Primary adrenal insufficiency (PAI) is potentially life threatening, but rare. In children, genetic defects prevail whereas adults suffer more often from acquired forms of PAI. The spectrum of genetic defects has increased in recent years with the use of next-generation sequencing methods and now has reached far beyond genetic defects in all known enzymes of adrenal steroidogenesis. Cofactor disorders such as P450 oxidoreductase (POR) deficiency manifesting as a complex form of congenital adrenal hyperplasia with a broad clinical phenotype have come to the fore. In patients with isolated familial glucocorticoid deficiency (FGD), in which no mutations in the genes for the ACTH receptor (MC2R) or its accessory protein MRAP have been found, non-classic steroidogenic acute regulatory protein (StAR) and CYP11A1 mutations have been described; and more recently novel mutations in genes such as nicotinamide nucleotide transhydrogenase (NNT) and thioredoxin reductase 2 (TRXR2) involved in the maintenance of the mitochondrial redox potential and generation of NADPH important for steroidogenesis and ROS detoxication have been discovered. In addition, whole exome sequencing approach also solved the genetics of some syndromic forms of PAI including IMAGe syndrome (CDKN1C), Irish traveler syndrome (MCM4), MIRAGE syndrome (SAMD9); and most recently a syndrome combining FGD with steroid-resistant nephrotic syndrome and ichthyosis caused by mutations in the gene for sphingosine-1-phosphate lyase 1 (SGPL1). This review intends do give an update on novel genetic forms of PAI and their suggested mechanism of disease. It also advocates for advanced genetic work-up of PAI (especially in children) to reach a specific diagnosis for better counseling and treatment.
Marco Janner, Amit V Pandey, Primus E Mullis and Christa E Flück
Objective: A severely virilized 46, XX newborn girl was referred to our center for evaluation and treatment of congenital adrenal hyperplasia (CAH) because of highly elevated 17α-hydroxyprogesterone levels at newborn screening; biochemical tests confirmed the diagnosis of salt-wasting CAH. Genetic analysis revealed that the girl was compound heterozygote for a previously reported Q318X mutation in exon 8 and a novel insertion of an adenine between nucleotides 962 and 963 in exon 4 of the CYP21A2 gene. This 962_963insA mutation created a frameshift leading to a stop codon at amino acid 161 of the P450c21 protein.
Aim and methods: To better understand structure–function relationships of mutant P450c21 proteins, we performed multiple sequence alignments of P450c21 with three mammalian P450s (P450 2C8, 2C9 and 2B4) with known structures as well as with human P450c17. Comparative molecular modeling of human P450c21 was then performed by MODELLER using the X-ray crystal structure of rabbit P450 2B4 as a template.
Results: The new three dimensional model of human P450c21 and the sequence alignment were found to be helpful in predicting the role of various amino acids in P450c21, especially those involved in heme binding and interaction with P450 oxidoreductase, the obligate electron donor.
Conclusion: Our model will help in analyzing the genotype–phenotype relationship of P450c21 mutations which have not been tested for their functional activity in an in vitro assay.
Vibor Petkovic, Mario Thevis, Didier Lochmatter, Amélie Besson, Andrée Eblé, Christa E Flück and Primus E Mullis
A heterozygous missense mutation in the GH-1 gene converting codon 77 from arginine (R) to cysteine (C), which was previously reported to have some GH antagonistic effect, was identified in a Syrian family. The index patient, a boy, was referred for assessment of his short stature (−2.5 SDS) at the age of 6 years. His mother and grandfather were also carrying the same mutation, but did not differ in adult height from the other unaffected family members. Hormonal examination in all affected subjects revealed increased basal GH, low IGF-I concentrations, and subnormal IGF-I response in generation test leading to the diagnosis of partial GH insensitivity. However, GH receptor gene (GHR) sequencing demonstrated no abnormalities. As other family members carrying the GH-R77C form showed similar alterations at the hormonal level, but presented with normal final height, no GH therapy was given to the boy, but he was followed through his pubertal development which was delayed. At the age of 20 years he reached his final height, which was normal within his parental target height. Functional characterization of the GH-R77C, assessed through activation of Jak2/Stat5 pathway, revealed no differences in the bioactivity between wild-type-GH (wt-GH) and GH-R77C. Detailed structural analysis indicated that the structure of GH-R77C, in terms of disulfide bond formation, is almost identical to that of the wt-GH despite the introduced mutation (Cys77). Previous studies from our group demonstrated a reduced capability of GH-R77C to induce GHR/GH-binding protein (GHBP) gene transcription rate when compared with wt-GH. Therefore, reduced GHR/GHBP expression might well be the possible cause for the partial GH insensitivity found in our patients. In addition, this group of patients deserve further attention because they could represent a distinct clinical entity underlining that an altered GH peptide may also have a direct impact on GHR/GHBP gene expression causing partial GH insensitivity. This might be responsible for the delay of growth and pubertal development. Finally, we clearly demonstrate that GH-R77C is not invariably associated with short stature, but that great care needs to be taken in ascribing growth failure to various heterozygous mutations affecting the GH–IGF axis and that careful functional studies are mandatory.
Marie-Anne Burckhardt, Sameer S Udhane, Nesa Marti, Isabelle Schnyder, Coya Tapia, John E Nielsen, Primus E Mullis, Ewa Rajpert-De Meyts and Christa E Flück
3β-hydroxysteroid dehydrogenase deficiency (3βHSD) is a rare disorder of sexual development and steroidogenesis. There are two isozymes of 3βHSD, HSD3B1 and HSD3B2. Human mutations are known for the HSD3B2 gene which is expressed in the gonads and the adrenals. Little is known about testis histology, fertility and malignancy risk.
To describe the molecular genetics, the steroid biochemistry, the (immuno-)histochemistry and the clinical implications of a loss-of-function HSD3B2 mutation.
Biochemical, genetic and immunohistochemical investigations on human biomaterials.
A 46,XY boy presented at birth with severe undervirilization of the external genitalia. Steroid profiling showed low steroid production for mineralocorticoids, glucocorticoids and sex steroids with typical precursor metabolites for HSD3B2 deficiency. The genetic analysis of the HSD3B2 gene revealed a homozygous c.687del27 deletion. At pubertal age, he showed some virilization of the external genitalia and some sex steroid metabolites appeared likely through conversion of precursors secreted by the testis and converted by unaffected HSD3B1 in peripheral tissues. However, he also developed enlarged breasts through production of estrogens in the periphery. Testis histology in late puberty revealed primarily a Sertoli-cell-only pattern and only few tubules with arrested spermatogenesis, presence of few Leydig cells in stroma, but no neoplastic changes.
The testis with HSD3B2 deficiency due to the c.687del27 deletion does not express the defective protein. This patient is unlikely to be fertile and his risk for gonadal malignancy is low. Further studies are needed to obtain firm knowledge on malignancy risk for gonads harboring defects of androgen biosynthesis.
Vibor Petkovic, Maria Consolata Miletta, Annemieke M Boot, Monique Losekoot, Christa E Flück, Amit V Pandey, Andrée Eblé, Jan Maarten Wit and Primus E Mullis
Short stature caused by biologically inactive GH is clinically characterized by lack of GH action despite normal-high secretion of GH, pathologically low IGF1 concentrations and marked catch-up growth on GH replacement therapy.
Design and methods
Adopted siblings (girl and a boy) of unknown family history were referred for assessment of short stature (−4.5 and −5.6 SDS) at the age of 10 and 8.1 years respectively. They had delayed bone ages (6.8 and 4.5 years), normal GH peaks at stimulation tests, and severely reduced IGF1 concentrations (−3.5 and −4.0 SDS). Genetic analysis of the GH1 gene showed a heterozygous P59S mutation at position involved in binding to GH receptor (GHR).
Isoelectric focusing analysis of secreted GH in patient serum revealed the presence of higher GH-P59S peak compared with that of wt-GH. Furthermore, computational simulation of GH-P59S binding to GHR suggested problems in correct binding of the mutant to the GHR. In vitro GHR binding studies revealed reduced binding affinity of GH-P59S for GHR (IC50, 30 ng/ml) when compared with the wt-GH (IC50, 11.8 ng/ml) while a significantly decreased ability of the mutant to activate the Jak2/Stat5 signaling pathway was observed at physiological concentrations of 25–100 ng/ml.
The clinical and biochemical data of our patients support the diagnosis of partial bioinactive GH syndrome. The higher amount of GH-P59S secreted in their circulation combined with its impact on the wt-GH function on GHR binding and signaling may alter GHR responsiveness to wt-GH and could ultimately explain severe short stature found in our patients.
Christa Flück, Anna Nordenström, S Faisal Ahmed, Salma R Ali, Marta Berra, Joanne Hall, Birgit Köhler, Vickie Pasterski, Ralitsa Robeva, Katinka Schweizer, Alexander Springer, Puck Westerveld, Olaf Hiort, Martine Cools and the COST Action BM1303 Working Group
The treatment and care of individuals who have a difference of sex development (DSD) have been revised over the past two decades and new guidelines have been published. In order to study the impact of treatments and new forms of management in these rare and heterogeneous conditions, standardised assessment procedures across centres are needed. Diagnostic work-up and detailed genital phenotyping are crucial at first assessment. DSDs may affect general health, have associated features or lead to comorbidities which may only be observed through lifelong follow-up. The impact of medical treatments and surgical (non-) interventions warrants special attention in the context of critical review of current and future care. It is equally important to explore gender development early and refer to specialised services if needed. DSDs and the medical, psychological, cultural and familial ways of dealing with it may affect self-perception, self-esteem, and psychosexual function. Therefore, psychosocial support has become one of the cornerstones in the multidisciplinary management of DSD, but its impact remains to be assessed. Careful clinical evaluation and pooled data reporting in a global DSD registry will allow linking genetic, metabolomic, phenotypic and psychological data. For this purpose, our group of clinical experts and patient and parent representatives designed a template for structured longitudinal follow-up. In this paper, we explain the rationale behind the selection of the dataset. This tool provides guidance to professionals caring for individuals with a DSD and their families. At the same time, it collects the data needed for answering unsolved questions of patients, clinicians, and researchers. Ultimately, outcomes for defined subgroups of rare DSD conditions should be studied through large collaborative endeavours using a common protocol.
Daniele Cassatella, Sasha R Howard, James S Acierno, Cheng Xu, Georgios E Papadakis, Federico A Santoni, Andrew A Dwyer, Sara Santini, Gerasimos P Sykiotis, Caroline Chambion, Jenny Meylan, Laura Marino, Lucie Favre, Jiankang Li, Xuanzhu Liu, Jianguo Zhang, Pierre-Marc Bouloux, Christian De Geyter, Anne De Paepe, Waljit S Dhillo, Jean-Marc Ferrara, Michael Hauschild, Mariarosaria Lang-Muritano, Johannes R Lemke, Christa Flück, Attila Nemeth, Franziska Phan-Hug, Duarte Pignatelli, Vera Popovic, Sandra Pekic, Richard Quinton, Gabor Szinnai, Dagmar l’Allemand, Daniel Konrad, Saba Sharif, Özlem Turhan Iyidir, Brian J Stevenson, Huanming Yang, Leo Dunkel and Nelly Pitteloud
Congenital hypogonadotropic hypogonadism (CHH) and constitutional delay of growth and puberty (CDGP) represent rare and common forms of GnRH deficiency, respectively. Both CDGP and CHH present with delayed puberty, and the distinction between these two entities during early adolescence is challenging. More than 30 genes have been implicated in CHH, while the genetic basis of CDGP is poorly understood.
We characterized and compared the genetic architectures of CHH and CDGP, to test the hypothesis of a shared genetic basis between these disorders.
Exome sequencing data were used to identify rare variants in known genes in CHH (n = 116), CDGP (n = 72) and control cohorts (n = 36 874 ExAC and n = 405 CoLaus).
Mutations in at least one CHH gene were found in 51% of CHH probands, which is significantly higher than in CDGP (7%, P = 7.6 × 10−11) or controls (18%, P = 5.5 × 10−12). Similarly, oligogenicity (defined as mutations in more than one gene) was common in CHH patients (15%) relative to CDGP (1.4%, P = 0.002) and controls (2%, P = 6.4 × 10−7).
Our data suggest that CDGP and CHH have distinct genetic profiles, and this finding may facilitate the differential diagnosis in patients presenting with delayed puberty.