Spectrum of mutations of the AAAS gene in Allgrove syndrome: lack of mutations in six kindreds with isolated resistance to corticotropin.

Familial glucocorticoid deficiency due to corticotropin (ACTH) resistance consists of two distinct genetic syndromes that are both inherited as autosomal recessive traits: isolated ACTH resistance (iACTHR), which may be caused by inactivating mutations of the ACTH receptor (the MC2R gene) or mutations in an as yet unknown gene(s), and Allgrove syndrome (AS). The latter is also known as triple-A syndrome (MIM 231550). In three large cohorts of AS kindreds, the disease has been mapped to chromosome 12; most recently, mutations in the AAAS gene on 12q13 were found in these AS families. AAAS codes for the WD-repeat containing ALADIN (for alacrima-achalasia-adrenal insufficiency-neurologic disorder) protein. We investigated families with iACTHR (n = 4) and AS (n = 6) and a Bedouin family with ACTHR and a known defect of the TSH receptor. Four AS families were of mixed extraction from Puerto Rico (PR); most of the remaining six families were Caucasian families from North America (NA). Sequencing analysis found no MC2R genetic defects in any of the kindreds. No iACTHR kindreds, but all of AS families, had AAAS mutations. The previously reported IVS14+1G-->A splice donor mutation was found in all PR families, apparently due to a founder effect; one NA kindred was heterozygous for this mutation. In the latter family, long-range PCR failed to identify a deletion or other rearrangements of the AAAS gene. No other heterozygote or transmitting parent had any phenotype that could be considered part of AS. The IVS14+1G-->A mutation results in a premature termination of the predicted protein; although it was present in all PR families (in the homozygote state in three of them), there was substantial clinical variation between them. One PR family also carried a novel splice donor mutation of the AAAS gene in exon 11, IVS11+1G-->A; the proband was a compound heterozygote. A novel point mutation, 43C-->A(Gln15Lys), in exon 1 of the AAAS gene was identified in the homozygote state in a Canadian AS kindred with a milder AS phenotype. The predicted amino acid substitution in this family is located in a sequence that may participate in the preservation of stability of ALADIN beta-strands, whereas the splicing mutation in exon 11 may interfere with the formation of WD repeats in this molecule. We conclude that 1) AAAS does not appear to be frequently mutated in families with iACTHR; 2) AAAS is mutated in AS families from PR (that had previously been mapped to 12q13) and NA; and, 3) there is significant clinical variability between patients with the same AAAS defect.

Natural history and incidence of premature thelarche in Puerto Rican girls aged 6 months to 8 years diagnosed between 1990 and 1995.

OBJECTIVE: To describe the natural history and estimate the incidence of premature thelarche in girls aged 6 months to 8 years living in Puerto Rico and diagnosed between 1990 and 1995. BACKGROUND: In the 1970s and 1980s, several pediatric endocrinologists, based on their clinical experience, acknowledged a dramatic increase in the number of cases of precocious sexual development in Puerto Rico. In 1987, the Puerto Rico Health Department developed the Registry of Premature Thelarche and Precocious Sexual Development, which began to operate in 1989. Data regarding the long-term outcomes of girls diagnosed with premature thelarche are insufficient. Knowledge about the natural history of this condition is relevant for predicting the long-term prognosis and therapeutic management of the affected population. METHODS: Of 2,716 cases of precocious sexual development reported to the Premature Thelarche and Precocious Sexual Development Registry, 1,916 (70.5%) were premature thelarche. The clinical characteristics and evolution during follow-up of premature thelarche cases were described and compared by age group at diagnosis. RESULTS: Incidences were 6.2 and 1.62 per 1,000 live births for girls aged < 2 years and 2 to 8 years, respectively. These estimates were 10 and 15 times higher than those reported in Olmsted, MN. When the average change in mammary tissue diameter during follow-up was evaluated, a slight reduction in girls aged < 2 years was observed; however, it remained constant for girls aged 2 to 8 years. CONCLUSIONS: The results of this study underscore the need to continue an active search of premature thelarche cases and to perform analytical investigations of precocious sexual development to expand the understanding of the etiology of this important public health problem.

Identification of phthalate esters in the serum of young Puerto Rican girls with premature breast development.

Premature breast development (thelarche) is the growth of mammary tissue in girls younger than 8 years of age without other manifestations of puberty. Puerto Rico has the highest known incidence of premature thelarche ever reported. In the last two decades since this serious public health anomaly has been observed, no explanation for this phenomenon has been found. Some organic pollutants, including pesticides and some plasticizers, can disrupt normal sexual development in wildlife, and many of these have been widely used in Puerto Rico. This investigation was designed to identify pollutants in the serum of Puerto Rican girls with premature thelarche. A method for blood serum analysis was optimized and validated using pesticides and phthalate esters as model compounds of endocrine-disrupting chemicals. Recovery was > 80% for all compounds. We performed final detection by gas chromatography/mass spectrometry. We analyzed 41 serum samples from thelarche patients and 35 control samples. No pesticides or their metabolite residues were detected in the serum of the study or control subjects. Significantly high levels of phthalates [dimethyl, diethyl, dibutyl, and di-(2-ethylhexyl)] and its major metabolite mono-(2-ethylhexyl) phthalate were identified in 28 (68%) samples from thelarche patients. Of the control samples analyzed, only one showed significant levels of di-isooctyl phthalate. The phthalates that we identified have been classified as endocrine disruptors. This study suggests a possible association between plasticizers with known estrogenic and antiandrogenic activity and the cause of premature breast development in a human female population.

Genetic heterogeneity of adrenocorticotropin (ACTH) resistance syndromes: identification of a novel mutation of the ACTH receptor gene in hereditary glucocorticoid deficiency.

Hereditary primary adrenal insufficiency syndromes due to ACTH resistance include hereditary glucocorticoid deficiency (HGD) and Allgrove's syndrome (AS). Patients with both conditions present in childhood with failure to thrive, weakness, and fatigue or adrenal crisis; patients with AS in addition have alacrima and achalasia (triple A syndrome). We studied four kindreds with HGD and four kindreds with AS for abnormalities of the ACTH receptor (ACTHR) gene. The ACTHR coding sequence in all AS kindreds and two HGD kindreds was normal. Analysis of the ACTHR gene of the proband in one of the HGD kindreds showed him to be homozygous for the previously described G221T transition causing a Ser74Ile substitution of the protein, which has been shown to inactivate the ACTHR in signal transduction. The proband in another HGD kindred was found to be a compound heterozygote with the G221T transition in one allele and a novel C818A transition in the other allele of ACTHR. The C818A transition caused the substitution of the highly conserved Pro273 by His in the receptor protein. In vitro expression of the mutated ACTHR in mouse melanoma M3 cells showed that at a medium ACTH concentration of 3 nM, cells transfected with the wild-type ACTHR produced twofold and threefold, respectively, of the amount of intracellular cAMP when compared to cells transfected with the ACTHR carrying the Pro273His and the Ser74Ile mutation, respectively, confirming that HGD in this kindred is caused by loss-of-function mutations of the ACTHR. These results showed that the genetic cause of the ACTH-resistant syndromes is heterogeneous.

Segregation of Allgrove (triple-A) syndrome in Puerto Rican kindreds with chromosome 12 (12q13) polymorphic markers.

Allgrove syndrome (AS), also known as triple-A syndrome, is a rare cause of congenital adrenal insufficiency due to adrenocorticotropic hormone resistance. It is inherited in an autosomal recessive manner and is associated with achalasia, alacrima, and other neurological abnormalities, including autonomic, sensory, and upper- and lower-motor neuropathy, deafness, and mental retardation. Although the etiology of AS remains unknown, recently the disease was linked to a chromosome 12 locus (corresponding cytogenetic band 12q13) in consanguineous families of European ancestry. In the present study, we investigated four nonconsanguineous families with documented inheritance of AS for linkage with the reported 12q13 locus. Eighteen subjects were studied, of whom five were affected by AS. DNA was extracted from peripheral blood lymphocytes and amplified by standard methods with primers from polymorphic sequence tagged sites (STSs) located in the chromosome 12q13 region. Two-point logarithm-of-odds (LOD) score analysis revealed a maximum LOD score of 1.7 for STSs D12S361 and D12S368 without any recombinants [recombination distance (theta) = 0]. Multipoint linkage analysis defined an area of estimated genetic distance less than 0.5 cM (approximately 500,000 bp) between STSs D12S361 and D12S359 that is most likely to contain the AS gene(s). We conclude that, in Puerto Rican families, AS segregates with polymorphic markers that have been mapped to the chromosome 12q13 locus, revealing the absence of heterogeneity for this syndrome in a genetically distinct population. Candidate genes in the region include those that code for several of the keratin proteins, transcription factors, and others.

Secondary sexual characteristics and menses in young girls seen in office practice: a study from the Pediatric Research in Office Settings network.

OBJECTIVE: To determine the current prevalence and mean ages of onset of pubertal characteristics in young girls seen in pediatric practices in the United States. METHODS: A cross-sectional study was conducted by 225 clinicians in pediatric practices belonging to Pediatric Research in Office Settings, a practice-based research network. After standardized training in the assessment of pubertal maturation, practitioners rated the level of sexual maturation on girls 3 through 12 years who were undergoing complete physical examinations. RESULTS: Data were analyzed for 17,077 girls, of whom 9.6% were African-American and 90.4% white. At age 3, 3% of African-American girls and 1% of white girls showed breast and/or pubic hair development, with proportions increasing to 27.2% and 6.7%, respectively, at 7 years of age. At age 8, 48.3% of African-American girls and 14.7% of white girls had begun development. At every age for each characteristic, African-American girls were more advanced than white girls. The mean ages of onset of breast development for African-American and white girls were 8.87 years (SD, 1.93) and 9.96 years (SD, 1.82), respectively; and for pubic hair development, 8.78 years (SD, 2.00) and 10.51 years (SD, 1.67), respectively. Menses occurred at 12.16 years (SD, 1.21) in African-American girls and 12.88 years (SD, 1.20) of age in white girls. CONCLUSIONS: These data suggest that girls seen in a sample of pediatric practices from across the United States are developing pubertal characteristics at younger ages than currently used norms. Practitioners may need to revise their criteria for referral of girls with precocious puberty, with attention to racial differences.

Compound heterozygous mutations of the luteinizing hormone receptor gene in Leydig cell hypoplasia.

The human LH receptor (hLHR) is a member of the G protein-coupled receptors characterized by the presence of seven-transmembrane (TM) helices. Inactivating mutations of the hLHR lead to Leydig cell hypoplasia (LCH), a form of male pseudohermaphroditism resulting from the failure of fetal testicular Leydig cell differentiation. We have identified three mutations of the hLHR in a patient with LCH: deletion of exon 8 (delta Exon 8), A872G transition resulting in Asn291Ser substitution in the extracellular domain, and C1847A transversion resulting in Ser616Tyr substitution in the seventh TM helix. Nucleotide sequencing, gene dosage, and allele-specific amplification analyses revealed that exon 8 deletion and the two missense mutations are present in different alleles of the hLHR. Constructs of mutated hLHR (hLHR-delta Exon8, hLHR-872/1847, hLHR-1847, and hLHR-872) were used to transfect 293 cells, and the properties of the hLHR expressed were examined. Ligand-binding assays failed to detect the expression of hLHR-delta Exon8. Transfectants expressing hLHR-872/1847 demonstrated greatly reduced ligand binding and ligand-induced cAMP accumulation in comparison to those expressing wild type hLHR. Similar reduction in cAMP accumulation was observed in transfectants expressing hLHR-1847, but not hLHR-872 alone. These findings suggest that, in addition to the 7-TM helices, the polypeptide encoded by exon 8 plays an important role in LHR expression and signal transduction. On the other hand, glycosylation of Asn291 may not be critical for these activities. These results also establish that LCH can result from impaired signal transduction due to compound heterozygous mutations. Implications of these mutations on structure-function relationship of the hLHR and the genotype-phenotype correlation in LCH are discussed.

Absence of molecular defect in the type II 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) gene in premature pubarche children and hirsute female patients with moderately decreased adrenal 3 beta-HSD activity.

To date the molecular basis and hormonal criteria for inherited mild late-onset 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) deficiency congenital adrenal hyperplasia (CAH) have not been defined. We have thus investigated the presence or absence of mutation in the type II 3 beta-HSD gene encoding adrenal/gonadal 3 beta-HSD in each of five premature pubarche children and hirsute female patients manifesting moderately decreased adrenal 3 beta-HSD activity. ACTH-stimulated hormonal levels in all patients compared with mean levels in pubertal stage-matched normal subjects were between 2.5 and 6.5 SD for 17-hydroxypregnenolone levels, and between 2.5 and 7 SD for dehydroepiandrosterone levels in all except one patient. 17-Hydroxypregnenolone to cortisol ratios were between 2.5 and 4.3 SD, and dehydroepiandrosterone to androstenedione ratios were between 3 and 8.6 SD. The type II 3 beta-HSD gene regions of a putative promoter, exons I, II, III, and IV, and exon-intron boundaries in all subjects were amplified by polymerase chain reaction and then sequenced. All patients had normal sequences of the type II 3 beta-HSD gene in both alleles. Three female patients heterozygotic for severe 3 beta-HSD deficiency CAH with one allele mutation of the gene demonstrated normal ACTH-stimulated hormone profiles. These data indicate that moderately decreased adrenal 3 beta-HSD activity resulting in modestly increased delta 5 precursor steroid levels and delta 5 to delta 4 steroid ratios in premature pubarche and hirsute patients is not caused by a mutation in the type II 3 beta-HSD gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Action of human growth hormone (hGH) on extrathyroidal conversion of thyroxine (T4) to triiodothyronine (T3) in children with hypopituitarism.

To study the action of human growth hormone (hGH) on peripheral metabolism of serum thyroxine (T4), an oral loading dose of levothyroxine (1.2 mg/m2) was administered to seven children with hypopituitarism before initiation of hGH therapy. Serum concentrations of triiodothyronine (T3), T4, reverse triiodothyronine (rT3), and thyroxine-binding globulin (TBG) capacity were measured sequentially for 6 days. The study was repeated after 4 wk of treatment with hGH. Serum concentrations of T4 were not affected by hGH therapy. In contrast, mean basal serum concentration of T3 increased significantly after treatment with hGH. Also, changes in serum concentrations of T3 and in the ratio of T3/T4 after an oral dose of levothyroxine were significantly augmented during hGH therapy. Serum concentrations of rT3 changed in the opposite direction of T3 during therapy. After treatment with hGH, the mean basal level of serum rT3 decreased, and increases in serum concentrations of rT3 after oral levothyroxine were significantly attenuated. No changes in mean serum concentrations of thyroid stimulating hormone (TSH) and TBG capacity were observed. These data suggest that administration of hGH to children with hypopituitarism enhances the extrathyroidal conversion of T4 to T3 and concomitantly decreases the serum concentration of rT3.