Roberto Lala

Pamidronate treatment of bone fibrous dysplasia in nine children with McCune-Albright syndrome

McCune‐Albright syndrome is a rare genetic disorder consisting of skin and bone dysplasia and peripheral endocrinopathies. Little data have been collected regarding bisphosphonate treatment of bone fibrous dysplasia in paediatric patients with this syndrome. The aim of our study was to investigate the therapeutic efficacy of pamidronate in these patients. Nine patients with moderate to severe forms of bone fibrous dysplasia were treated with pamidronate intravenously (0.5‐1 mg/ kg/daily for 2‐3 d) at 0.5‐1‐y intervals. Patients were treated over a time period of 0.5‐3.5 y. During treatment no spontaneous fracture occurred. Bone pain and gait abnormality due to pain disappeared after 2‐3 therapeutic cycles. Cranial asymmetry and limb length discrepancy remained unchanged. Elevated serum alkaline phosphatase and urine hydroxyproline values were reduced by the treatment, demonstrating drug activity at the lesional level. The effectiveness of pamidronate was also seen at the non‐lesional level through an increase in bone density. Radiographic and scintigraphic evidence of lesion healing was not attained. Pamidronate treatment can ameliorate the course of bone fibrous dysplasia in children and adolescents with McCune‐Albright syndrome.

Arginine potentiates the GHRH- but not the pyridostigmine-induced GH secretion in normal short children. Further evidence for a somatostatin suppressing effect of arginine.

To investigate the mechanism underlying the GH‐releasing effect of arginine (ARG), we studied the interactions of ARG (0.5 g/kg infused i. v. over 30 min) with GHRH (1 μg/kg i. v.) and with pyridostigmine (PD, 60 mg orally) on GH secretion in 15 children and adolescents with familial short stature (5.1‐15.4 years). In a group of eight subjects ARG induced a GH increase not statistically different to that observed after GHRH (peak, mean±SEM: 38.0±10.4 vs 64.0±14.4 mU/1). The combined administration of ARG and GHRH led to GH levels (101±15.2 mU/1) higher than those observed after GHRH (P < 0.025) or ARG alone (P < 0.001) and overlapping with those recorded after combined PD and GHRH administration (111±22.4 mU/1). In the other seven subjects, ARG and PD administration induced a similar GH response either when administered alone (25.2±13.6 and 27.8±4.0 mU/1, respectively) or in combination (33.8±5.4 mU/1). In conclusion, our results show that in children ARG administration potentiates GHRH‐ but not PD‐induced GH increase. These findings agree with the hypothesis that the GH‐releasing effect of both ARG and PD is mediated via the same mechanism, namely, by suppression of endogeneous somatostatin release. Combined administration of either ARG or PD with GHRH has a similar striking GH‐releasing effect which is clearly higher than that of GHRH alone.

A Novel GNAS1 Mutation, R201G, in McCune–Albright Syndrome

ACTIVATING MISSENSE mutations of the GNAS1 gene, encoding the alpha subunit of the stimulatory G protein, Gs, have been identified in patients with the McCune– Albright syndrome (MAS, characterized by polyostotic fibrous dysplasia [FD], café au lait skin pigmentation, and endocrine disorders). The reduced GTPase activity of the mutated protein leads to overstimulation of adenylyl cyclase. Recent studies on the nature of FD suggest that the excess generation of cAMP resulting from activity of the mutated Gs alpha may represent a common (albeit not necessarily the only) pathogenetic mechanism for the diverse, skeletal, and nonskeletal manifestations of MAS. Similar mutations of the GNAS1 gene also occur in nonMAS associated FD of bone. MAS was diagnosed in an 8-year-old male presenting with precocious puberty, facial deformities, and typical café au lait spots with a “coast of Maine” profile. Extensive involvement of the cranial vault was apparent on X-ray examination, and a sample of parietal bone demonstrated changes typical of the sclerotic/pagetoid variant of FD. The craniofacial lesions were apparently progressive and were treated with pamidronate which was reported to reduce bone pain as well as to normalize the levels of serum alkaline phosphatase, osteocalcin, and hydroxyproline. At the age of 13, acromegalic bone changes and growth hormone oversecretion were detected. Genomic DNA was extracted from a surgical sample of the FD parietal bone that was obtained under an institutionally approved protocol for the use of human tissue in research (National Institutes of Health Protocol #97-DK0055). Mutation analysis was performed by sequencing the polymerase chain reaction (PCR) amplification product (exon 8) in both directions and by sequencing the PCR product obtained with peptide nucleic acid (PNA) inhibition of the normal allele amplification, a novel highly sensitive method especially suited for mutation analysis of mosaic populations. With both assays, a novel C → G (R201G) mutation was detected (Fig. 1). With the exception of a single case (of polyostotic FD) in which an R201S mutation was identified previously, R201C and R201H have been the mutations found consistently in MAS patients and in non-MAS FD of bone. Thus, of the predicted missense mutations of codon 201, only R201P and R201L remain undetected to date (although R201L has been observed in an isolated, non-MAS endocrine tumor (Table 1). The diversity of the amino acids encoded by the missense mutations detected so far (basic, uncharged polar, nonpolar) demonstrates that substitution of the R201 per se, rather than the characteristics of the substituting amino acid, is the critical molecular event leading to reduced GTPase activity. It is thought that R201 is an essential component of the “timing device” that regulates GTPase. Bourne et al. have reported that two of the single-point missense mutations in R201 of Gs alpha (R201C and R201H), as found in human tumors, appear to have reduced kcat-GTP, although the methodology and exact values were not presented. They further reported as a personal communication, the work of A.G. Gilman’s laboratory in which replacement of the R201 with mutations requiring more than one base pair changes (Val, Ala, Lys, and Glu) in bacterial recombinant Gs alpha also had reduced kcatGTP. To our knowledge, the kcat-GTP activity of the remaining single base pair missense mutations (Gly, Ser, Leu, and Pro) have not been studied. Nevertheless, the loss of the highly basic side chain of arginine (which can also be ADP-

McCune-Albright syndrome: A longitudinal clinical study of 32 patients

We report the diagnostic clinical features and their long term evolution in 32 patients with McCune-Albright syndrome. Patient data are made up of two periods: the first, classified as personal history, is from birth until the time when the diagnosis of McCune-Albright syndrome was made; the second, classified as clinical observation, is from the first observation until the end of follow up. The total duration of these two periods was 9.6+/-2.9 yr; mean age at first observation was 5.7 yr (range 0.7-11 yr). The probability of manifesting main clinical signs according to age was calculated: almost all had skin dysplasia at birth, 50% probability of peripheral precocious puberty in females at 4 years and 50% of bone dysplasia at 8 years of age were found. Other clinical signs had diagnostic relevance when preceding the main signs leading to diagnosis of McCune-Albright syndrome even without specific genetic investigation. The most important clinical manifestations have different evolutions: skin lesions increase in dimensions according to body growth; precocious puberty in females evolves rapidly but periods of regression can be seen in some patients; bone dysplasia in most patients evolves with an increase both in the number of affected bones and in the severity of lesions.

Familial tumoral calcinosis and testicular microlithiasis associated with a new mutation of GALNT3 in a white family.

Background: Familial tumoral calcinosis (FTC) is a rare autosomal recessive disease characterised by the development of multiple calcified masses in periarticular soft tissues; GALNT3 gene mutations have recently been described in an African American and in a Druse Arab family with FTC. Objective: To report the clinical and histological features caused by a new GALNT3 mutation in a white family. Results: Homozygosity for the nonsense mutation Lys463X was found in both affected siblings, who displayed a classic phenotype, the male also having testicular microlithiasis. He is the first subject described with testicular microlithiasis in FTC. Conclusions: The high testicular expression of GALNT3 suggests that the gene alteration could act locally by causing deposition of calcium, and the testis may be an underestimated site of calcification in FTC. Autoimmune diseases are present in several members of the family. Although immune disorders have been described in FTC, autoimmunity does not segregate with the GALNT3 mutation in this family.

Molecular Analysis of the GNAS1 Gene for the Correct Diagnosis of Albright Hereditary Osteodystrophy and Pseudohypoparathyroidism

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 Gsα protein deficiency, PTH resistance, and typical Albright hereditary osteodystrophy (AHO). Heterozygous mutations in the GNAS1 gene encoding the Gsα 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 Gsα 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.

An R201H activating mutation of the GNAS1 (Gsα) gene in a corticotroph pituitary adenoma

In the pituitary gland, activating mutations of the GNAS1 (Gsα) gene at Gln227 have been identified in adrenocorticotrophin secreting, growth hormone secreting, and prolactin secreting adenomas. To date, mutations at the codon encoding R201, typically underlying the McCune-Albright syndrome and isolated fibrous dysplasia of bone, have been demonstrated only in growth hormone secreting pituitary adenomas. In this study, a polymerase chain reaction amplified target sequence in exon 8 of the GNAS1 gene was sequenced, identifying the first R201 mutation seen in an isolated basophilic adenoma which generated Cushings disease in a child. This case adds Cushings disease to the range of human diseases caused by R201 mutations of the GNAS1 gene.

Sperm count of young men surgically treated for cryptorchidism in the first and second year of life: fertility is better in children treated at a younger age.

INTRODUCTION Recent data has indicated the usefulness of performing orchiopexy in the first years of life. In this study, we evaluated testicular function in young men operated on for cryptorchidism in the first year of life. To our knowledge, this is the first report on the effects of such an early treatment. MATERIALS AND METHODS Testicular function was assessed in a group of young men operated for cryptorchidism during the first year of life (Group A, n=13) and during the second year of life (Group B, n=16). RESULTS Total sperm counts were clearly higher in Group A (52.3+/-14.3 million/ml vs. 30.4+/-23.5 million/ml, p=0.005) as was sperm motility (36.2+/-8.7 vs. 23.1+/-15.7%, p=0.009). A clear inverse relationship was found between age at orchiopexy and total sperm count (r=-0.394, p=0.034) and sperm motility (r=-0.382, p=0.041). The relationship between volume of testes, position at surgery, uni/bilaterality of cryptorchidism, evidence of Ad spermatogonia at biopsy performed during surgery and treatment with LHRH and hCG performed before surgery and fertility was not significant. The latter findings may be partially explained by the low number of patients participating in the study and need further investigation. CONCLUSIONS We obtained, for the first time, results showing the benefit of treating cryptorchidism during the first year of life rather than in the second year or even later.

Early Hormonal and Surgical Treatment of Cryptorchidism

PURPOSE We investigated the efficacy of early gonadotropin treatment of cryptorchidism for promoting testicular descent and ameliorating testicular histology. MATERIALS AND METHODS We treated 319 cryptorchid testes in 281 boys 4 months to 3 years old with luteinizing hormone-releasing hormone and human chorionic gonadotropin sequential therapy. Surgery was done on the 207 testes that did not respond to medical treatment. Microscopic biopsies were performed in 134 of these 207 testes. Histological findings were compared to those of 30 cryptorchid testes in boys younger than 1 year who underwent surgery without previous hormonal treatment. RESULTS Combined luteinizing hormone-releasing hormone and human chorionic gonadotropin treatment induced scrotal descent of a percentage of cryptorchid testes depending on clinical position. Therapeutic success was greater when testes were in a lower position and results were not age dependent. Hormonal treatment of cryptorchidism during the first year of life stimulated spermatogonia maturation. CONCLUSIONS When administered at the end of age 6 months, hormonal treatment can be considered an effective and timely substitution for gonadotropin and testosterone insufficiency in cryptorchid infants. Therefore, we recommend this therapeutic procedure combined with surgery in the first year of life.

Bone mass and metabolism in thalassemic children and adolescents treated with different iron-chelating drugs

We evaluated bone mineral density (BMD) and bone turnover in 22 homozygous prepubertal beta-thalassemic patients treated with desferrioxamine. Ten patients underwent treatment with desferrioxamine for the whole study period, while 12 patients stopped desferrioxamine and were then treated with deferiprone (L1). Lumbar and femoral BMD and bone metabolism markers were examined at baseline and after 1 and 3 years of follow up. All patients were prepubertal at baseline and they all became pubertal over the 3 years of follow up. At baseline, the mean lumbar Z score value was −2.048 SD ± 0.75; the Z score was less than −2 SD in 13 children, within −1 and −2 SD in 6, and within 0 and −1 SD in only 3 subjects. A significant BMD increase (P ≪ 0.0001) was observed at both the lumbar (+8.466%/year) and the femoral level (average of +3.46%/year at neck and +5.83%/year at the intertrochanteric region) after 3 years, without any significant difference being shown between patients treated with desferrioxamine and those treated with L1. The mean Z score SD values increased to −1.957 ± 0.975 at 1 year (not significantly different from baseline) and to −1.864 ± 1.221 at 3 year follow up (P ≪ 0.05 vs baseline); an increase in bone turnover was also observed. These findings show that low BMD, a hallmark of beta-thalassemia, improves significantly when puberty begins; this increase involves different skeletal sites, regardless of pharmacological treatment with different iron-chelating drugs.