Madelung-Like Deformity in Pseudohypoparathyroidism Type 1b

Abstract

Context:

Pseudohypoparathyroidism (PHP) types 1a and 1b are distinguished by clinical, biochemical, and molecular features. We report extended kindred with PHP 1b in which many affected members also had growth plate defects, including brachydactyly and a Madelung-like deformity.

Design:

Analyses included clinical examination, assessment of mineral metabolism, thyroid function, skeletal radiography, and analysis of the GNAS and STX16 genes.

Setting:

Patients were studied in an academic medical center.

Results:

We studied 37 members of a family in which PHP 1b occurred in 23 individuals. Ten of 17 affected patients who were examined had brachydactyly E, including two subjects with Madelung-like defects. Five of 16 subjects had subclinical hypothyroidism; no subject showed sc ossification or short stature. None of the unaffected members had brachydactyly or an elevated serum level of PTH or TSH. Levels of immunoactive erythrocyte Gα_s were normal in two affected subjects tested. Linkage analysis indicated linkage between PTH resistance and the GNAS gene locus; however, no mutations were identified in GNAS exons 1–13. Methylation analysis of genomic DNA from affected subjects showed loss of maternal epigenotype in exon 1A with normal methylation of the differentially methylated regions for XLGαs and NESP55, and PCR demonstrated heterozygosity for a 3.0-kb deletion in the STX16 gene.

Conclusion:

The segregation of brachydactyly with PHP 1b in this family indicates that an imprinting defect in GNAS can lead to growth plate defects, including brachydactyly and Madelung deformity. These features suggest that GNAS signaling plays a more extensive role in chondrocyte maturation than previously thought.

Pseudohypoparathyroidism (PHP) is a condition in which biochemical hypoparathyroidism results from decreased responsiveness of target tissues to PTH rather than PTH deficiency. In PHP type 1a, subjects have short stature; round facies; brachydactyly; mild mental retardation; and sc ossification, a clinical constellation termed Albright hereditary osteodystrophy (AHO). PHP type 1a results from heterozygous mutations on the maternal allele of the imprinted GNAS gene (20q13.2-q13.3) that reduce expression or function of the Gα_s protein (1). By contrast, subjects with GNAS mutations on paternally inherited alleles have many but not all of the features of AHO (2) and lack hormonal resistance, a condition termed pseudopseudohypoparathyroidism. The tissue- and patient-specific expression of hormone resistance in patients with AHO has been attributed to tissue-specific genomic imprinting of GNAS, a highly complex genetic locus. GNAS derives considerable genetic plasticity by use of four alternative first exons, NESP55, XLαs, exon 1A, and exon 1, which splice onto exons 2–13 of GNAS. Transcripts starting with exon 1 encode Gα_s and are expressed from both the maternal and paternal alleles in most tissues, and loss of one functional GNAS allele (i.e. haploinsufficency) does not cause hormone resistance in these tissues because 50% of normal Gα_s activity is sufficient to ensure normal transmembrane signal transduction. By contrast, in some tissues (i.e. imprinted tissues) transcription occurs only from the maternal allele. Mutations that reduce expression or function of the maternal GNAS allele lead to a state in which little if any Gα_s protein is produced, whereas mutations in a paternal allele have no effect.

Epigenetic defects that reduce expression of Gα_s from the maternal GNAS allele account for the development of PTH resistance in patients with PHP type 1b (3). Subjects with PHP type 1b typically lack features of AHO and show normal expression of Gα_s in tissues in which transcription of Gα_s is biallelic (1). In most familial cases, the epigenetic defect results from microdeletions in the STX16 gene located approximately 220 kb centromeric of GNAS exon 1A (3) or deletions removing differentially methylated regions (DMR) of GNAS exon NESP55 or the antisense transcript (4, 5).

Recent studies have revealed that some patients with sporadic PHP type 1b may have mild features of AHO, including sc ossifications and brachydactyly (6–9). We evaluated an extended, multigenerational African-American family in which many members had PHP type 1b. Many affected subjects had brachydactyly, and two subjects had Madelung-like deformities. Our findings provide further evidence for the role of GNAS signaling in regulating growth plate physiology.

Subjects and Methods

Population

We studied an African-American kindred in which PHP type 1 occurred in four generations (Fig. 1). We reviewed the records of 37 family members, including 23 affected members (17 females and six males) and 14 unaffected subjects (10 females and four males). Height was measured using a stadiometer, and growth charts from the Centers for Disease Control and Prevention were used to determine height Z scores. A subject was considered affected if he or she had an abnormally elevated serum level of intact PTH. We evaluated all the family members, but due to sampling problems, it was not possible to perform all studies on all subjects. The protocol was approved by the appropriate institutional review boards.

Fig. 1.

Pedigree of family. Legend describes the relevant phenotypic features of male (square) and female (circle) subjects. /, Deceased individual. The numbers beneath each symbol indicate the pedigree number (see Supplemental Table 1, published on The Endocrine Society's Journals Online web site at http://jcem.endojournals.org).

Linkage analysis

Linkage was determined using previously described single-nucleotide polymorphisms in the PTH gene (10) and the gene encoding the PTH/PTHrP receptor (PTHR1) (10) and using microsatellites and single-nucleotide polymorphisms in GNAS (11).

Molecular analysis of the GNAS gene

The GNAS gene was analyzed by Sanger sequencing and by restriction endonuclease digestion as previously described (2, 11). We also analyzed bisulfite-modified genomic DNA after amplification by nested PCR using primers specific for DMR located in exon 1A, XLαs, and NESP55 (11). The second round of PCR employed primers that were designed with 5′ GC tags that enhanced the GC content of PCR products and facilitated successful direct-cycle sequencing (12). All primer sequences are available upon request.

Analysis of the STX16 gene

We performed PCR in a 100-μl reaction volume containing 500 ng of genomic DNA, oligonucleotide primers that annealed to sequences within exon 2 (5′-CTGATGACCGTATGGCACTG-3′) and exon 7 (5′-GCTCTGGAGTTCACCCTCAA-3′) of the STX16 gene, and TaKaRa LA Taq DNA polymerase (Takara Bio Inc., Japan), with the following cycling conditions: 94 C for 1 min followed by 30 cycles of 98 C for 5 sec, 68 C for 15 min, and 72 C for 10 min. PCR products were analyzed by PAGE.

Quantification of Gα_s protein in erythrocyte cell membranes

Semiquantitative immunoblot analysis of erythrocyte Gα_s protein was performed (11) using membranes from two affected family members (1000 and 1005) and two normal subjects. A pool of erythrocyte membranes from 10 normal subjects was used to generate a dose-response curve (curve not shown). Quantification of the bands was performed by phosphor imager analysis.

Results

Clinical and endocrinological evaluation

Twenty-three patients had biochemical evidence of PTH resistance, but none had short stature, round facies, obesity, or sc ossifications. Ten of 17 patients with PHP 1b who were available for analysis had brachydactyly E, often limited to the fourth metacarpals or metatarsals; no subject had brachydactyly of the first digit (i.e. brachydactyly D). Brachydactyly was not present in any unaffected subjects. Two subjects (V-7 and V1–7) had Madelung-like deformities that affected both the distal ulna and radius (Fig. 2).

Fig. 2.

Hand radiographs. A, Radiograph of subject VI-2 shows shortening of distal phalanx of digit V and marked chondrodysostosis consistent with Madelung-type deformity (arrows). B, Radiograph of subject V-7 shows marked chondrodysostosis consistent with Madelung-type deformity (lower arrows); shortening of metacarpal V and shortening of second middle phalanx of the index finger (arrow) consistent with brachydactyly E. C, Radiograph of subject V5 shows no evidence of brachydactyly.

Biochemical and molecular studies

Linkage analysis indicated discordance between PHP type 1b and specific alleles for genes encoding PTH and the PTH/PTHrP receptor (data not shown) but showed strong linkage (limit of detection = 4.17 at θ = 0.00, peak D205100) between the GNAS locus and PHP type 1. Analysis of the coding regions and flanking intronic sequences of the GNAS gene using genomic DNA from two affected subjects revealed no mutations, and levels of immunoactive Gα_s protein in erythrocytes membranes were normal (93 and 112% of normal pool values, respectively). Restriction endonuclease analysis of genomic DNA from affected family members indicated an abnormal methylation pattern for the exon 1A DMR region (data not shown) that in turn indicated that the NgoMIV sites on both alleles were unmethylated. These results are consistent with a loss of methylation of the exon 1A region on the maternal allele. Comprehensive analysis of bisulfite-treated DNA from four affected subjects (V-14, V-13, III-3, and VI-2) showed complete conversion of all C's to T's within the amplified region of the exon 1A DMR, indicating that both GNAS alleles were unmethylated. By contrast, the bisulfite-treated DNA sequences of the NESP55 and XLαs DMR were similar to those of normal control DNA, indicating that the methylation defect was limited to exon 1a (data not shown). Similar methylation analyses in two unaffected subjects (III-3 and V-20) showed normal methylation patterns at all DMR.

PCR analysis of STX16

All 14 affected PHP type 1b subjects who were tested were heterozygous for the 3-kb deletion in STX16, thus confirming the basis for PHP 1b in this family. One unaffected subject, III-3, who had normal serum calcium and PTH levels and normal methylation patterns for GNAS also carried the microdeletion but was unavailable for determination of brachydactyly. This individual transmitted the STX16 mutation to her affected son, IV-13, and likely inherited the mutation from her father, II-1, an obligate carrier (Fig. 1).

Discussion

We evaluated an extensive multigenerational family in which many members had PHP type 1b (Fig. 1). Although we were unable to perform molecular and biochemical studies on all patients, we believe that affected members of this family had PHP type 1b based on the following findings: 1) normal sequence of the GNAS gene; 2) normal levels of immunoactive erythrocyte Gα_s; and 3) abnormal methylation patterns in GNAS that are consistent with an epigenetic defect. The abnormal imprinting was limited to the DMR of exon 1A, and a previously described 3-kb STX16 microdeletion (13) was identified in all affected subjects who were available for analysis. Our linkage studies directly eliminated the PTH and PTHR1 genes as potential second gene defects, and the size of the kindred essentially excludes a primary role for another gene defect unless in complete linkage disequilibrium with GNAS. Thus, although we were unable to analyze genomic DNA from every member of this extended kindred, our results establish the 3-kb STX16 microdeletion as the basis for PHP type 1b in affected members of this extended kindred.

Brachydactyly was present in nearly 60% of the patients with PHP 1b who were tested and in none of the unaffected relatives. The brachydactyly was of the type E form and in many cases was limited to shortening of the fourth metacarpal and was not associated with short stature. In addition, two of the affected subjects in this study had Madelung-like defects.

The classical Madelung wrist deformity is characterized by particular prominence of the lower end of the ulna, marked shortening and bowing of the radius, and palmar and ulnar deviation of the carpal bones. The Madelung deformity is a dyschondrosteosis that results from premature fusion of the physis with cessation of longitudinal growth. The physeal lesion is always located in the ulnar zone of the distal radius but varies in the anteroposterior plane, which in part determines the precise appearance of the deformity in the patient (14, 15). The Madelung deformity, or defects that resemble the Madelung deformity, occurs in several syndromes, some of which are also associated with mesomelic short stature. Leri-Weill syndrome and Langer mesomelic dwarfism are due to heterozygous and homozygous mutations, respectively, of the short stature homeobox gene (SHOX) located on pseudoautosomal regions of the X and Y chromosomes. Other causes of Madelung deformity include Turner syndrome, multiple exostoses syndrome, multiple epiphysial dysplasia, and dysostosis multiplex of the mucopolysaccharidoses (16).

The Madelung deformity occurs when a major disruption to normal growth plate architecture occurs and leads to a reduction in the number of chondrocyte columns. The columns that remain show a breakdown of normal columnar architecture because nests of chondrocytes replace the abnormal columns. On a cellular level, within the nests, chondrocytes exist at varying stages of maturation, inappropriately sited adjacent to cells of other developmental stages. In addition, there is an apparent increase in the size of the hypertrophic zone and accelerated fusion of the physis. These histological features are reminiscent of the accelerated program of differentiation into hypertrophic chondrocytes that characterizes the severe metaphyseal dysplasia that occurs in mice and humans that lack functional alleles encoding the PTH1R or PTHrP (17–19). More recently heterozygous mutations in PTHrP have been identified as one basis for familial brachydactyly E and short stature (20, 21), providing further confirmation of the important role of the PTHrP-PTH1R signaling pathway in the growth plate. The occurrence of brachydactyly in patients with AHO, whether associated with a GNAS mutation on maternal or paternal allele, suggests that haploinsufficiency rather than imprinting accounts for a deficiency in Gα_s protein that impairs PTHrP signaling in the growth plate. Nevertheless, a previous report demonstrated modest preferential expression of the maternal GNAS allele in a bone sample from one of 19 human subjects (22), thus providing limited evidence that imprinting can influence GNAS expression in bone in at least some patients. Partial imprinting of GNAS may also occur in other tissues that had previously been considered to exhibit biallelic expression of Gα_s (23). Thus, the development of brachydactyly in PHP 1b may be limited to those patients in whom Gα_s expression is influenced by an imprinting mechanism in growth plate cells. A similar mechanism may also explain the development of the Madelung deformity in the patients we describe here.

In summary, our findings confirm previous case reports that described brachydactyly as evidence of mild AHO in at least some patients with sporadic PHP 1b (6–9, 24). Moreover, the coexistence of a Madelung-like deformity in at least some of the PHP 1b patients represents a novel association and extends the list of conditions that can cause this unusual growth plate defect.