The PAX8 gene, mapped on 2q12-q14, encodes for a transcription factor involved in thyroid cell proliferation and differentiation. Five mutations in PAX8 have been so far described in both sporadic and rare familial forms of thyroid dysgenesis with proposed autosomal dominant inheritance, all associated with thyroid hypoplasia and/or dysfunction. Fifty-four subjects with congenital hypothyroidism detected during neonatal screening and associated with an ultrasound or scintiscan picture of thyroid dysgenesis were investigated for PAX8 mutations. The entire PAX8 coding region with exon-intron boundaries was amplified from genomic DNA, and a mutational screening was performed by denaturing HPLC followed by direct sequencing when denaturing HPLC elution abnormalities appeared. A new heterozygous deletion (c.989_992delACCC) in exon 7 causing a frameshift with premature stop codon after codon 277 was identified in a subject with thyroid hypoplasia. This mutation is the only one so far identified that lies outside the paired domain. The predicted mutant protein completely lacks the C-terminal region but contains the paired box, octapeptide, and homeodomain. It retains the ability to bind a paired-domain sequence in vitro but is transcriptionally inactive. These results provide evidence that the C-terminal region is essential for transcriptional activity. The new mutation has been inherited from the completely euthyroid mother. It was also present in a brother with slightly elevated TSH only. Thus, it is associated with thyroid dysgenesis in the proband and both euthyroidism and compensated hypothyroidism in her family. This suggests that other factors/genes may modulate phenotypic expression.

Pseudohypoparathyroidism (PHP) is a heterogeneous disease characterized by PTH resistance and classified as types Ia, Ib, Ic, and II, according to its different pathogenesis and phenotype. PHP-Ia patients show G s␣ protein deficiency, PTH resistance, and typical Albright hereditary osteodystrophy (AHO). Heterozygous mutations in the GNAS1 gene encoding the G s␣ protein have been identified both in PHP-Ia and in pseudopseudohypoparathyroidism (PPHP), a disorder with isolated AHO. A single GNAS1 mutation may be responsible for both PHP-Ia and PPHP in the same family when inherited from the maternal and the paternal allele, respectively, suggesting that GNAS1 is an imprinted gene. To evaluate whether molecular diagnosis is a useful tool to characterize AHO and PHP when testing for G s␣ activity and PTH resistance is not available, we have performed GNAS1 mutational analysis in 43 patients with PTH resistance and/or AHO. Sequencing of the whole coding region of the GNAS1 gene identified 11 mutations in 18 PHP patients, eight of which have not been reported previously. Inheritance was ascertained in 13 cases, all of whom had PHP-Ia: the mutated alleles were inherited from the mothers, who had AHO (PPHP), consistent with the proposed imprinting mechanism. GNAS1 molecular analysis confirmed the diagnosis of PHP-Ia and PPHP in the mutated patients. Our results stress the usefulness of this approach to obtain a complete diagnosis, expand the GNAS1 mutation spectrum, and illustrate the wide mutation heterogeneity of PHP and PHP-Ia. PHP (OMIM 103580) was the first human disease to be ascribed to deficient responsiveness to a hormone by otherwise normal target organs (1). It consists of a heterogeneous group of endocrine disorders, whose common feature is resistance to PTH, as shown by hypocalcemia, hyperphosphatemia, and elevation of serum PTH despite normal renal function (2). PTH stimulates the formation of intracellular cAMP by adenylyl cyclase through the activation of G s protein (3, 4). G s protein belongs to the superfamily of heterotrimeric G proteins formed of three subunits (␣, ␤, ␥) encoded by separate genes (5). They mediate signal transduction across cell membranes by coupling extracellular receptors to intracellular effector proteins. The activity of hormone-sensitive adenylate cyclase is regulated by at least two G proteins, one stimulatory (G s ) and one inhibitory (G i ). G protein specificity is defined by its specific ␣-subunit. G s␣ , which binds GTP and stimulates adenylyl cyclase, has GTPase activity and couples multiple receptors to the stimulation of adenylyl cyclase, including those for PTH, TSH, and LH/FSH.

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 alpha-subunit of the stimulatory G protein (Gsalpha). 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.