Potential for Optimization of Growth Hormone Treatment in Children with Growth Hormone Deficiency (GHD), Small for Gestational Age (SGA), and Turner Syndrome (TS) in Germany - Data from the PATRO® Children Study.

INTRODUCTION: Growth hormone (GH) treatment in children with growth hormone deficiency (GHD), short children born small for gestational age (SGA), and Turner syndrome (TS) is well established. However, a variety of parameters are still under discussion to achieve optimal growth results and efficiency of GH use in real world treatment. METHODS: German GH-treatment naïve patients of the PATRO Children database were grouped according to their start of treatment into groups of 3 years from 2007 to 2018. Time trends in age, gender, GH dose, height standard deviation score (SDS), first year growth response, and Index of Responsiveness (IoR) were investigated in children with GHD, short children born SGA, and TS starting GH treatment in the German patient population of the PATRO Children database from 2007 - 2018 to determine specific parameters for GH treatment optimization. RESULTS: All patient groups started GH treatment at a relatively high chronological age (2007 - 2009: GHD 8.33 ± 3.19, SGA 7.32 ± 2.52, TS 8.65 ± 4.39) with a slight but not significant trend towards younger therapy start up to 2016 to 2018 (GHD 8.04 ± 3.36, SGA 6.67 ± 2.65, TS 7.85 ± 3.38). In the GHD and SGA groups female patients were underrepresented compared to male patients (GHD 32.3 %, SGA 43.6 %) with no significant change over the 4 time periods. Patients with GHD started GH treatment at a low dose (0.026 mg/kg/day). In SGA and TS patients GH therapy was started below the registered dose recommendation (0.030 mg/kg/day and 0.0337 mg/kg/day, respectively). In the first year of treatment the mean GH dose was increased moderately (GHD: 0.0307, SGA: 0.0357, TS: 0.0408 mg/kg/day). There was no significant change of GH dosing over time from 2007 - 2018. The IoR was comparable between time-groups for all three diagnoses. DISCUSSION: This study shows potential for improvement of GH treatment results in GHD, SGA, and TS patients in terms of early dose adjustment and younger age at start of treatment. This is in accordance with important parameters used in prediction models.

Cross-sectional analysis: clinical presentation of children with persistently low ALP levels.

OBJECTIVES: Low activity of serum alkaline phosphatase (ALP) is a hallmark of hypophosphatasia (HPP), but low readings of ALP are not always recognized in clinical routine. Understanding the clinical presentations associated with low ALP may contribute to a timelier diagnosis of HPP. METHODS: Data from paediatric patients with low ALP, excluding patients in intensive care and with oncological/haematological disorders, were analysed. Most recent ALP values, previous diagnoses, medication and relevant symptoms were extracted from patient records at nine specialised centres and analysed descriptively. A relationship between body height and ALP values was scrutinised by linear regression. RESULTS: Of 370 children, 15 (4.1%) had a diagnosis of HPP. In the subgroup without a diagnosis of HPP, 241 (67.9%) out of 355 patients had one or more medical conditions known to be associated with low serum ALP. Of those, hypothyroidism, malnutrition and steroid administration were most frequent. Characteristic symptoms, particularly, short stature, muscle weakness and delay of motor development were more frequent and ALP values were lower in patients with documented HPP diagnosis compared to patients without diagnosis of HPP (Ø z-scores: -2.52) (interquartile range [IQR] = 0.20) vs. -1.96 (IQR = 0.87). A weak positive linear relationship between z-scores of ALP and body height was identified (p<0.001). CONCLUSIONS: This analysis of paediatric patient records elucidates a wide range of disorders associated with low ALP activity. In case of additional specific symptoms, HPP should always be considered as a differential diagnosis.

Increased Insulin Concentrations During Growth Hormone Treatment in Girls With Turner Syndrome Are Ameliorated by Hormone Replacement Therapy.

Objective: Turner syndrome (TS) is characterized by complete or partial loss of one sex chromosome and is commonly associated with short stature, metabolic changes (such as central obesity, abnormal glucose tolerance and high triglycerides) and premature ovarian insufficiency (POI). Primary management of TS during childhood and adolescence comprises treatment with human growth hormone (hGH) and, in cases with early loss of ovarian function, hormone replacement therapy (HRT). Given that metabolic parameters are altered when HRT is applied during menopause, we analyzed whether metabolic changes might be positively or negatively affected within 10 years after HRT and/or hGH in girls with TS. Design: Observational study. Methods: Data were collected from the medical records of 31 girls with TS attending two endocrinologic centers in Germany between 2000 and 2020. Descriptive statistics are reported as the mean ± SEM or percentages. Results: The mean age at first presentation was 99.06 ± 8.07 months, the mean height was 115.8 ± 3.94 cm, and the mean BMI 19.0 ± 0.99 was kg/m2. Treatment with hGH was given to 96.8% of the girls, starting at an average age of 99.06 ± 8.70 months, and was continued for 67.53 ± 6.28 months. HRT was administered to 80.6% of all patients and was started at a mean age of 164.4 ± 4.54 months. During the follow-up, we did not observe any significant absolute changes in lipid parameters, but we detected beneficial effects of childhood hGH: significantly lower cholesterol (-0.206/month; p = 0.006), lower low density lipoprotein cholesterol (-0.216/month; p = 0.004), and higher high density lipoprotein cholesterol (+0.095/month; p = 0.048). Insulin concentrations, showed a significant increase attributable to hGH treatment (+0.206/month; p = 0.003), which was ameliorated by concomitant or subsequent HRT (-0.143/month; p = 0.039). Conclusion: Treatment with hGH and HRT is provided to most girls with TS. Metabolic effects are associated with both modalities. Monitoring of metabolic changes appears to be important to detect unfavorable effects, and could guide treatment adjustment and duration.

Safety and Effectiveness of Omnitrope®, a Biosimilar Recombinant Human Growth Hormone: More Than 10 Years' Experience from the PATRO Children Study.

INTRODUCTION: Omnitrope® was approved as a biosimilar recombinant human growth hormone (rhGH) in 2006. OBJECTIVE: The purpose of this work was to evaluate the long-term safety and effectiveness of Omnitrope® in PATRO Children - an ongoing, international, longitudinal, non-interventional study in children who require rhGH treatment. METHODS: The study population includes infants, children, and adolescents receiving Omnitrope®. Adverse events (AEs) are monitored for safety and rhGH effectiveness is evaluated by calculation of the height standard deviation score (HSDS), height velocity (HV), and HVSDS using height measurements and country-specific references. RESULTS: As of November 2017, 6,009 patients from 298 centers across 14 countries were enrolled in PATRO Children. Overall, 57.7% of patients had growth hormone deficiency (GHD), 25.8% were born small for gestational age (SGA), and 4.8% had Turner syndrome (TS). In total, 84.1% were rhGH treatment naïve at study entry. The mean duration of Omnitrope® treatment in the study was 36.1 months (range 0-133.7). Overall, 10,360 AEs were reported in 2,750 patients (45.8%). Treatment-related AEs were reported in 396 patients (6.6%; 550 events), and serious AEs (SAE) in 636 patients (10.6%; 1,191 events); 50 SAEs in 37 patients (0.6%) were considered treatment related. Following 5 years of therapy in patients who were rhGH treatment naïve at study entry, improvement from baseline in mean HSDS was +1.85 in GHD, +1.76 in SGA, and +1.0 in TS patients. In total, 912 (17.9%) patients reached adult height (n = 577 GHD, n = 236 SGA, n = 62 TS). CONCLUSIONS: This analysis of PATRO Children indicates that biosimilar rhGH is well tolerated and effective in real-world clinical practice.

Diabetes screening in overweight and obese children and adolescents: choosing the right test.

Type 2 diabetes can occur without any symptoms, and health problems associated with the disease are serious. Screening tests allowing an early diagnosis are desirable. However, optimal screening tests for diabetes in obese youth are discussed controversially. We performed an observational multicenter analysis including 4848 (2668 female) overweight and obese children aged 7 to 17 years without previously known diabetes. Using HbA1c and OGTT as diagnostic criteria, 2.4% (n = 115, 55 female) could be classified as having diabetes. Within this group, 68.7% had HbA1c levels ≥48 mmol/mol (≥6.5%). FPG ≥126 mg/dl (≥7.0 mmol/l) and/or 2-h glucose levels ≥200 mg/dl (≥11.1 mmol/l) were found in 46.1%. Out of the 115 cases fulfilling the OGTT and/or HbA1c criteria for diabetes, diabetes was confirmed in 43.5%. For FPG, the ROC analysis revealed an optimal threshold of 98 mg/dl (5.4 mmol/l) (sensitivity 70%, specificity 88%). For HbA1c, the best cut-off value was 42 mmol/mol (6.0%) (sensitivity 94%, specificity 93%). CONCLUSIONS: HbA1c seems to be more reliable than OGTT for diabetes screening in overweight and obese children and adolescents. The optimal HbA1c threshold for identifying patients with diabetes was found to be 42 mmol/mol (6.0%). What is Known: • The prevalence of obesity is increasing and health problems related to type 2 DM can be serious. However, an optimal screening test for diabetes in obese youth seems to be controversial in the literature. What is New: • In our study, the ROC analysis revealed for FPG an optimal threshold of 98 mg/dl (5.4 mmol/l, sensitivity 70%, specificity 88%) and for HbA1c a best cut-off value of 42 mmol/mol (6.0%, sensitivity 94%, specificity 93%) to detect diabetes. Thus, in overweight and obese children and adolescents, HbA1c seems to be a more reliable screening tool than OGTT.

Low association between fasting and OGTT stimulated glucose levels with HbA1c in overweight children and adolescents.

BACKGROUND: Diabetes and prediabetes are defined based on different methods such as fasting glucose, glucose at 2-hour in oral glucose tolerance test (OGTT), and glycated hemoglobin A1c (HbA1c). These parameters probably describe different deteriorations in glucose metabolism limiting the exchange between each other in definitions of diabetes. OBJECTIVE: To investigate the relationship between OGTT and HbA1c in overweight and obese children and adolescents living in Germany. METHODS: Study population: Overweight and obese children and adolescents (n = 4848; 2668 female) aged 7 to 17 years without known diabetes. The study population was stratified into the following subgroups: normal glucose tolerance, prediabetes, diabetes according to OGTT and/or HbA1c categories, confirmed diagnosis of diabetes. RESULTS: In the entire study group fasting plasma glucose (FPG) correlated weakly to 2-hour glucose (r = 0.26), FPG correlated weakly to HbA1c (r = 0.18), and 2-hour glucose correlated weakly to HbA1c (r = 0.17, all P < .001). Patients with confirmed diabetes showed a very high correlation between FPG and 2-hour glucose (r = 0.73, n = 50). Moderate correlations could be found for patients with impaired fasting glucose (2-hour glucose vs HbA1c: r = 0.30, n = 436), for patients with diabetes according to OGTT and/or HbA1c (FPG vs 2-hour glucose: r = 0.43; 2-hour glucose vs HbA1c: r = -0.30, n = 115) and for patients with confirmed diabetes (2-hour glucose vs HbA1c: r = -0.47, all P < .001). CONCLUSIONS: Because FPG, 2-hour glucose, and HbA1c correlated only weakly we propose that these parameters, particularly in the normal range, might reflect distinct aspects of carbohydrate metabolism.

Acceptance of a reusable self-injection device for recombinant human growth hormone: final data from a questionnaire-based, cross-sectional, international, multicenter, observational study in pediatric patients.

BACKGROUND: A questionnaire-based survey was conducted to assess attitudes toward a reusable self-injection system (SurePal™) among pediatric patients with growth disturbances who were prescribed treatment with Omnitrope(®) within routine clinical practice. METHODS: This was a multicenter, observational study, incorporated into the noninterventional PAtients TReated with Omnitrope(®) (PATRO) Children study. Included subjects, or their caregivers, completed a questionnaire on the following five main areas: attractiveness of SurePal™, training received, using the device, the low drug wastage system, and experience versus other devices used previously (pretreated patients). Responses were based on a 5-point scale, with 2 being the best possible outcome and -2 the worst possible outcome. RESULTS: In total, 550 patients were included in this study (338 from France, 169 from Germany, and 43 from the UK). The mean age ± standard deviation of participants was 10.8±3.5 years; the majority (57%) were male and growth hormone treatment naïve (88%). Almost half (49.8%) of children prepared their SurePal™ for injection themselves and 45.5% performed injections themselves. As patients progressed into their teens, the majority (≥75%) favored preparing SurePal™ and performing injections themselves, rather than seeking assistance. The attractiveness of SurePal™ was rated as excellent/good by 84.7% of patients overall; this rating was similarly high (≥79%) across countries and age-groups. Preparing (88.8%) and using (83.3%) SurePal™ were rated as very easy/easy by most patients; these ratings were similarly high, irrespective of country or age-group. The dose-memory function was rated as very helpful/helpful by 66.2% of patients. Among 246 patients who reported using the low drug-waste feature, 87.4% found it helpful. Among pretreated patients (n=64), 78.2% reported that SurePal™ was much better/better than their previous device. CONCLUSION: These data confirm the ease of use and patient preference for SurePal™ among pediatric patients with growth disturbances.

Acceptability of the reusable SurePal™ self-injection device for Omnitrope(®) among pediatric patients: results from a questionnaire-based, cross-sectional, multicenter observational study.

BACKGROUND: SurePal™ is a reusable self-injection system that has been developed to support daily administration of Omnitrope(®) (Sandoz, Kundl, Austria). A questionnaire-based cross-sectional survey was conducted to evaluate acceptability of, and preference for, SurePal™ in pediatric patients who were prescribed treatment with Omnitrope(®) within routine clinical care. METHODS: This multicenter, observational study was incorporated into the ongoing non-interventional PATRO (PAtients TReated with Omnitrope(®)) Children study. Patients (or caregivers) were provided with a questionnaire that included five main topics; attractiveness of the device, training received, using SurePal™, the low drug wastage system, and experience versus other devices used previously (where applicable). Questions were scored on a 5-point scale, with -2 being the worst possible outcome (eg, very hard/very poor) and 2 being the best possible outcome (eg, very easy/excellent). RESULTS: A total of 186 patients were included in this study (Germany, n=154; UK, n=32). The attractiveness of SurePal™ was rated as excellent/good by 87.1% of patients. Overall, 86.5% of patients found that using their SurePal™ was very easy/easy. Almost all patients (96.2%) found that preparing their SurePal™ for injection was very easy/easy, and 89.2% found that injecting with SurePal™ was very easy/easy. 85.5% of patients recorded that the dose memory function was helpful, and 87.6% that taking their SurePal™ apart after an injection was very easy/easy. Of the 88 patients who recorded that they had used the low drug waste feature, 89.8% found the feature to be helpful. Among pre-treated patients (n=42), 81% recorded that SurePal™ was much better/better than their previously used device. CONCLUSION: This questionnaire-based cross-sectional survey in pediatric patients confirms the ease of use and patient preference for SurePal™, a reusable self-injection system that has been developed to support daily administration of Omnitrope(®).

Treatment of central precocious puberty and early puberty with GnRH analog in girls with Williams-Beuren syndrome.

OBJECTIVE: Little has been published on treatment of precocious puberty in girls with Williams-Beuren syndrome (WBS), a condition occurring frequently in this group. We analyzed our own data on growth/age at menarche of now adult female patients with WBS being diagnosed with central precocious puberty or early puberty. Data of patients treated with gonadotropin-releasing hormone (GnRH) analog (n=13) were compared with those not treated (control group, n=11). PATIENTS: Longitudinal data on the somatic development of 24 now adult female patients were analyzed. RESULTS: Medium final height was 157.2±6.5 cm compared to 151.4±5.6 cm in the control group. No significant difference could be found in the discrepancy of genetic target height and final height. Prepubertally girls were normal weight in both groups; in adulthood the majority of patients were overweight/obese. Menarche commenced 11 months after cessation of therapy. CONCLUSION: As already known from other studies, hormonal suppression via GnRH analog was well tolerated.

Hormonal 'minipuberty' influences the somatic development of boys but not of girls up to the age of 6 years.

OBJECTIVE: Hormonal 'minipuberty' refers to a transient sex-specific surge of LH, FSH, testosterone (T) and estradiol (E2) in the first few months of life. We hypothesized a potential long-term effect of this hormonal surge on somatic parameters in the following years and therefore designed this longitudinal study. DESIGN: A hierarchical multiple regression analysis was used to analyse the potential influence of hormone concentrations during minipuberty on anthropometric measurements conducted in the first 6 years of life. PATIENTS: Thirty-five healthy babies (17 male, 18 female) were the participants. MEASUREMENTS: Testosterone, E2, SHBG, LH and FSH were measured at the ages of four, eight and 20 weeks. Anthropometric measurements were taken eight times in the first 12 months, then every 6 months up to the age of 6 years. RESULTS: A significant negative effect was found in boys between testosterone and LH levels at 8 weeks and body weight up to the age of 6 years and BMI up to 6 years (LH) and 3 years (T), respectively. A further negative effect was found between E2 levels at the age of 20 weeks and body weight as well as body length in the years that followed. A positive effect was observed between E2 at the age of 4 weeks and skinfold thickness up to the age of 6 years in boys. No significant effects were found in girls. CONCLUSIONS: The findings seem to reflect an up to now unknown long-term influence of the physiological early hormonal surge on the subsequent male but not female somatic development.

No significantly increased frequency of the inversion polymorphism at the WBS-critical region 7q11.23 in German parents of patients with Williams-Beuren syndrome as compared to a population control.

BACKGROUND: Typical Williams-Beuren syndrome (WBS) is commonly caused by a ~1.5 Mb - ~1.8 Mb heterozygous deletion of contiguous genes at chromosome region 7q11.23. The majority of WBS cases occurs sporadically but few familial cases of autosomal dominant inheritance have been reported. Recent data demonstrated the existence of the paracentric inversion polymorphism at the WBS critical region in 7q11.23 in some of the progenitors transmitting the chromosome which shows the deletion in the affected child. In parents having a child affected by WBS the prevalence of such a structural variant has been reported to be much higher (~25- ~30%) than in the general population (~1- ~6%). However, in these previously reported studies only a limited number of randomly selected patients and non transmitting parents of WBS patients were used as controls, but without specification of any clinical data. Therefore we have undertaken a German population-based molecular cytogenetic investigation. We evaluated the incidence of the paracentric inversion polymorphism at 7q11.23 analyzing interphase nuclei of lymphocytes using a three color fluorescence in situ hybridization (FISH) probe. RESULTS: FISH analysis was carried out on couples with a child affected by WBS as compared to a population sample composed of different normal individuals: Control group I: couples with two healthy children, control group II: couples with fertility problems, planning ICSI and control group III: couples with two healthy children and one child with a chromosome aberration, not involving region 7q11.23. The three color FISH assay showed that the frequency of the paracentric inversion polymorphism at 7q11.23 in couples with a child affected by WBS was 20.8% (5 out of 24 pairs) as compared to 8.3% (2 out of 24 pairs, control group I), 25% (4 out of 16 pairs, control group II) and 9.1% (1 out of 11 pairs, control group III), respectively (total 7 out of 51 pairs, 13.8%). The frequencies differed between the groups, but this was statistically not significant (p > 0.05, Fisher's test). CONCLUSION: Our results do not support the hypothesis that the paracentric inversion polymorphism at 7q11.23 is a major predisposing factor for the WBS deletion.

A large-scale mutation search reveals genetic heterogeneity in 3M syndrome.

The 3M syndrome is a rare autosomal recessive disorder recently ascribed to mutations in the CUL7 gene and characterized by severe pre- and postnatal growth retardation. Studying a series of 33 novel cases of 3M syndrome, we have identified deleterious CUL7 mutations in 23/33 patients, including 19 novel mutations and one paternal isodisomy of chromosome 6 encompassing a CUL7 mutation. Lack of mutations in 10/33 cases and exclusion of the CUL7 locus on chromosome 6p21.1 in six consanguineous families strongly support the genetic heterogeneity of the 3M syndrome.

Sex hormone testosterone affects language organization in the infant brain.

Using a phonological discrimination paradigm, we show that the brain responses of 4-week-old infants systematically vary as a function of biological sex and testosterone level. Females who are generally low on testosterone demonstrated a clear phonological discrimination effect with a bilateral distribution. In male infants this effect systematically varied as a function of testosterone level. Males with high testosterone showed no discrimination effect, whereas males with low testosterone displayed a discrimination effect, which was clearly left-lateralized. The present data provide evidence for a strong influence of testosterone on language function and lateralization already present during the first weeks of life.

Long-term GnRH agonist treatment for female central precocious puberty does not impair reproductive function.

Depot gonadotropin releasing hormone (GnRH) agonist (GnRHa) therapy is the treatment of choice for patients with central precocious puberty (CPP). It is still unclear whether long-term exposure to GnRHa is associated with impaired reproductive function in adulthood. The present study was performed on 46 women, former CPP patients, 12.5+/-3.7 years after the discontinuation of treatment with depot GnRHa. In a structured interview, we assessed general health status, clinical signs possibly associated with hyperandrogenism, menstrual cycle, gynaecological diseases and reproductive function. It appears that long-term treatment with depot GnRHa is safe and does not impair reproductive function. The risk of former CPP patients to develop hirsutism and/or polycystic ovary syndrome does not seem to be increased compared to the normal population but this issue needs to be addressed in further long-term follow-up studies.

Prevalence of autoantibodies associated with thyroid and celiac disease in Ullrich-Turner syndrome in relation to adult height after growth hormone treatment.

A prospective, multicenter study of patients with Ullrich-Turner syndrome (UTS) was conducted to estimate the prevalence of autoantibodies to tissue transglutaminase (tTg), thyroid stimulating hormone receptor (TSH-R), thyroglobulin (TG) and thyroid peroxidase (TPO) in relation to adult height after long-term growth hormone (GH) treatment. Out of 347 near-adult (> 16 years) patients with UTS from 96 German centers, whose longitudinal growth was documented within the Pharmacia International Growth Study (KIGS), 188 returned for a standardized follow-up visit at a median chronological age of 18.7 (16.0-23.6) years (bone age > 15 years). Serum samples of 120 patients were obtained for central measurements of TSH, thyroxine (T4) and free T4 and autoantibodies by standard immunoassays. Information regarding thyroid disease, karyotype and anthropometric data was extracted from the KIGS database. Thirty-six percent of the patients with UTS had positive TG and/or TPO autoantibodies and 4% had positive tTg autoantibodies, whereas 2% had positive TG and/or TPO autoantibodies as well as positive tTg autoantibodies. TSH-R autoantibodies were undetectable in all patients. The detection of autoantibodies was unrelated to a specific karyotype. Median height standard deviation scores (SDS, UTS) at start of GH treatment (0.43; -1.07, 1.85) and at follow-up (1.36; -0.11, 2.57) were comparable in all patients independent of their antibody status. The total deltaheight SDS, however, was higher in patients with negative autoantibody titers (1.08; -0.03, 2.25) compared to those with positive antibody titers (0.68; -0.44, 1.82; p < 0.01). Our study confirms the high prevalence of autoantibodies in patients with UTS predisposing them to autoimmune thyroid disease and celiac disease, and indicates for the first time that autoimmune pathologies may interfere with GH therapy and thus compromise final height. Therefore, medical care for patients with UTS should routinely include screening for these autoimmune disorders in order to assure early detection and appropriate treatment.

Early manifestation of calcinosis cutis in pseudohypoparathyroidism type Ia associated with a novel mutation in the GNAS gene.

OBJECTIVE: To clarify the molecular defect for the clinical finding of congenital hypothyroidism combined with the manifestation of calcinosis cutis in infancy. CASE REPORT: The male patient presented with moderately elevated blood thyrotropin levels at neonatal screening combined with slightly decreased plasma thyroxine and tri-iodothyronine concentrations, necessitating thyroid hormone substitution 2 weeks after birth. At the age of 7 months calcinosis cutis was seen and the patient underwent further investigation. Typical features of Albright's hereditary osteodystrophy (AHO), including round face, obesity and delayed psychomotor development, were found. METHODS AND RESULTS: Laboratory investigation revealed a resistance to parathyroid hormone (PTH) with highly elevated PTH levels and a reduction in adenylyl cyclase-stimulating protein (Gsalpha) activity leading to the diagnosis of pseudohypoparathyroidism type Ia (PHP Ia). A novel heterozygous mutation (c364T > G in exon 5, leading to the amino acid substitution Ile-106 --> Ser) was detected in the GNAS gene of the patient. This mutation was not found in the patient's parents, both of whom showed normal Gsalpha protein activity in erythrocytes and no features of AHO. A de novo mutation is therefore likely. CONCLUSIONS: Subcutaneous calcifications in infancy should prompt the clinician to a thorough search for an underlying disease. The possibility of AHO and PHP Ia should be considered in children with hypothyroidism and calcinosis cutis. Systematic reviews regarding the frequency of calcinosis in AHO are warranted.

The residue E351 is essential for the activity of human 21-hydroxylase: evidence from a naturally occurring novel point mutation compared with artificial mutants generated by single amino acid substitutions.

Congenital adrenal hyperplasia (CAH) [OMIM 201910] is a group of autosomal recessive disorders, caused in 90-95% of cases by a deficiency of steroid 21-hydroxylase due to mutations in the CYP21A2 gene. The functional and structural effects of a novel rare missense mutation (E351K) in CYP21A2 found in a male patient with simple virilizing CAH were studied. The novel E351K point mutation is located in the ERR triad of the 21-hydroxylase. The ERR triad is a glutamine-arginine-arginine motif conserved in all cytochrome P450 sequences. The glutamate and first arginine residue are invariant in all P450 cytochrome enzymes, whereas the second arginine residue is present as arginine, histidine, or asparagine. Although the ERR triad is involved in some way to heme binding by the cytochrome P450 monooxygenases, the E351K mutation leads to severe but not complete loss of CYP21 enzyme activity. The functional analysis in COS-7 cells revealed a reduced conversion of 17-hydroxyprogesterone to 11-deoxycortisol of 1.1+/-0.5% (SD) and of progesterone to 11-deoxycorticosterone of 1.2+/-0.3% of wild-type activity. Analyzing the artificial mutants (E351D, E351I) of the E351 residue did not show a restoration of the in vitro 21-hydroxylase activity. These effects could be readily explained by structural changes induced by the mutations, which were rationalized by a three-dimensional-model structure of the CYP21 protein. The combination of in vitro enzyme function and computerized protein analysis of the E351 residue of the CYP21 protein provides experimental evidence for the ERR triad being a fundamental structural element of cytochrome P450 enzymes.

Thirteen novel mutations in the NR0B1 (DAX1) gene as cause of adrenal hypoplasia congenita.

X-linked adrenal hypoplasia congenita (AHC) is a rare developmental disorder associated with primary adrenal insufficiency and combined primary and secondary male hypogonadism. It is caused by deletions or mutations of the NR0B1 (DAX1) gene encoding DAX1, an atypical orphan member of the nuclear receptor superfamily. The continuous molecular genetic analysis of male patients with primary adrenal insufficiency revealed 13 novel mutations within the coding region of the NR0B1 gene which are predicted to inactivate the DAX1 function. These were three nonsense mutations (c.312C>A, p.Cys104X, c.670C>T, p.Gln224X; and c.873G>A, p.Trp291X), five duplications (c.269_270dup, c.421_422dup, c.895_896dup, c.989dup, c.999_1000dup), and five deletions (c.483del, c.745_746del, c.734_740del, c.1092del, and c.1346del). All of the mutations resulted in a premature stop codon destroying the ligand binding domain of the predictive DAX1 protein.

Congenital adrenal hyperplasia due to 11-hydroxylase deficiency: functional characterization of two novel point mutations and a three-base pair deletion in the CYP11B1 gene.

Congenital adrenal hyperplasia is a group of autosomal recessive disorders second most often caused by deficiency of steroid 11-hydroxylase (CYP11B1) due to mutations in the CYP11B1 gene. We studied the functional and structural consequences of two novel missense mutations (W116C, L299P) and an in-frame 3-bp deletion (DeltaF438) in the CYP11B gene, detected in three unrelated families. All patients are suffering from classical CYP11B1 deficiency. In vitro expression studies in COS-7 cells revealed a decreased CYP11B1 activity in the W116C mutant to 2.9 +/- 0.9% (sd) for the conversion of 11-deoxycortisol to cortisol. The L299P mutant reduced the enzymatic activity to 1.2 +/- 0.9%, whereas the DeltaF438 mutation resulted in no measurable residual CYP11B1 activity. Introduction of these mutations in a three-dimensional model structure of the CYP11B1 protein provides a possible explanation for the in vitro measured effects. We hypothesize that the W116C mutation influences the conformational change of the 11-hydroxylase protein necessary for substrate access and product release. The L299P mutation causes a change in the position of the I helix relative to the heme group, whereas the DeltaF438 mutation results in a steric disarrangement of the heme group relative to the enzyme. Studying the enzyme function in vitro helps to understand the phenotypical expression and disease severity of 11-hydroxylase deficiency, which is the basis for accurate genetic counseling, prenatal diagnosis, and treatment. Moreover, the combination of in vitro enzyme function and molecular modeling provides new insights in cytochrome P450 structural-functional relationships.

Uterine size in women with Turner syndrome after induction of puberty with estrogens and long-term growth hormone therapy: results of the German IGLU Follow-up Study 2001.

BACKGROUND: To evaluate the factors influencing uterine size in young adult women with Turner syndrome (TS) after long-term growth hormone (GH) treatment. METHODS: Cross-sectional study. Out of 188 women with TS from 96 German centres, whose longitudinal growth was documented within KIGS (Pfizer International Growth Database), data on uterine size were collected voluntarily at a standardized follow-up visit: 75 TS women (ages: 15.8-30.8 years) with complete data were included. Classification according to karyotype: 45,X (78.6%), 45,X/46,XX (5.4%), 45,X/46,iXq (8%), 45,X/46,XY (8%). Puberty was induced with estrogens in all women. At follow-up, 66 were on cyclic estrogens and progestins. RESULTS: 13/66 (19.6%) TS women who received estrogens had a reduced uterine length <5 cm. Calculating the data in standard deviation scores (SDS), only women with 45,X/46,XX karyotype had normal median uterine length and volume of 0.6 and 1.59 SDS respectively. An incomplete breast development (Tanner stage B 3) was found in women with 45,X karyotype (n = 11; 18.6%) and with 45,X/46,XY (n = 2). CONCLUSIONS: Only TS women with karyotype 45,X/46,XX had normal uterine sizes, whereas 26% of the TS women with karyotype 45,X had a uterine length <-2 SDS, and 18% a volume <-2 SDS.