Silvia Parajes, Angel OK Chan, W M But, Ian T Rose, Angela E Taylor, Vivek Dhir, Wiebke Arlt and Nils Krone
Cytochrome P450 side-chain cleavage enzyme (CYP11A1) catalyses the first and rate-limiting step of steroidogenesis, the conversion of cholesterol to pregnenolone. CYP11A1 deficiency is commonly associated with adrenal insufficiency, and in 46,XY individuals, with variable degrees of disorder of sex development (DSD).
Patient and methods
The patient was born with hyperpigmentation, micropenis, penoscrotal hypospadias, and mild cryptorchidism. Biochemical and hormonal findings were normal except for low testosterone and low-borderline cortisol. However, no short synacthen test was undertaken. Development was unremarkable apart from an episode labeled as sepsis with documented hyperkalemia and elevated C-reactive protein at age 15 days. Diagnosis of 46,XY DSD was made at age 2.5 months. Progression of hyperpigmentation prompted further investigations and the diagnosis of adrenal insufficiency was established at 2 years with raised ACTH, normal renin activity, and failure of cortisol to respond to short synacthen test. Genetic analyses were performed. The novel CYP11A1 mutations were characterized in vitro and in silico.
The patient was compound heterozygous for two novel CYP11A1 mutations, p.R360W and p.R405X. p.R360W retained 30–40% of wild-type activity. In silico analyses confirmed these findings and indicated that p.R405X is severe.
This study demonstrates the pathogenicity of two novel CYP11A1 mutations found in a patient with delayed diagnosis of CYP11A1 deficiency. Patients with partial deficiencies of steroidogenic enzymes are at risk to be misdiagnosed if adrenal function is not assessed. The adrenocortical function should be routinely assessed in all patients with DSD including severe hypospadias of unknown origin to prevent life-threatening adrenal crises.
Brigitte Delemer, Jean-Pierre Aubert, Pierre Nys, Frédéric Landron and Stéphane Bouée
To document the initial management of hypothyroidism in France with respect to diagnostic setting, investigations, and therapeutic approach.
Observational study of the management by primary care practitioners (PCPs) and endocrinologists of patients diagnosed with, and treated for, hypothyroidism during the enrollment period or the previous 6 months.
A representative sample of PCPs and endocrinologists enrolled up to five consecutive patients and reported sociodemographic, clinical, therapeutic, and laboratory data. Data were submitted at baseline and at the first measurement of TSH after starting the treatment.
The analysis population comprised 1255 patients (mean (s.d.) age 52.8 (16.3) years; 84% female). Hypothyroidism was suspected on clinical grounds in 77% of patients, with goiter in 16%. Autoimmune thyroiditis, supported by positive anti-thyroid antibodies, was the most frequent diagnosis (59%), followed by iatrogenic causes (28%), of which thyroidectomy was the most common. The median baseline TSH was 8.6 mIU/l, suggesting a high incidence of subclinical hypothyroidism. Imaging studies were requested in over 75% of patients, with ultrasound performed in 98% and scintigraphy performed in 19% of these patients. Both groups of physicians treated their patients almost exclusively with levothyroxine. Endocrinologists were more likely than PCPs to provide counseling on how to take medication correctly.
This observational study of a large cohort of patients with newly diagnosed hypothyroidism in France illustrates current practice and indicates some areas where physician education may be required to optimize adherence to guidelines and cost-effectiveness.
Earn H Gan, Katie MacArthur, Anna L Mitchell and Simon H S Pearce
Autoimmune Addison's disease (AAD) is a rare condition with a complex genetic basis. A panel of rare and functionally defective genetic variants in the sialic acid acetylesterase (SIAE) gene has recently been implicated in several common autoimmune conditions. We performed a case–control study to determine whether these rare variants are associated with a rarer condition, AAD.
We analysed nine SIAE gene variants (W48X, M89V, C196F, C226G, R230W, T312M, Y349C, F404S and R479C) in a United Kingdom cohort of 378 AAD subjects and 387 healthy controls. All samples were genotyped using Sequenom iPlex chemistry to characterise primer extension products.
A heterozygous rare allele at codon 312 (312*M) was found in one AAD patient (0.13%) but was not detected in the healthy controls. The commoner, functionally recessive variant at codon 89 (89*V) was found to be homozygous in two AAD patients but was only found in the heterozygous state in controls. Taking into account all nine alleles examined, 4/378 (1.06%) AAD patients and 1/387 (0.25%) healthy controls carried the defective SIAE alleles, with a calculated odds ratio of 4.13 (95% CI 0.44–97.45, two-tailed P value 0.212, NS).
We demonstrated the presence of 89*V homozygotes and the 312*M rare allele in the AAD cohort, but overall, our analysis does not support a role for rare variants in SIAE in the pathogenesis of AAD. However, the relatively small collection of AAD patients limits the power to exclude a small effect.
Leo Niskanen, Lawrence A Leiter, Edward Franek, Jianping Weng, Taner Damci, Manuel Muñoz-Torres, Jean-Paul Donnet, Lars Endahl, Trine Vang Skjøth and Allan Vaag
Insulin degludec/insulin aspart (IDegAsp) is a soluble co-formulation of insulin degludec (70%) and insulin aspart (IAsp: 30%). Here, we compare the efficacy and safety of IDegAsp, an alternative IDegAsp formulation (AF: containing 45% IAsp), and biphasic IAsp 30 (BIAsp 30).
Sixteen-week, open-label, randomised, treat-to-target trial.
Insulin-naive subjects with type 2 diabetes (18–75 years) and a HbA1c of 7–11% were randomised to twice-daily IDegAsp (n=61), AF (n=59) or BIAsp 30 (n=62), all in combination with metformin. Insulin was administered pre-breakfast and dinner (main evening meal) and titrated to pre-breakfast and pre-dinner plasma glucose (PG) targets of 4.0–6.0 mmol/l.
Mean HbA1c after 16 weeks was comparable for IDegAsp, AF and BIAsp 30 (6.7, 6.6 and 6.7% respectively). With IDegAsp, 67% of subjects achieved HbA1c <7.0% without confirmed hypoglycaemia in the last 4 weeks of treatment compared with 53% (AF) and 40% (BIAsp 30). Mean fasting PG was significantly lower for IDegAsp vs BIAsp 30 (treatment difference (TD): −0.99 mmol/l (95% confidence interval: −1.68; 0.29)) and AF vs BIAsp 30 (TD: −0.88 mmol/l (−1.58; −0.18)). A significant, 58% lower rate of confirmed hypoglycaemia was found for IDegAsp vs BIAsp 30 (rate ratio (RR): 0.42 (0.23; 0.75)); rates were similar for AF vs BIAsp 30 (RR: 0.92 (0.54; 1.57)). IDegAsp and AF had numerically lower rates of nocturnal confirmed hypoglycaemia vs BIAsp 30 (RR: 0.33 (0.09; 1.14) and 0.66 (0.22; 1.93) respectively).
IDegAsp provided comparable overall glycaemic control to BIAsp 30 with a significantly lower rate of hypoglycaemia.
Sally Tantawy, Lin Lin, Ilker Akkurt, Guntram Borck, Dietrich Klingmüller, Berthold P Hauffa, Heiko Krude, Heike Biebermann, John C Achermann and Birgit Köhler
Steroidogenic factor 1 (SF-1, NR5A1) is a key transcriptional regulator of many genes involved in the hypothalamic–pituitary–gonadal axis and mutations in NR5A1 can result in 46,XY disorders of sex development (DSD). Patients with this condition typically present with ambiguous genitalia, partial gonadal dysgenesis, and absent/rudimentary Müllerian structures. In these cases, testosterone is usually low in early infancy, indicating significantly impaired androgen synthesis. Further, Sertoli cell dysfunction is seen (low inhibin B, anti-Müllerian hormone). However, gonadal function at puberty in patients with NR5A1 mutations is unknown.
Subjects and methods
Clinical assessment, endocrine evaluation, and genetic analysis were performed in one female and one male with 46,XY DSD who showed spontaneous virilization during puberty. The female patient presented at adolescence with clitoral hypertrophy, whereas the male patient presented at birth with severe hypospadias and entered puberty spontaneously. Molecular analysis of NR5A1 was performed followed by in vitro functional analysis of the two novel mutations detected.
Testosterone levels were normal during puberty in both patients. Analysis of NR5A1 revealed two novel heterozygous missense mutations in the ligand-binding domain of SF-1 (patient 1: p.L376F; patient 2: p.G328V). The mutant proteins showed reduced transactivation of the CYP11A promoter in vitro.
Patients with 46,XY DSD and NR5A1 mutations can produce sufficient testosterone for spontaneous virilization during puberty. Phenotypic females (46,XY) with NR5A1 mutations can present with clitoromegaly at puberty, a phenotype similar to other partial defects of androgen synthesis or action. Testosterone production in 46,XY males with NR5A1 mutations can be sufficient for virilization at puberty. As progressive gonadal dysgenesis is likely, gonadal function should be monitored in adolescence and adulthood, and early sperm cryopreservation considered in male patients if possible.
Andrii Dinets, Mykola Hulchiy, Anastasios Sofiadis, Mehran Ghaderi, Anders Höög, Catharina Larsson and Jan Zedenius
Increased incidence of papillary thyroid carcinoma (PTC) is observed as a consequence of radiation exposure in connection to the Chornobyl nuclear plant accident in 1986. In this study, we report a cohort of adult Ukrainian patients diagnosed with PTC from 2004 to 2008 following exposure at the age of 18 years or younger.
In total, 70 patients were identified and clinically characterized. The common BRAF 1799T>A mutation was assessed by pyrosequencing, the RET/PTC1 and RET/PTC3 (NCOA4) rearrangements by RT-PCR, and the expression of Ki-67 (MIB-1 index), BCL2, cyclin A, and cyclin D1 by immunohistochemistry.
In total, 46/70 (66%) cases carried a BRAF mutation and/or a RET/PTC rearrangement. A BRAF mutation was detected in 26 tumors, RET/PTC1 in 20 cases, and RET/PTC3 in four cases. In four of these cases, BRAF mutation and RET/PTC rearrangement were coexisting. The BRAF mutation was underrepresented among PTCs with accompanying chronic lymphocytic thyroiditis (CLT) compared with PTCs without this feature (12 vs 44%). MIB-1 proliferation index determined by double staining with leukocyte common antigen was low (mean 0.8%; range 0.05–4.5%). Moreover, increased expression of cyclin A was observed in PTCs with a tumor size >2 cm compared with PTCs ≤2 cm (1.2 vs 0.6%). BCL2 and cyclin D1 showed frequent expression but without associations to clinical characteristics or amplification of the CCND1 locus.
Our results suggest that this cohort has frequent BRAF mutation, RET/PTC1 rearrangement, and low proliferation index. Furthermore, BRAF 1799T>A was underrepresented in PTCs with CLT, and cyclin A expression was associated with increased PTC tumor size.
Maria Thunander, Carina Törn, Christer Petersson, Birger Ossiansson, Jan Fornander and Mona Landin-Olsson
C-peptide is a main outcome measure in treatment trials of diabetes. C-peptide also has a role in the classification of diabetes, which is often difficult in adults and this is also increasingly recognised in adolescents and elders.
We aimed to describe the levels of C-peptide in relation to age and body mass index (BMI) in a large population-based cohort of adults with newly diagnosed diabetes and compare the capabilities of C-peptide, age and BMI to discriminate between autoimmune and non-autoimmune diabetes.
Subjects and methods
Blood samples from 1180 patients were analysed regarding islet cell antibody, glutamic acid decarboxylase antibody and fasting C-peptide (FCP). Receiver operating characteristics (ROC) curves were analysed to check the ability of age, BMI and C-peptide to discriminate between autoantibody-positive (Ab+) and -negative (Ab−) diabetes.
Mean FCP was 0.73±0.5 (range 0.13–1.80) nmol/l in the Ab+ and 1.42±0.9 (range 0.13–8.30) nmol/l in the Ab−. FCP was 0.02 nmol/l higher per year increase in age at diagnosis of diabetes. Mean BMI was 26.0±4.8 (range 18.0–39.0) kg/m2 in the Ab+ and 28.9±5.3 (range 15.5–62.6) kg/m2 in the Ab−. FCP increased with age also within each BMI group. The highest area under the curve (AUC) in the ROC analysis was found for C-peptide, followed by age and BMI (0.78, 0.68 and 0.66 respectively).
At diagnosis of diabetes, C-peptide was superior to age and BMI in discriminating between autoimmune and non-autoimmune diabetes. C-peptide increased significantly with BMI and age, latter also within each BMI group. Most of the adults had normal or high levels of C-peptide at presentation of diabetes among the autoimmune patients.
Anne Hege Straume, Kristian Løvås, Hrvoje Miletic, Karsten Gravdal, Per Eystein Lønning and Stian Knappskog
Background and objectives
Testicular Leydig cell tumours (LCTs) are rare, steroid-secreting tumours. Elevated levels of aromatase (CYP19 or CYP19A1) mRNA have been previously described in LCTs; however, little is known about the mechanism(s) causing CYP19 over-expression. We report an LCT in a 29-year-old male with elevated plasma oestradiol caused by enhanced CYP19 transcription.
Design and methods
First, we measured the intra-tumour expression of CYP19 and determined the use of CYP19 promoters by qPCR. Secondly, we explored CYP19 and promoter II (PII) for gene amplifications and activating mutations in PII by sequencing. Thirdly, we analysed intra-tumour expression of steroidogenic factor 1 (SF-1 (NR5A1)), liver receptor homologue-1 (LRH-1 (NR5A2)) and cyclooxygenase-2 (COX2 (PTGS2)). Finally, we analysed SF-1 for promoter mutations and gene amplifications.
Similar to what has been recorded in normal Leydig cells, we first found the bulk of tumour CYP19 transcripts to be PII derived, excluding promoter shift as a cause of enhanced transcription. Secondly, we excluded CYP19 and PII gene amplifications, and activating mutations in PII, as causes of elevated CYP19 mRNA. We found SF-1 mRNA to be up-regulated in the tumour, while LRH-1 and COX2 were down-regulated. The finding of elevated SF-1 levels in the tumour was confirmed by immunohistochemistry. The elevated level of SF-1 was not due to promoter mutations or amplifications of the SF-1 gene.
Our results strongly suggest that the elevated levels of SF-1 have induced PII-regulated CYP19 transcription in this tumour. These findings are of relevance to the understanding of CYP19 up-regulation in general, which may occur in several tissues, including breast cancer.
Mariam Elbornsson, Galina Götherström, Ingvar Bosæus, Bengt-Åke Bengtsson, Gudmundur Johannsson and Johan Svensson
Few studies have determined the effects of more than 5–10 years of GH replacement in adults on bone mineral content (BMC) and bone mineral density (BMD).
In this prospective, single-centre, open-label study, the effects of 15 years of GH replacement on BMC and BMD, measured using dual-energy X-ray absorptiometry, were determined in 126 hypopituitary adults (72 men) with adult-onset GH deficiency (GHD). Mean age was 49.4 (range 22–74) years at the initiation of the study.
The mean initial GH dose of 0.63 (s.e.m. 0.03) mg/day was gradually lowered to 0.41 (0.01) mg/day after 15 years. The mean serum IGF1 SDS increased from −1.69 (0.11) at baseline to 0.63 (0.16) at the study end (P<0.001 vs baseline). The 15 years of GH replacement induced a sustained increase in total body BMC (+5%, P<0.001) and BMD (+2%, P<0.001). Lumbar (L2–L4) spine BMC increased by 9% (P<0.001) and BMD by 5% (P<0.001). In femur neck, a peak increase in BMC and BMD of 7 and 3%, respectively, was observed after 7 years (both P<0.001). After 15 years, femur neck BMC was 5% above the baseline value (P<0.01), whereas femur neck BMD had returned to the baseline level. In most variables, men had a more marked response to GH replacement than women.
Fifteen-year GH replacement in GHD adults induced a sustained increase in total body and lumbar (L2–L4) spine BMC and BMD. In femur neck, BMC and BMD peaked at 7 years and then decreased towards baseline values.
Rebecca M Reynolds, Javier Labad, Alison V Sears, Rachel M Williamson, Mark W J Strachan, Ian J Deary, Gordon D O Lowe, Jackie F Price and Brian R Walker
Both type 2 diabetes and glucocorticoid therapy are highly prevalent. Although people with type 2 diabetes may be more susceptible to adverse effects of glucocorticoids, and it is recommended that glucocorticoid therapy is avoided for fear of worsening glycaemic control, the extent to which this advice is followed and the consequences when glucocorticoids are prescribed are poorly documented. The aim was to assess the characteristics of people with type 2 diabetes prescribed glucocorticoids in a real-world setting and to quantify resulting adverse effects.
Cross-sectional cohort study.
Cardiometabolic variables, body fat distribution, cognitive function and mood were studied in the 1066 participants of the Edinburgh Type 2 Diabetes Study, of whom 162 (15%) were taking systemic, topical or inhaled glucocorticoids.
Glucocorticoid therapy was more common in women and in smokers but was not avoided in patients with diabetic complications or cardiovascular risk factors. People taking glucocorticoids were more centrally obese with slightly higher HbA1c and total serum cholesterol but were no more likely to have hepatic steatosis or hypertension. Glucocorticoid treatment was associated with substantially lower mood and greater anxiety. Women taking glucocorticoid therapy were twice as likely to report depressive symptoms compared with those not taking treatment. Glucocorticoid therapy was also associated with poorer cognitive function among those with subclinical atherosclerosis, as indicated by low ankle–brachial pressure index.
Glucocorticoids are prescribed commonly for people with type 2 diabetes despite being associated with adverse indices of glycaemic control, cardiovascular risk factors, mood and cognitive function.