The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 4 1650-1655
Copyright © 2004 by The Endocrine Society
Brachydactyly in 14 Genetically Characterized Pseudohypoparathyroidism Type Ia Patients
Luisa de Sanctis,
Sergio Vai,
Maria Rita Andreo,
Damiano Romagnolo,
Leandra Silvestro and
Carlo de Sanctis
Department of Pediatric Sciences, University of Torino (L.d.S., D.R., L.S.), Torino; Department of Pediatrics and Neonatology, ASL6 (S.V.), Ciriè (Torino); and Division of Pediatric Endocrinology, Regina Margherita Children’s Hospital (M.R.A., C.d.S.), Torino, Italy
Address all correspondence and requests for reprints to: Dr. Luisa de Sanctis, Department of Pediatric Sciences, University of Torino, Piazza Polonia 94, Torino 10126, Italy. E-mail: ldesanct{at}pediatria.unito.it.
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Abstract
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Brachydactyly, classically described as shortening of III, IV, and V metacarpals and I distal phalanx, is the typical and most specific sign of Albright’s hereditary osteodystrophy, a peculiar phenotype reported in subjects with pseudohypoparathyroidism type Ia (PHP-Ia) caused by mutations in the GNAS gene, which encodes for the 
-subunit of the stimulatory G protein (Gs). It has been reported in 70% of PHP subjects from routine radiological examinations, but there are no specific data for hand alterations in genetically characterized PHP-Ia subjects. We evaluated the metacarpophalangeal pattern profile in 14 GNAS-mutated PHP-Ia subjects and determined the prevalence and patterns of left hand bone shortening. To search for genotype/phenotype correlations, we compared metacarpophalangeal pattern profiles in subjects with identical mutations. Shortening below –2 SD score (SDS) was present in at least one bone in each subject, with a prevalence of 100%; however, great variability existed between subjects and between hand bone segments. Between subjects, shortening ranged from –2 to –10.4 SDS and involved 1–19 hand bones (5.3–100%). Between segments, III–IV metacarpals were the most compromised (–10.4 and –10.0 SDS, respectively); V metacarpals and I–IV distal phalanges were the most frequently shortened (85.7%). Overall, bone length median values revealed shortening below –2 SDS in all metacarpals and all distal phalanges, i.e. brachymetacarpia and brachytelephalangy, that cluster together. These segments were shortened in 64.3–85.7% of patients, significantly differing from proximal and middle phalanges, which were shortened in 21.4–50%. Even if these hand alterations were a constant and typical finding in our PHP-Ia population, cluster analysis in subjects with the same genotypes did not generally show a genotype/phenotype correlation. Variability between subjects may be the result of complex interactions between GNAS defects and other genetic or epigenetic factors. In conclusion, hand shortening analysis in 14 genetically characterized patients showed typical brachymetacarpia and brachytelephalangy. Further studies in PHP-Ia subjects without GNAS mutations and in other brachydactyly syndromes will determine whether the pattern described is also specific.
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Introduction
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BRACHYDACTYLY, DESCRIBED AS relative shortening and widening of specific long bones in the hands, usually the III, IV, and V metacarpals and the I distal phalanx (1), is the typical and most specific sign of Albright’s hereditary osteodystrophy (AHO). This peculiar phenotype also includes small stature, obesity with rounded face, sc ossifications, and mental retardation and is described in some subjects with pseudohypoparathyroidism (PHP) (2, 3). PHP embraces a heterogeneous group of disorders, whose common feature is resistance to PTH. Types Ia (PHP-Ia), Ib, Ic, and II are distinguished on the basis of their pathogenesis and phenotypes (4, 5).
PHP-Ia is caused by dominant mutations in the guanine nucleotide-binding protein -stimulating polypeptide (GNAS) gene that encodes for Gs protein. Subjects show Gs protein deficiency and resistance to hormones that act through Gs activation via the cAMP-adenylyl cyclase pathway (i.e. PTH, TSH, and FSH/LH) and AHO (6, 7, 8, 9, 10, 11, 12, 13). Gs protein deficiency and GNAS mutations have also been identified in patients with isolated AHO phenotype, a clinical disorder called pseudopseudohypoparathyroidism (PPHP) (14, 15). A single GNAS mutation can be responsible for both PHP-Ia and PPHP in the same family when inherited from the maternal and paternal alleles, respectively. This has suggested that other genetic factors, such as genomic imprinting, may influence endocrine function (16, 17, 18). Among PHP-Ia/PPHP patients, AHO expression, too, varies greatly, as some subjects with GNAS mutations show few of its features (12, 19). The typical brachydactyly has been reported in 70% of PHP by routine radiological examinations (1).
The metacarpophalangeal pattern profile (MPP) introduced by Poznanski et al. (20) is an analytical tool for evaluating significant absolute and relative differences in the lengths of hand bones. It has been used to identify brachydactyly and to compare hand bone shortening in several disorders, such as PHP and PPHP, brachydactyly type E, Turner’s syndrome, and acrodysostosis (21, 22). There are no specific data for hand alterations in genetically characterized PHP-Ia patients.
In the present study we evaluated the prevalence and the entity of length shortening in the left hand bones of 14 GNAS-characterized PHP-Ia subjects. To search for genotype/phenotype correlation, MPPs from subjects harboring identical mutations, either in the same family or unrelated, were compared.
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Subjects and Methods
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Patients
Fourteen genetically characterized PHP-Ia subjects, five males and nine females, aged 4–26 yr, from 13 families (patients 8 and 9 are dizygotic twins) were included (Table 1
). Informed consent was obtained from all patients or their families.
Posteroanterior left hand radiographs were used to measure metacarpal and proximal, middle, and distal phalanx lengths. We decided to use the left hand because considerable asymmetry (42%) has been described in PHP subjects (21). SD units relative to the norm for sex and age (z-scores) (20), with median, minimal, and maximal z-score values and the percentage of bones below –2 SD score (SDS) were calculated. The z-scores of the length of each bone were used to construct MPPs according to the method of Poznanski et al. (20); the z-score is plotted on the ordinate, whereas the location by row is determined on the abscissa.
To evaluate the presence of a genotype/phenotype correlation, MPPs from subjects with identical mutations, either from the same family or unrelated, were compared. Left hand radiographs were also obtained from the mothers of patients 3, 7–9, and 11, where c.344C
T, c.366delC, c.555delC and c.565_568delGACT mutations were identified, respectively, and PPHP was diagnosed by the presence of an isolated AHO phenotype. The subjects’ hand pattern profiles were constructed and compared with those of their mutated offspring. Moreover, MPPs from cases 10–12, harboring the c.565_568delGACT mutation, were compared.
Hierarchical cluster analysis was used to cluster individuals and variables (23, 24, 25). Euclidean quadratic distance was used to estimate similarity between bones and product-moment correlation between subjects, using average linkage between groups (see the supplemental data published on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). Differences between proportions were tested by Fisher’s exact test ( = 0.05) (26). SPSS 9.0 (http://www.spss.com) was used to store, compute, and analyze data.
Molecular analysis
GNAS gene analysis was performed on genomic DNA isolated from peripheral blood leukocytes with the phenol/chloroform method. Exons 1–13, corresponding to the whole GNAS-coding sequence, and intron/exon boundaries were amplified by using 13 pairs of previously described GNAS-specific primers (12, 19, 27). Gel-purified fragments obtained with the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA) were directly sequenced with a semiautomated detection system (ABI 373A sequencer, PE Applied Biosystems, Foster City, CA). DNA sequence abnormalities were confirmed by restriction analysis of genomic PCR products with the appropriate endonucleases. The same technique was used to trace segregation of the mutation in the available subjects of each family and to test 50–55 unrelated normal individuals (27).
GNAS causal mutations in 12 of 14 subjects have been previously described (27). In the remaining two patients GNAS gene analysis identified two new causal mutations. The first, c.348_349insT, is a single base insertion at codon 115 in exon 5 identified in patient 6 (Table 1
), who showed resistance to PTH and TSH, as well as several AHO clinical signs (obesity, rounded face, and mental retardation). It causes a frameshift with the creation of a premature stop codon after 22 codons. The second, c.861_862delTG, is a TG deletion at codon 287 in exon 11 identified in heterozygosis in patient 13 (Table 1), who had resistance to PTH and TSH associated with AHO signs (brachydactyly, obesity, rounded face, and mental retardation). It causes a frameshift with the creation of a premature stop codon after 11 codons.
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Results
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All patients have resistance to PTH and TSH (patient 9 also has resistance to FSH/LH) and several clinical AHO signs; in particular, brachydactyly was observed in 11 of 14 patients, clinically or by routine left hand x-ray examination (Table 1).
Shortening below –2 SDS was found in 152 of 266 bones (57.1%), and each subject showed one or more shortened bones (Table 2).
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TABLE 2. Age and hand bones length expressed in SDS (Z scores) with median, minimum, and maximal values and percentage below –2 SDS in 14 genetically characterized PHP-Ia patients
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Great variability was observed in bone-shortening entity and prevalence between patients and between segments. Between subjects, bone lengths varied from –10.4 SDS (patient 10) to normal values, whereas the prevalence of shortening below –2 SDS ranged from 5.3% in patient 9 to 100% in patients 10, 12, and 14. Between segments, the most severely impaired were III and IV metacarpals (reaching –10.4 and –10.0 SDS), whereas shortening prevalences ranged from 21.4% for II proximal phalanx to 85.7% for V metacarpals and I and IV distal phalanges (Table 2).
Overall, bone length median values in the 14 patients revealed shortening below –2 SDS in all metacarpals and all distal phalanges, i.e. brachymetacarpia and brachytelephalangy. Good correlations were observed for metacarpals (r ranging from 0.70–0.91) and distal phalanges (r ranging from 0.68–0.95) only (Fig. 1; matrix correlations not shown).
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FIG. 1. Hierarchical cluster analysis of all hand bones in the 14 PHP-Ia patients. Distance was computed according to quadratic Euclidean distance method and aggregation using average linkage between groups. Dendrogram shows how bones are homogeneous and aggregate according to their expected involvement; metacarpals and distal phalanges group differs from proximal and middle phalanges group.
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With a cut-off value of 50% of bones below –2 SDS, shortening percentages significantly differ between the metacarpals and distal phalanges groups (100%) and between the proximal and middle groups (22%; Table 2).
MPPs from patients 3, 7–9, and 11 and their mothers, harboring c.344CT, c.366delC, c.555delC and c.565_568-delGACT mutations, respectively (Figs. 2 and 4), and MPPs from cases 10–12 from different families and harboring the c.565_568delGACT mutation (Figs. 3 and 4) were considered. Pattern profile comparison in subjects harboring c.344CT and c.366delC mutations with their mutated mothers showed significant dissimilarity, with product-moment correlations of –0.34 and –0.29, respectively (Fig. 2, A and B, and Fig. 4). Comparison in the family with c.555delC mutation identified in the dizygotic twins and their mother demonstrated mild similarities between the siblings (r = 0.45), in contrast to dissimilarities in parent to child pattern profiles (r = 0.24 and 0.04 for patients 8 and 9 with their mother, respectively; Figs. 2C and 4). On the other hand, in patient 11 with the c.565_568delGACT mutation and his mutated mother, the profile similarity was considerable, with an r of 0.90 (Figs. 2D and 4). The c.565_568delGACT mutation displayed by subjects from different families was associated with similar MPP for patient 10 vs. patient 12 and for patient 11 vs. patient 12 (r = 0.41 and 0.45, respectively), but with dissimilar patterns for patient 10 vs. patient 11 (r = 0.09; Figs. 3 and 4).
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FIG. 2. MPP comparisons between subjects belonging to the same family and harboring the same mutation (A, c.555delC mutation; B, c.344CT (P115L) mutation; C, c.363–366delC mutation; D, c.565–568delGACT mutation). r is Pearson’s product-moment coefficient.
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FIG. 4. Hierarchical cluster analysis of the 14 PHP-Ia patients and of four GNAS mutated mothers based on MPPs. Distance was computed according to correlation method (Pearson’s r), and aggregation was determined using average linkage between groups. Dendrogram shows how cases generally do not aggregate according to their mutation.
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FIG. 3. MPP comparisons between unrelated subjects harboring the same mutation (c.565–568delGACT). r is Pearson’s product-moment coefficient.
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Discussion
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The prevalence and type of hand shortening in AHO have only been reported in molecularly uncharacterized patients, clinically defined as PHP/PPHP subjects. In some of these subjects, hand shortness is immediately apparent to an experienced physician, but it must usually be determined with appropriate and objective techniques. Archibald’s sign has been used to evaluate a relative shortening of metacarpals, but is also positive in about 10% of normal subjects and can be unreliable, because it is dependent on the shortening of adjacent bones; it is particularly useless when subtle degrees of shortening of several metacarpals are present. By routine radiological examination, a 70% brachydactyly prevalence has been described in 20 PHP subjects by Merzou et al. (1), with involvement for the most part of the IV, but also of III and V, metacarpals; involvement of the I distal phalanx was not present in their series. The MMP is an analytical method that reveals absolute and relative significant differences in hand bone length. It has been used by Poznanski et al. (20) to characterize brachydactyly in a heterogeneous group of patients, including either PHP or PPHP subjects. The I distal phalanx of the thumb was described as the most commonly shortened bone (75%), followed by the IV metacarpal, which was shortened in 65%.
In this study GNAS gene analysis allowed identification of the causal mutation and characterization of the PHP-Ia subtype in 14 AHO patients resistant to PTH, TSH, and FSH/LH. Therefore, we decided to analyze MPPs in this genetically defined subset of patients to determine whether GNAS mutations have typical and specific effects on skeletal phenotype.
Clinical or routine radiological examinations demonstrated brachydactyly in 11 of 14 patients (78.5%), and MMP analysis revealed a significant shortening below – 2 SDS of one or more bones in all subjects.
There were great differences in hand shortening: some subjects displayed severe shortening (even reaching –10.4, –10.0, –9.3 and –9.2 SDS) of all hand bones (i.e. patients 10, 12, and 14), a finding similar to that described in acrodysostosis (21), whereas others had shortening below –2 SDS for only one or a few bones (i.e. patients 9 and 6). Shortening prevalence below –2 SDS ranged from 5.3–100% of the bones. Shortening of the hand segments also varied greatly, both in severity, where metacarpals were the most impaired, and in prevalence, where the metacarpals and distal phalanges group was shortened in 64.3–85.7% of cases, and the proximal and middle phalanges group was shortened in 21.4–50.0% of cases.
Median z-scores indicated shortening below –2 SDS in all metacarpals (ranging from –2.0 to –3.5 SDS) and all distal phalanges (ranging from –2.6 to –4.5 SDS), not only in the classically reported III, IV, and V metacarpals and the I distal phalanx (1, 21). The V metacarpal and I–IV distal phalanges were the most frequently affected (85.7%), with no differences in shortening prevalence, whereas data in PHP patients have previously shown 75% prevalence for the I distal phalanx and 65% for the IV metacarpal (21). This pattern profile was both a typical and constant finding, as in subjects with milder hand involvement (patients 9 and 6), metacarpals and distal phalanges are relatively more impaired than other segments. Thus, brachymetacarpia, which denotes shortening of the metacarpals, and brachytelephalangy, which denotes shortening of the distal phalanges, characterized the hands of the 14 PHP-Ia subjects.
As in PHP/PPHP subjects brachydactyly has been described as a progressive sign (20), and our patients included both adults as well as children, even aged 4 or 5 yr, we looked for significant age-related differences. In the younger subjects (patients 1, 2, 4, 6, 7, 12, and 13), in whom brachydactyly could be not fully established because final height had not been reached, shortening was always evident, ranging from severe and involving all hand segments (patient 12) to mild (patient 6). The great variability observed in the adult group was also present in the younger patients. In subjects with mild shortening (patients 6 and 13), MPP analysis showed the same pattern profile as in the other subjects, with relatively major impairment of metacarpals and distal phalanges. In these patients, hand involvement could be progressive and thus worsen the already typical profile.
No significant MPP differences were observed between the sexes.
As hand shortening was constant in our patients, we evaluated whether the variability between subjects and between segments could be related to individual GNAS mutations, i.e. whether same genotypes correlated with specific skeletal abnormalities. Cluster analysis, used to compare the type of hand shortening in subjects with the same mutations from the same family or unrelated subjects, revealed that they did not generally display greatly similar patterns, indicating that each mutation was unlikely to be responsible for a specific MPP alteration. Thus, even if GNAS mutations seem both necessary and sufficient to produce the specific bone length alterations, a genotype/phenotype correlation cannot be determined by considering shortening as a phenotypic marker. MPP variability is probably the result of complex interactions between GNAS gene defects and other genetic or epigenetic factors.
In conclusion, the molecular characterization of the PHP-Ia condition in 14 patients allowed exploration of the phenotype relationship. Hand shortening evaluation with MPP analysis showed typical brachymetacarpia and brachytelephalangy, i.e. absolute or relative shortening of the metacarpals and distal phalanges in the 14 PHP-Ia patients. Brachydactyly assessment with formal MPP analysis should therefore be considered when PHP-Ia is suspected, as it is the only reliable way to disclose this typical pattern. However, as up to 30% of patients with clinically confirmed PHP-Ia do not have GNAS mutations (13, 27), it would be interesting to evaluate MPPs in these subjects as well as in other genetically defined brachydactyly syndromes to elucidate whether the pattern described in GNAS-mutated PHP-Ia subjects is also specific.
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Footnotes
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Abbreviations: AHO, Albright’s hereditary osteodystrophy; GNAS, guanine nucleotide-binding protein -stimulating polypeptide; MPP, metacarpophalangeal pattern profile; PHP-Ia, pseudohypoparathyroidism type Ia; PPHP, pseudopseudohypoparathyroidism; SDS, SD score.
Received May 16, 2003.
Accepted January 8, 2004.
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Abstract
Introduction
