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Objective: Congenital adrenal hyperplasia (CAH) is an inborn error of metabolism and a common disorder of sex development where >90% of all cases are due to 21-hydroxylase deficiency. Novel and rare pathogenic variants account for 5% of all clinical cases. Here, we sought to investigate the functional and structural effects of four novel (p.Val358Ile, p.Arg369Gln, p.Asp377Tyr, and p.Leu461Pro) and three combinations of CYP21A2 variants (i.e. one allele containing two variants p.[Ile172Asn;Val358Ile], p.[Val281Leu;Arg369Gln], or p.[Asp377Tyr;Leu461Pro]) identified in patients with CAH., Methods: All variants were reconstructed by in vitro site-directed mutagenesis, the proteins were transiently expressed in COS-1 cells and enzyme activities directed toward the two natural substrates (17-hydroxyprogesterone and progesterone) were determined. In parallel, in silico prediction of the pathogenicity of the variants based on the human CYP21 X-ray structure was performed., Results: The novel variants, p.Val358Ile, p.Arg369Gln, p.Asp377Tyr, and p.Leu461Pro exhibited residual enzymatic activities within the range of non-classic (NC) CAH variants (40-82%). An additive effect on the reduction of enzymatic activity (1-17%) was observed when two variants were expressed together, as identified in several patients, resulting in either NC or more severe phenotypes. In silico predictions were in line with the in vitro data except for p.Leu461Pro., Conclusions: Altogether, the combination of clinical data, in silico prediction, and data from in vitro studies are important for establishing a correct genotype and phenotype correlation in patients with CAH., (Copyright © 2019 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

We present the functional and structural effects of seven novel (p.Leu12Met, p.Arg16Cys, p.Ser101Asn, p.Ser202Gly, p.Pro267Leu, p.Gln389_Ala391del, and p.Thr450Met) and two previously reported but not studied (p.Ser113Phe and p.Thr450Pro) CYP21A2 mutations. Functional analyses were complemented with in silico prediction of mutation pathogenicity based on the recently crystallized human CYP21A2 structure. Mutated proteins were transiently expressed in COS-1 cells and enzyme activities towards 17-hydroxyprogesterone and progesterone were determined. Residual enzyme activities between 43% and 97% were obtained for p.Arg16Cys, p.Ser101Asn, p.Ser202Gly, p.Pro267Leu, and p.Thr450Met, similar to the activities of the well-known nonclassic mutations p.Pro453Ser and p.Pro482Ser, whereas the p.Leu12Met variant showed an activity of 100%. Conversely, the novel p.Ser113Phe, p.Gln389_Ala391del, and p.Thr450Pro mutations drastically reduced the enzyme function below 4%. The K m values for all novel variants were in the same order of magnitude as for the wild-type protein except for p.The450Met. The maximum velocity was decreased for all mutants except for p.Leu12Met. We conclude that p.Leu12Met is a normal variant; the mutations p.Arg16Cys, p.Ser101Asn, p.Ser202Gly, p.Pro267Leu, and p.Thr450Met could be associated with very mild nonclassic CAH, and the mutations p.Ser113Phe, p.Gln389_Ala391del, and p.Thr450Pro are associated with classic CAH. The obtained residual activities indicated a good genotype-phenotype correlation., Competing Interests: The authors declare that there are no competing interests regarding the publication of this paper.

Background: Permanent neonatal diabetes mellitus (PNDM) is a rare disorder, characterized by uncontrolled hyperglycemia diagnosed during the first 6 months of life. In general, PNDM has a genetic origin and most frequently it results from heterozygous mutations in KCNJ11, INS and ABCC8 genes. Homozygous or compound heterozygous inactivating mutations in GCK gene as cause of PNDM are rare. In contrast, heterozygosis for GCK inactivating mutations is frequent and results in the maturity-onset diabetes of young (MODY), manifested by a mild fasting hyperglycemia usually detected later in life. Therefore, as an autosomal recessive disorder, GCK-PNDM should be considered in families with history of glucose intolerance or MODY in first relatives, especially when consanguinity is suspected., Results: Here we describe two patients born from non-consanguineous parents within a family. They presented low birth weight with persistent hyperglycemia during the first month of life. Molecular analyses for KCNJ11, INS, ABCC8 did not show any mutation. GCK gene sequencing, however, revealed that both patients were compound heterozygous for two missense combined in a novel GCK-PNDM genotype. The p.Asn254His and p.Arg447Gly mutations had been inherited from their mothers and fathers, respectively, as their mothers are sisters and their fathers are brothers. Parents had been later diagnosed as having GCK-MODY., Conclusions: Mutations' in silico analysis was carried out to elucidate the role of the amino acid changes on the enzyme structure. Both p.Asn254His and p.Arg447Gly mutations appeared to be quite damaging. This is the first report of GCK-PNDM in a Brazilian family.

The syndrome of resistance to thyroid hormone (RTH β) is an inherited disorder characterized by variable tissue hyposensitivity to 3,5,30-L-triiodothyronine (T(3)), with persistent elevation of free-circulating T(3) (FT(3)) and free thyroxine (FT(4)) levels in association with nonsuppressed serum thyrotropin (TSH). Clinical presentation is variable and the molecular analysis of THRB gene provides a short cut diagnosis. Here, we describe 2 cases in which RTH β was suspected on the basis of laboratory findings. The diagnosis was confirmed by direct THRB sequencing that revealed 2 novel mutations: the heterozygous p.Ala317Ser in subject 1 and the heterozygous p.Arg438Pro in subject 2. Both mutations were shown to be deleterious by SIFT, PolyPhen, and Align GV-GD predictive methods., (© Georg Thieme Verlag KG Stuttgart · New York.)

The objective of this study was to review the growth curves for Turner syndrome, evaluate the methodological and statistical quality, and suggest potential growth curves for clinical practice guidelines. The search was carried out in the databases Medline and Embase. Of 1006 references identified, 15 were included. Studies constructed curves for weight, height, weight/height, body mass index, head circumference, height velocity, leg length, and sitting height. The sample ranged between 47 and 1,565 (total = 6,273) girls aged 0 to 24 y, born between 1950 and 2006. The number of measures ranged from 580 to 9,011 (total = 28,915). Most studies showed strengths such as sample size, exclusion of the use of growth hormone and androgen, and analysis of confounding variables. However, the growth curves were restricted to height, lack of information about selection bias, limited distributional properties, and smoothing aspects. In conclusion, we observe the need to construct an international growth reference for girls with Turner syndrome, in order to provide support for clinical practice guidelines.

Background and Aim: Turner syndrome (TS) patients have an increased risk of weight gain and metabolic syndrome. To date, it is unknown what factors are involved in this metabolic process, even though it is recognized that TS patients are frequently born small-for-gestational age. The aim of this study was to evaluate the correlation between lipid and glucose profiles with being overweight and birth weight and length in TS patients., Study Design: This was a cross-sectional study., Subjects and Outcome Measures: Serum glucose, insulin (HOMA-IR), total cholesterol, and triglycerides were measured in 64 patients with TS. Data regarding birth weight and length and current body mass index (BMI) were also evaluated., Results: Total cholesterol showed a significant negative correlation with birth weight and a positive correlation with BMI; triglycerides showed significant negative correlation with birth weight and length and a positive correlation with BMI; and HOMA-IR showed a significant negative correlation with birth weight and length. Low birth weight and a high BMI were predictive for 28% of total cholesterol and triglycerides; and low birth weight for 22% of HOMA-IR., Conclusions: Lipid profile was correlated with a high current BMI and low birth weight and length in TS patients and glucose profile only with low birth weight. Thus far, growth retardation may play a role in metabolic derangements in this group of patients, being considered another example of fetal programming., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

This study analyzes the body composition of young adult women with Turner syndrome (TS) either treated or not treated with recombinant human growth hormone (rhGH) and compares them with a group of healthy women. Fifty-two non-treated TS patients (23.0 ± 5.8 years), 30 treated with rhGH (21.5 ± 1.5 years), and 133 healthy young adult women (22.9 ± 3.2 years) were evaluated regarding height (H) and weight, body mass index (BMI), brachial perimeter and tricipital cutaneous fold (fat and lean areas at the arm), sitting height (SRH = sitting height/H × 100), leg length (leg/H), waist and hip circumferences (waist/hip), and bioimpedance (percentages of water, lean mass, and fat mass). Age at start of rhGH therapy varied from 7.8 to 15.1 years (10.0 ± 1.3 years), duration of treatment from 2.8 to 8.2 years (3.7 ± 1.5 years), and the mean dose was 0.42 mg/kg/w (from 0.32 to 0.50 mg/kg/w). Body composition (except height) did not differ between TS groups, but there were differences when compared to the control group: weight and sitting height were lower in TS patients; and BMI, SHR, and leg/H were higher. There was an association between all groups with regards to BMI, waist, SHR, and leg/H, but not in percentage of fat mass. SHR was positively correlated with BMI, waist, hip, and percentage of fat mass. This sample of TS patients (with and without rhGH therapy) did not differ in BMI or body composition. However, there were differences between patients with TS patients and normal healthy women. Regardless of rhGH therapy, TS patients should be monitored, particularly for sitting height, SHR, leg length, leg/H, and waist/hip.

Background: Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is caused by deletions, large gene conversions or mutations in CYP21A2 gene. The human gene is located at 6p21.3 within a locus containing the genes for putative serine/threonine Kinase RP, complement C4, steroid 21-hydroxylase CYP21 tenascin TNX, normally, in a duplicated cluster known as RCCX module. The CYP21 extra copy is a pseudogene (CYP21A1P). In Brazil, 30-kb deletion forming monomodular alleles that carry chimeric CYP21A1P/A2 genes corresponds to ~9% of disease-causing alleles. Such alleles are considered to result from unequal crossovers within the bimodular C4/CYP21 locus. Depending on the localization of recombination breakpoint, different alleles can be generated conferring the locus high degree of allelic variability. The purpose of the study was to investigate the variability of deleted alleles in patients with 21-hydroxylase deficiency., Methods: We used different techniques to investigate the variability of 30-kb deletion alleles in patients with 21-hydroxylase deficiency. Alleles were first selected after Southern blotting. The composition of CYP21A1P/A2 chimeric genes was investigated by ASO-PCR and MLPA analyses followed by sequencing to refine the location of recombination breakpoints. Twenty patients carrying at least one allele with C4/CYP21 30-kb deletion were included in the study., Results: An allele carrying a CYP21A1P/A2 chimeric gene was found unusually associated to a C4B/C4A Taq I 6.4-kb fragment, generally associated to C4B and CYP21A1P deletions. A novel haplotype bearing both p.P34L and p.H62L, novel and rare mutations, respectively, was identified in exon 1, however p.P30L, the most frequent pseudogene-derived mutation in this exon, was absent. Four unrelated patients showed this haplotype. Absence of p.P34L in CYP21A1P of normal controls indicated that it is not derived from pseudogene. In addition, the combination of different approaches revealed nine haplotypes for deleted 21-hydroxylase deficiency alleles., Conclusions: This study demonstrated high allelic variability for 30-kb deletion in patients with 21-hydroxylase deficiency indicating that a founder effect might be improbable for most monomodular alleles carrying CYP21A1P/A2 chimeric genes in Brazil.

The aim of this study was to evaluate the physical measurements and body composition of female patients with the classic form of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. Twenty-eight girls with CAH were classified according to both hormonal control (well or not well controlled) and the clinical form of the disease (simple virilizing or salt-wasting). In the control group, 112 healthy individuals were included, divided into two subgroups (male and female). Both patients and controls were subdivided by age into three groups according to pubertal stage: < or =10 years (prepubertal), 11-15 years (pubertal), and > or =15 years (postpubertal). Anthropometrical evaluations and bioelectrical impedance were used to obtain the physical measurement and body composition data. The patients with the simple virilizing form presented higher values for BMI, waist, arm fat area, and fat mass percentage. The not well controlled group presented shorter leg length. Values obtained for BMI as well as for arm fat area, brachial circumference, waist, hip, bi-iliac diameter and fat mass percentage were significantly higher in the patients than in the controls, whereas leg length, hand size and the percentages of water and lean mass were lower. Alterations in body composition were observed in all age groups, mainly by increase of fat mass with age. After puberty, impairments in limb measurements (leg, hand and foot) were more evident. Patients with CAH presented differences in anthropometric parameters but mainly in body composition. Hence, more comprehensive and careful anthropometric evaluation during monitoring of patients with CAH is recommended.

The aim of this work was to analyse C4 genotypes, C4 protein levels, phenotypes and genotypes in patients with the classical form of 21-hydroxylase deficiency. Fifty-four patients from 46 families (36 female, 18 male; mean age 10.8 years) with different clinical manifestations (31 salt-wasting; 23 simple-virilizing) were studied. Taq I Southern blotting was used to perform molecular analysis of the C4/CYP21 gene cluster and the genotypes were defined according to gene organization within RCCX modules. Serum C4 isotypes were assayed by enzyme-linked immunosorbent assay. The results revealed 12 different haplotypes of the C4/CYP21 gene cluster. Total functional activity of the classical pathway (CH50) was reduced in individuals carrying different genotypes because of low C4 concentrations (43% of all patients) to complete or partial C4 allotype deficiency. Thirteen of 54 patients presented recurrent infections affecting the respiratory and/or the urinary tracts, none of them with severe infections. Low C4A or C4B correlated well with RCCX mono-modular gene organization, but no association between C4 haplotypes and recurrent infections or autoimmunity was observed. Considering this redundant gene cluster, C4 seems to be a well-protected gene segment along the evolutionary process.

Although autoimmune thyroid disease (AITD) is frequent in Turner's syndrome (TS), followup studies are scant, and there are none regarding subclinical thyroiditis. We investigated thyroid function and morphology in 17 patients with TS (mean age 14.6 years) with transient and asymptomatic variations of TSH and/or thyroid hormones. Our 2-year follow-up included measurements of TSH, free T4, T3 and TPO and Tg antibodies, ultrasound (US) (first and last evaluations) and scintigraphy (first evaluation). Thyroid volume was evaluated relative to the patients' stature. Fourteen had abnormal hormones, including four with hypothyroidism and one with hyperthyroidism, ten had positive antibodies, and all had abnormalities on US; uptake was normal in 14/16. Abnormal hormones were independent of antibodies, number of US findings, age, time of disease and volume. At the end of the follow-up, antibodies were associated with a high number of abnormal US features, particularly heterogeneous texture. Our results indicate that recurring thyroid hormone variations in TS are due to chronic AITD.

Short stature is the main factor of emotional impact in girls and women with Turner's syndrome (TS). Growth hormone, alone or associated with sex steroids, allows better adult height. The results of spontaneous final height (FH) in TS can help to evaluate the real cost-benefit of any treatment to improve FH in patients from the same population. The aim of this study was to determine spontaneous FH in women TS and to look for factors which influence it. We evaluated 58 patients with TS who attained FH. Data of weight and length at birth, parents' heights, karyotype, spontaneous puberty and sex hormone replacement were obtained. Mean FH was 144.8 cm and target height 157.0 cm. FH was correlated only to maternal height. The deficit in FH was lower than that found in other Brazilian studies but similar to that described in the literature. This study may help verify the efficacy of therapeutic actions on FH of Brazilian women with TS.

Objective: To evaluate the growth and body composition of pre-pubertal diabetic children, and to check for influence of the age of diabetes onset and length, sex, insulin requirement and glycosylated hemoglobin., Patients and Methods: 59 diabetic children (39 M; 29 F), age 1.2-11.5 years, and 67 controls (36 M; 31 F), age 1.2-11.7 years were included. Weight, height, body mass index (BMI), arm circumference, skin folds, fat mass and muscle areas were evaluated and transformed into standard deviation scores (SDS)., Results: Among the diabetic children the mean height SDS was -0.13 (+/- 0.97) while in the control group it was 0.28 (+/- 0.86) (p= 0.013). The difference between the first and the current height SDS showed that the height SDS decreased significantly (p< 0.001) and multiple regression analysis indicated correlation with the duration of the disease. The mean arm fat SDS also revealed difference (p< 0.001). The means for weight, BMI, addition of 3 skinfolds and muscle mass did not demonstrate difference between the groups., Conclusions: The diabetic children showed reduction of height SDS during the period studied and they were significantly shorter than the controls, even though their statures were within the population standards. The arm fat area also showed an increase in relation with the controls.

Glucocorticoids are essential in the treatment of patients with congenital adrenal hyperplasia (CAH). The opposite actions of glucocorticoids and androgens in bone mass achievement justify a study of bone mineral density (BMD) in these patients. We evaluated BMD in patients with CAH due to classic 21-hydroxylase (CYP21A2) deficiency and investigated the involvement of clinical and laboratory factors in the BMD. This study assessed the clinical and laboratory factors involved in BMD of 45 patients at the Pediatric Unit of Endocrinology, UNICAMP, who had been diagnosed as having classical CAH due to CYP21A2 deficiency including molecular characterization. The sample consisted of 28 females and 17 males; 23 salt-wasting (SW) and 22 simple virilizing (SV) cases, with average of 9.9 years (ranges, 5.1-16.3 years) when bone densitometry was performed. The DEXA method was used for calculating the areal BMD Z score in L2-L4. The variables were analyzed with reference to the BMD for chronological age (BMD/CA), height age (BMD/HA), and bone age (BMD/BA). The mean Z score for BMD/CA was 0.08 +/- 1.21 (-2.55 to 2.64); it was 0.29 +/- 1.33 (-2.01 to 4.00) for BMD/HA, and -0.90 +/- 1.24 (-3.41 to 1.92) for BMD/BA. The BMD/CA was significantly lower in females and in patients on treatment for a long period and of more advanced chronological age. Weight and body mass index (BMI) Z scores showed a positive correlation with advanced BA. The higher the weight and BMI Z scores, the higher the BMD/HA. The BMD/BA values were significantly higher in the group in which BA was closer to CA. The BMD/BA value was significantly lower when compared to the value obtained with height and chronological ages. Sex, duration of treatment, weight, BMI, and bone age have an effect on areal BMD in patients with CAH due to CYP21A2 deficiency, which may be underestimated when evaluated in relation to CA and HA.

The authors report a case of a virilizing adrenal tumor that developed in a 2-year-old child with Beckwith-Wiedemann syndrome (BWS). He had a fetal diagnosis of omphalocele and a history of neonatal adrenal cysts. The importance of prenatal diagnosis of BWS and postnatal follow-up of tumors is discussed. The differential diagnosis of adrenal pathologies occurring in BWS also is reviewed.