Mercedes Maceiras

Three New SF-1 (NR5A1) Gene Mutations in Two Unrelated Families with Multiple Affected Members: Within-Family Variability in 46,XY Subjects and Low Ovarian Reserve in Fertile 46,XX Subjects

Background: Three novel heterozygous SF-1 gene mutations affecting multiple members of two unrelated families with a history of 46,XY disorders of sex development (DSD) and 46,XX ovarian insufficiency are described. Methods: Clinical and mutational analysis of the SF-1 gene in 9 subjects of two families. Results: Family 1 had 2 affected 46,XY DSD subjects. One, born with severe perineal hypospadias, was raised as a male, and presented normal adolescence. The other, born with ambiguous genitalia, uterus, and mild testicular dysgenesis, was raised as a female. A W279X heterozygous mutation and an intronic deletion (g3314-3317delTCTC (IVS 4 + 8) was found in the SF-1 gene. In family 2, 4/6 affected siblings had 46,XY DSD or hypospadias. An affected 46,XX sister had normal sexual development but increased FSH levels. The 37-year-old affected mother had entered menopause. An Y183X heterozygous mutation was detected. Conclusion: An extreme within-family phenotypic variability, ranging from severe prenatal undervirilization to normal pubertal development, was observed in 46,XY-affected siblings, indicating that other unknown factors might be involved in the phenotype. Low ovarian reserve and preserved fertility in 46,XX subjects can be observed in heterozygous SF-1 gene mutations.

Steroid 21‐hydroxylase gene mutational spectrum in 454 Argentinean patients: genotype–phenotype correlation in a large cohort of patients with congenital adrenal hyperplasia

Objective  To report genotype–phenotype correlation in a large cohort of patients.

Preserved Fertility in a Patient with a 46,XY Disorder of Sex Development due to a New Heterozygous Mutation in the NR5A1/SF-1 Gene: Evidence of 46,XY and 46,XX Gonadal Dysgenesis Phenotype Variability in Multiple Members of an Affected Kindred

In humans, steroidogenic factor 1 (NR5A1/SF-1) mutations have been reported to cause gonadal dysgenesis, with or without adrenal failure, in both 46,XY and 46,XX individuals. We have previously reported extreme within-family variability in affected 46,XY patients. Even though low ovarian reserve with preserved fertility has been reported in females harboring NR5A1 gene mutations, fertility has only been observed in one reported case in affected 46,XY individuals. A kindred with multiple affected members presenting gonadal dysgenesis was studied. Four 46,XY individuals presented severe hypospadias at birth, one of them associated with micropenis and cryptorchidism. The other 3 developed spontaneous male puberty, and 1 has fathered 5 children. Four 46,XX patients presented premature ovarian failure (one of them was not available for the study) or high follicle-stimulating hormone levels. Mutational analysis of the NR5A1 gene revealed a novel heterozygous mutation, c.938G→A, predicted to cause a p.Arg313Hys amino acid change. A highly conserved amino acid of the ligand-binding domain of the mature protein is affected, predicting abnormal protein function. We confirm that preserved fertility can be observed in patients with a 46,XY disorder of sex development due to heterozygous mutations in the NR5A1 gene.

Differences in Serum GH Cut-Off Values for Pharmacological Tests of GH Secretion Depend on the Serum GH Method Clinical Validation from the Growth Velocity Score during the First Year of Treatment

Background: The serum GH cut-off value for pharmacological tests of GH secretion (PhT GH) depends on the type of test and also on the method used for determining serum GH. Cut-off serum GH values as different as 5–10 ng/ml, have been reported, and have been validated biochemically. We have used the growth velocity (GV)-standard deviation score (SDS) during the first year of treatment with rhGH to validate these cut-offs on a biological basis. Methods: Fifty pre-pubertal patients with short stature (height ≤–2 SDS and GV ≤–1.2 SDS) were studied. GH deficiency (GHD) was diagnosed in 39 patients, on the basis of clinical and auxological parameters and on the serum concentration of IGF-1, and non-GHD in the other 11 patients. Two PhT GH (arginine and clonidine) were carried out in the 50 patients. Serum GH was determined by two different methods: one detecting most of serum GH isoforms, named Total GH (HGH Bio-Tech, MAIA Clone), and another one, only detecting the 22 kDa GH, named 22K GH (GH-22K IFMA, Wallac). Results: Basal data: all patients with GHD and with non-GHD had maximal serum GH response (MaxR) values below and above the cut-off, respectively, for the serum Total GH and 22K GH. The mean 22K GH/Total GH ratio was similar to previous publications. Post-rhGH treatment data: the two groups improved their height SDS during the first year of treatment, particularly patients with GHD. A receiver-operator curve was used to define the best threshold for post-treatment GV-SDS that separates GHD from non-GHD patients. This value was 1.91 GV-SDS. A negative correlation between first year treatment GV-SDS and pre-treatment serum GH MaxR was found for the two assays (p < 0.001). Then, the best cut-off GV-SDS, previously calculated with the receiver-operator curve (1.91 SDS) was used to interpolate the corresponding serum GH values, as determined by the two methods. For Total GH, the value was 10.8 ng/ml, and for 22K GH, it was 5.4 ng/ml. Conclusion: The cut-off values calculated by biological means to separate GHD from non-GHD were remarkably similar to those calculated biochemically (10.0 and 4.8 ng/ml, respectively) for Total and 22K GH. This is a biological validation for using different cut-off values, appropriate for each assay, to diagnose GHD.

Age-specific thyroid hormone and thyrotropin reference intervals for a pediatric and adolescent population.

Abstract Background: Establishment of reliable reference intervals remains valuable for confirming validity and advancing standardization across methods and populations. Moreover, knowledge of the measurement uncertainty (U) and of the reference change value (RCV) has important applications in clinical chemistry. Methods: Starting from the information available in the laboratory data base (29,901 subjects) an initial selection was carried out by eliminating all subjects with a clinical or laboratory pathological report; data from 7581 0- to 20-year-old subjects (53.87% girls) remained in the study. These subjects, divided into nine age groups, were used to define reference distribution percentiles (2.5th, 50th and 97.5th) of serum thyrotropin (TSH), triiodothyronine (T3), thyroxine (T4), and free T4 (fT4), as well as U and RCV of these assays. Results: In early infancy, T4 and fT4 values were higher than in the older age groups. Serum T4 95th percentile reference value, useful for the diagnosis of hyperthyroidism, was 142.9 in 20-year-old boys and 230.4 nmol/L in early infants and serum T3 95th percentile was 2.6 and 3.5 nmol/L, respectively, while fT4 2.5th percentile reference value, useful for the diagnosis of hypothyroidism, was 9.6 and 13.0 pmol/L, respectively. Serum TSH 97.5th percentile showed less age variation, 4.38–4.88 mIU/L. Performance of the four assays resulted in approximately 20% Us, reflecting simple and complex imprecision, trueness, analytical and functional sensitivity. RCV of serum TSH (58.6%) was larger than for thyroid hormones (28.3%–34.7%), probably due to the high biological variation of this hormone. Conclusions: We have established reference interval for TSH and thyroid hormones, as well as Us for assessing reliability of measurements, and RCVs to alert users on the presence of clinical significant changes.

Serum IGF-I and IGFBP-3 Reference Values from a Chemiluminescent Assay in Normal Children and Adolescents of Hispanic and Italian Origin: Presence of Sexual Dimorphism in IGF-I Values

Serum IGF-I and IGFBP-3 assays are used to monitor rhGH treatment. Some discrepancies in results obtained by means of different assays have been reported. The aim of this study was to establish normal ranges for circulating IGF-I and IGFBP-3 in children and adolescents of Hispanic and Italian origin. Circulating levels of IGF-I and IGFBP-3 were measured in 169 Hispanic and Italian prepubertal children and 66 adolescents of both sexes, using a chemiluminescent assay. Serum levels of IGF-I and IGFBP-3 increased from early childhood into adolescence. After pubertal peaks of IGF-I and IGFBP-3, slight decreases were observed with increasing age. Furthermore, serum IGF-I levels were significantly higher in girls than in boys, suggesting a sexual dimorphism in serum IGF-I values in late prepuberty and early puberty. Differences in IGF-I and IGFBP-3 absolute values between our study and previous studies suggest the need to establish reference ranges for each ethnic group.

Cut-off values of serum growth hormone (GH) in pharmacological stimulation tests (PhT) evaluated in short-statured children using a chemiluminescent immunometric assay (ICMA) calibrated with the International Recombinant Human GH Standard 98/574.

Hospital de Pediatr i a Prof. Dr. Juan P. Garrahan, Combate de los Pozos 1881, Ciudad Autonoma de Buenos Aires, CP 1245, Argentina, Phone: +54 11 43084300, Fax: +54 11 43086700, mE- ail: echaler@yahoo.com Juan M. Lazzati, Mercedes Maceiras, Marco A. Rivarola and Alicia Belgorosky: Hospital de Pediatr i a Prof. Dr. Juan P. Garrahan , Buenos Aires , Argentina Gabriela Ballerini, Mauro Frusti, Ignacio Bergada and Gabriela Ropelato: Hospital de Ni n os Ricardo Gutierrez , Buenos Aires , Argentina

Unique Dominant Negative Mutation in the N-Terminal Mitochondrial Targeting Sequence of StAR, Causing a Variant Form of Congenital Lipoid Adrenal Hyperplasia

CONTEXT Steroid acute regulatory (StAR) protein is a mitochondria-targeted protein that is part of the transduceosome complex crucial for transport of cholesterol to mitochondria. Recessive mutations cause classic and nonclassic congenital lipoid adrenal hyperplasia. OBJECTIVE The aim of this study was to report the clinical, hormonal, genetic, and functional data of a novel heterozygous mutation in the StAR gene found in a 46,XY patient with ambiguous genitalia and neonatal severe steroidogenic deficiency. PATIENT Undetectable serum steroids with high ACTH and plasma renin activity but normal acute GnRH response were found in infancy. After gonadectomy (at 3 yr of age), serum LH and testosterone were undetectable, whereas FSH was normal but increased slowly afterward. Estrogen replacement therapy, started at 10.2 yr of age, suppressed gonadotropins (for 2 yr). However, after 1 month off estrogens, the patient showed castrated levels. At 11.9 yr old, after fludrocortisone withdrawal because of hypertension, plasma renin activity and aldosterone remained normal, suggesting mineralocorticoid recovery by a StAR-independent mechanism. RESULTS We found a de novo heterozygous IVS-2A>G StAR mutation and the reported heterozygous p.G146A SF1 polymorphism with normal CYP11A1, FDXR, FDX1, VDAC1, and TSPO genes. The mutant StAR transcript lacked exon 2, resulting in the in-frame loss of amino acids 22 to 59 in the N-terminal mitochondrial targeting signal. In vitro, the mutant protein exhibited reduced StAR activity in a dominant-negative manner and almost no mitochondria localization. CONCLUSIONS A misfolded p.G22_L59del StAR might interfere with wild-type StAR activity by blocking the transduceosome complex, causing an autosomal dominant form of StAR deficiency, explaining the clinical phenotype. We speculated that estrogen might have modulated mineralocorticoid function and pubertal maturation in a human natural model lacking endogenous steroid production.

Is BaF3 bioassay useful to identify patients with bioinactive growth hormone

ABSTRACT We analyzed the ability of the BaF3 cell line bioassay to select patients with biologically inactive GH. We first evaluated the biological response of the Ba/F3-hGHR cells to rhGH additional doses from 10 to 5000 pg/ml. The concentration points corresponding to the linear part of the curve were selected. We then analyzed a group of sera, diluted like the standard, including the entire range of GH concentrations that can be analyzed by bioassay. The serum/standard area below the curve ratio was calculated. Serum GH immunoactivity determined by IMMULITE/GH bioactivity ratios was calculated. Our experimental data showed that GH-bioactivity/GH-immunoactivity ratios below 0.303 are indicative of a bioinactive GH molecule. This bioassay would recognize only extreme cases of GH bioinactivity, and it would not be a useful tool in the search for patients with altered forms of GH.

Hydrocortisone treatment in girls with congenital adrenal hyperplasia inhibits serum dehydroepiandrosterone sulfate and affects the GH-IGF-I system.

Sex hormones are modulators of the GH/ IGF-I system. We have hypothesized that the inhibition of DHEAS in treated girls with congenital adrenal hyperplasia (CAH) might affect this modulation. We analyzed serum IGF-I, IGFBP-3 and DHEAS in 17 prepubertal (Pp) and 32 pubertal (Pu) girls with CAH, under hydrocortisone replacement therapy, in the presence of normal (Gr1) or high (Gr2) serum testosterone (T) and androstenedione (A) levels. All groups had appropriate normal controls. Serum DHEAS in patients with CAH was significantly lower than in the respective controls (p < 0.04), except for Pp CAH Gr2. Serum IGF-I, but not serum IGFBP-3, in CAH subgroups was significantly higher than in the respective controls (p < 0.05), except for Pp CAH Gr2. It is concluded that glucocorticoid treatment of girls with CAH results in hypofunction of the adrenal zona reticularis. Low levels of serum DHEAS could be involved in the regulation of IGF-I biological response in target tissues. Additional studies are necessary to confirm these findings.