2002 Paternal imprinting of G(s) in the human thyroid as the basis of TSH resistance in pseudohypoparathyroidism type 1a. Biochem Biophys Res Commun 296:67–72[CrossRef][Medline]
4. Weinstein LS, Liu J, Sakamoto A, Xie T, Chen M 2004 GNAS: normal and abnormal functions. Endocrinology 145:5459–5464[CrossRef][Medline]
5. Weinstein LS, Shenker A, Gejman PV, Merino MJ, Friedman E, Spiegel AM 1991 Activating mutations of the stimulatory G protein in the McCune-Albright syndrome. N Engl J Med 325:1688–1695[Abstract]
6. Albright BA, Hampton AO, Smith P 1937 Syndrome characterized by osteitis fibrosa disseminata, areas of pigmentation and endocrine dysfunction, with precocious puberty in females. N Engl J Med 216:727–741
7. Hayward BE, Barlier A, Korbonits M, Grossman AB, Jacquet P, Enjalbert A, Bonthron DT 2001 Imprinting of the G(s) gene GNAS1 in the pathogenesis of acromegaly. J Clin Invest 107:R31–R36
8. Kappy MS, Allen DB, Geffner ME 2005 Principles and practice of pediatric endocrinology. Springfield, IL: Charles C. Thomas
9. Linglart A, Gensure RC, Olney RC, Juppner H, Bastepe M 2005 A novel STX16 deletion in autosomal dominant pseudohypoparathyroidism type Ib redefines the boundaries of a cis-acting imprinting control element of GNAS. Am J Hum Genet 76:804–814[CrossRef][Medline]
10. Bastepe M, Frohlich LF, Linglart A, Abu-Zahra HS, Tojo K, Ward LM, Juppner H 2005 Deletion of the NESP55 differentially methylated region causes loss of maternal GNAS imprints and pseudohypoparathyroidism type Ib. Nat Genet 37:25–27[CrossRef][Medline]
11. Juppner H, Linglart A, Frohlich LF, Bastepe M 2006 Autosomal-dominant pseudohypoparathyroidism type Ib is caused by different microdeletions within or upstream of the GNAS locus. Ann NY Acad Sci 1068:250–255[CrossRef][Medline]
12. Miric A, Vechio JD, Levine MA 1993 Heterogeneous mutations in the gene encoding the -subunit of the stimulatory G protein of adenylyl cyclase in Albright hereditary osteodystrophy. J Clin Endocrinol Metab 76:1560–1568[Abstract]
13. Krawczak M, Reiss J, Cooper DN 1992 The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences. Hum Genet 90:41–54[Medline]
14. Nalla VK, Rogan PK 2005 Automated splicing mutation analysis by information theory. Hum Mutat 25:334–342[CrossRef][Medline]
15. Buratti E, Chivers M, Kralovicova J, Romano M, Baralle M, Krainer AR, Vorechovsky I 2007 Aberrant 5' splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization. Nucleic Acids Res 35:4250–4263[Abstract/Free Full Text]
16. Mantovani G, Bondioni S, Lania AG, Corbetta S, de Sanctis L, Cappa M, Di Battista E, Chanson P, Beck-Peccoz P, Spada A 2004 Parental origin of Gs mutations in the McCune-Albright syndrome and in isolated endocrine tumors. J Clin Endocrinol Metab 89:3007–3009[Abstract/Free Full Text]
17. Bastepe M, Juppner H 2005 GNAS locus and pseudohypoparathyroidism. Horm Res 63:65–74[CrossRef][Medline]
18. Kumar D, Duggan MB, Mueller RF, Karbani G 1997 Familial aplasia/hypoplasia of pelvis, femur, fibula, and ulna with abnormal digits in an inbred Pakistani Muslim family: a possible new autosomal recessive disorder with overlapping manifestations of the syndromes of Fuhrmann, Al-Awadi, and Raas-Rothschild. Am J Med Genet 70:107–113[CrossRef][Medline]
19. Goldfarb CA, Manske PR, Busa R, Mills J, Carter P, Ezaki M 2005 Upper-extremity phocomelia reexamined: a longitudinal dysplasia. J Bone Joint Surg 87:2639–2648[Abstract/Free Full Text]
20. Kobayashi K, Takahashi N, Jimi E, Udagawa N, Takami M, Kotake S, Nakagawa N, Kinosaki M, Yamaguchi K, Shima N, Yasuda H, Morinaga T, Higashio K, Martin TJ, Suda T 2000 Tumor necrosis factor stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction. J Exp Med 191:275–286[Abstract/Free Full Text]

This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Lietman, S. A. Articles by Levine, M. A. Search for Related Content PubMed PubMed Citation Articles by Lietman, S. A. Articles by Levine, M. A. Related Collections Pediatric Endocrinology Calcium and Bone Metabolism