The Spectrum of Genetic Defects in Congenital Adrenal Hyperplasia in the Population of Cyprus: A Retrospective Analysis.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is caused by mutations in the CYP21A2 gene. The study refers to CAH patients of Greek-Cypriot ancestry between years 2007 and 2018. One hundred and twenty patients with various degrees of CAH were categorized and genotyped. The patients were categorized in 4 mutation groups based on their clinical and biochemical findings. The majority of patients (85.0%) belonged to the non-classic (NC)-CAH form and the disorder was more often diagnosed in females (71.7%). The most severe classic salt-wasting (SW) form was identified in 11 neonates (9.2%). Seven (5.8%) children were also identified with the simple virilizing (SV) form and a median presentation age of 5 years [interquartile range (IQR) 3.2-6.5]. In the 240 nonrelated alleles, the most frequent mutation was p.Val281Leu (60.0%) followed by c.655 A/C>G (IVS2-13A/C>G) (8.8%), p.Pro453Ser (5.8%), DelEx1-3 (4.6%), p.Val304Met (4.6%), and p.Gln318stop (4.2%). Other less frequent mutations including rare deletions were also identified. Following our recent report that the true carrier frequency of CYP21A2 in Greek-Cypriots is 1:10, this study reports that the CAH prevalence is predicted around 1.7 cases per 10 000 people. Therefore, the up-to-date 120 CAH patients identified by our group make only the 6.9% of the ones estimated (approximately 1750) to exist in the Greek Cypriot population. The compiled data from a coherent population such as the Greek-Cypriot could be valuable for the antenatal diagnosis, management and genetic counselling of the existing and prospect families with CAH.

Familial Mediterranean fever associated with MEFV mutations in a large cohort of Cypriot patients.

Familial Mediterranean fever (FMF) is caused by mutations in the MEFV gene and the spectrum of mutations among Greek-Cypriots with FMF-related symptoms was examined. Sequence analysis for exons 2, 3, 5, and 10 of the MEFV gene was performed in a cohort of 593 patients. A total of 70 patients carried mutations in the homozygote or compound heterozygote state, 128 were identified with one MEFV mutation and 395 had no mutations. Of the 268 identified alleles, p.Val726Ala (27.61%) was the most frequent followed by p.Met694Val (19.40%). The missense mutations p.Arg761His (3.73%) and p.Ala744Ser (2.24%) were identified as the rarest. An interesting finding is the high frequency (18.28%) of the complex p.Phe479Leu-p.Glu167Asp that was identified in 49 of the mutated alleles. The MEFV genotypes did not follow a binomial distribution and proved not to satisfy the HWE (P < 0.001). The high percentage (66.61%) of patients with unidentified mutations could be due to mutations in the rest of the coding or noncoding MEFV gene or due to mutations in other genes that are also causing Hereditary Recurrent Fevers. Results from this work indicate the high incidence of FMF in Cyprus and describe the spectrum of the mutations which occur in the country.

Estrogen receptor signaling and targets: Bones, breasts and brain (Review).

Estrogens are involved in a number of physiological functions, including in the development of the brain, growth, reproduction and metabolism. The biological actions of estrogens are achieved by binding to estrogen receptors (ERs) in numerous types of tissues. ERα and ERß belong to the nuclear receptor superfamily and the G­protein coupled ER1 (GPER1) is a membrane receptor. The primary biologically active estrogen, 17ß­estradiol demonstrates a high affinity for ERs. Mechanistically, estrogens bind to the ERs in the nucleus, and the complex then dimerize and bind to estrogen response elements (EREs) located in the promoter regions of the target genes. This is referred to as the genomic mechanism of ERs' function. Furthermore, ERs can also act through kinases and other molecular interactions leading to specific gene expression and functions, referred to as the non­genomic mechanism. While ERα and ERß exert their functions via both genomic and non­genomic pathways, GPER1 exerts its function primarily via the non­genomic pathways. Any aberrations in ER signaling can lead to one of a number of diseases such as disorders of growth and puberty, fertility and reproduction abnormalities, cancer, metabolic diseases or osteoporosis. In the present review, a focus is placed on three target tissues of estrogens, namely the bones, the breasts and the brain, as paradigms of the multiple facets of the ERs. The increasing prevalence of breast cancer, particularly hormone receptor­positive breast cancer, is a challenge for the development of novel antihormonal therapies other than tamoxifen and aromatase inhibitors, to minimize toxicity from the long treatment regimens in patients with breast cancer. A complete understanding of the mechanism of action of ERs in bones may highlight options for novel targeted treatments for osteoporosis. Likewise, the aging of the brain and related diseases, such as dementia and depression, are associated with a lack of estrogen, particularly in women following menopause. Furthermore, gender dysphoria, a discordance between experienced gender and biological sex, is commonly hypothesized to emerge due to discrepancies in cerebral and genital sexual differentiation. The exact role of ERs in gender dysphoria requires further research.

Genetic diagnosis of endocrine disorders in Cyprus through the Cyprus Institute of Neurology and Genetics: an ENDO-ERN Reference Center.

The report covers the current and past activities of the department Molecular Genetics-Function and Therapy (MGFT) at the Cyprus Institute of Neurology and Genetics (CING), an affiliated Reference Center for the European Reference Network on Rare Endocrine Conditions (Endo-ERN).The presented data is the outcome of > 15 years long standing collaboration between MGFT and endocrine specialists from the local government hospitals and the private sector. Up-to-date > 2000 genetic tests have been performed for the diagnosis of inherited rare endocrine disorders. The major clinical entities included Congenital Adrenal Hyperplasia (CAH) due to pathogenic variants in CYP21A2 gene and Multiple Endocrine Neoplasia (MEN) type 2 due to pathogenic variants in the RET proto-oncogene. Other rare and novel pathogenic variants in ANOS1, WDR11, FGFR1, RNF216, and CHD7 genes were also found in patients with Congenital Hypogonadotropic Hypogonadism. Interestingly, a few patients with Disorders of Sexual Differentiation (DSD) shared rare pathogenic variants in the SRD5A2, HSD17B3 and HSD3B2 while patients with Glucose and Insulin Homeostasis carried theirs in GCK and HNF1A genes. Lastly, MGFT over the last few years has established an esteemed diagnostic and research program on premature puberty with emphasis on the implication of MKRN3 gene on the onset of the disease and the identification of other prognosis biomarkers.As an Endo-ERN member MGFT department belongs to this large European network and holds the same humanistic ideals which aim toward the improvements of health care for patients with rare endocrine conditions in respect to improved and faster diagnosis.

Obesogens in Adolescence: Challenging Aspects and Prevention Strategies.

Childhood obesity has become a global epidemic, with significant increases in prevalence over recent decades. While excessive calorie consumption and physical inactivity are known factors, emerging research highlights the role of endocrine-disrupting chemicals (EDCs), particularly obesogens, in obesity's pathogenesis. This review explores the historical context of the environmental obesogens hypothesis, their sources, mechanism of action, impact on prenatal and postnatal development, and epigenetics. Additionally, it discusses the long-term consequences of childhood obesity and proposes prevention strategies that will mitigate negative health effects. Obesogens were found to disrupt hormonal balance and metabolic processes through various mechanisms such as altering gene expression, hormonal interference, and inflammation. Especially significant was exposure during critical windows of development, which correlates with an increased risk of obesity in childhood or adolescence. Long-term effects of childhood obesity include chronic health conditions and psychosocial issues. A comprehensive approach is necessary to address childhood obesity encompassing genetic, environmental, and lifestyle factors. Prevention strategies should focus on reducing obesogen exposure, promoting healthy lifestyles, and implementing regulatory policies. Future research should investigate obesogens-diet interactions, microbiome impacts, and combined obesogens effects. Long-term human studies are also crucial for validating findings from animal models and allowing for informed decision-making to combat the obesity pandemic.

Stress, Thyroid Dysregulation, and Thyroid Cancer in Children and Adolescents: Proposed Impending Mechanisms.

Stress is a potential catalyst for thyroid dysregulation through cross-communication of the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-thyroid (HPT) axes. Stress and stressors exposure motivates molecular mechanisms affecting compound feedback loops of the HPT axis. While there is evidence of connection between stress and thyroid dysregulation, the question whether this connection is implicated in the development of thyroid cancer (TC) remains unanswered. In view of the rising incidence of TC in both adults and children alongside the increasing stress in our modern society, there is a need to understand possible interrelations between stress, thyroid dysregulation, and TC. Prolonged glucocorticoid secretion due to stress interferes with immune system response by altering the cytokines, inducing low-grade chronic inflammation, and suppressing function of immune-protective cells. Chronic inflammation is a risk factor linked to TC. The role of autoimmunity has been a matter of controversy. However, there is epidemiological connection between autoimmune thyroid disease (AITD) and TC; patients with AITD show increased incidence in papillary thyroid carcinoma (PTC), and those with TC show a high prevalence of intrathyroidal lymphocyte infiltration and thyroid autoantibodies. Timing and duration-dependent exposure to specific endocrine disrupting chemicals (EDCs) has an impact on thyroid development, function, and proliferation, leading to thyroid disease and potentially cancer. Thyroid hormone imbalance, chronic inflammation, and EDCs are potential risk factors for oxidative stress. Oxygen free radicals are capable of causing DNA damage via stimulation of the mitogen-activating protein kinase or phosphatidylinositol-3-kinase and/or nuclear factor kB pathways, resulting in TC-associated gene mutations such as RET/PTC, AKAP9-BRAF, NTRK1, RAASF, PIK3CA, and PTEN. Stressful events during the critical periods of prenatal and early life can influence neuroendocrine regulation and induce epigenetic changes. Aberrant methylation of tumor suppressor genes such as P16INK4A, RASSF, and PTEN is associated with PTC; histone H3 acetylation is shown to be higher in TC, and thyroid-specific noncoding RNAs are downregulated in PTC. This review focuses on the above proposed mechanisms that potentially lead to thyroid tumorigenesis with the aim to help in the development of novel prognostic and therapeutic strategies for TC.

The pathogenic p.Gln319Ter variant is not causing congenital adrenal hyperplasia when inherited in one of the duplicated CYP21A2 genes.

Objective: The study aimed to identify the pathogenic status of p.Gln319Ter (NM_000500.7: c.955C>T) variant when inherited in a single CYP21A2 gene (bimodular RCCX haplotype) and to discriminate between a non-causing congenital adrenal hyperplasia (CAH) allele when inherited in a duplicated and functional CYP21A2 gene context (trimodular RCCX haplotype). Methods: 38 females and 8 males with hyperandrogenemia, previously screened by sequencing and identified as carriers for the pathogenic p.Gln319Ter, were herein tested by multiplex ligation-dependent probe amplification (MLPA) and a real-time PCR Copy number Variation (CNV) assay. Results: Both MLPA and real-time PCR CNV analyses confirmed a bimodular and pathogenic RCCX haplotype with a single CYP21A2 in 19/46 (41.30%) p.Gln319Ter carriers and who in parallel all shared elevated 17-OHP levels. The remaining 27 individuals that also carried the p.Gln319Ter exhibited low 17-OHP levels as a result of their carriership of a duplicated CYP21A2 with a trimodular RCCX haplotype. Interestingly, all of these individuals also carried in linkage disequilibrium with p.Gln319Ter two single nucleotide polymorphisms, the c.293-79G>A (rs114414746) in intron 2 and the c.*12C>T (rs150697472) in the 3'-UTR. Therefore, these variants can be used to distinguish between pathogenic and non-pathogenic genomic contexts of the c.955T (p.Gln319) in the genetic diagnosis of congenital adrenal hyperplasia (CAH). Conclusion: The employed methodologies identified a considerable number of individuals with non-pathogenic p.Gln319Ter from the individuals that typically carry the pathogenic p.Gln319Ter in a single CYP21A2. Therefore, it is extremely important the detection of such haplotypes for the prenatal diagnosis, treatment and genetic counseling in patients with CAH.

Reduced serum concentrations of biomarkers reflecting Leydig and Sertoli cell function in male patients with congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) is a recessive condition that affects the adrenal glands. Despite life-long replacement therapy with glucocorticoids and mineralocorticoids, adult patients with CAH often experience impaired gonadal function. In pubertal boys and in men with CAH, circulating testosterone is produced by the adrenal glands as well as the testicular, steroidogenic cells. In this European two-center study, we evaluated the function of Leydig and Sertoli cells in 61 boys and men with CAH, primarily due to 21-hydroxylase deficiency. Despite conventional hormone replacement therapy, our results indicated a significant reduction in serum concentrations of both Leydig cell-derived hormones (i.e. insulin-like factor 3 (INSL3) and testosterone) and Sertoli cell-derived hormones (i.e. inhibin B and anti-Müllerian hormone) in adult males with CAH. Serum concentrations of INSL3 were particularly reduced in those with testicular adrenal rest tumors. To our knowledge, this is the first study to evaluate circulating INSL3 as a candidate biomarker to monitor Leydig cell function in patients with CAH.

Molecular modelling of novel ADCY3 variant predicts a molecular target for tackling obesity.

Severe early­onset obesity is mainly attributed to single gene variations of the hypothalamic leptin­melanocortin system, which is critical for controlling the balance between appetite and energy expenditure. Adenylate cyclase 3 (ADCY3), a transmembrane enzyme localized in primary neuronal cilia, is a key genetic candidate, which appears to have an essential role in regulating body weight. The present study aimed to identify ADCY3 genetic variants in severely obese young patients of Greek­Cypriot origin by genomic sequencing. Apart from previously reported variants, the novel and probably pathogenic variant c.349T>A, causing a p.Leu117Met substitution within one of the two pseudo­symmetric halves of the transmembrane part of the protein, was reported. Molecular modelling analysis used to delineate bonding interactions within the mutated protein structure strongly suggested a change in interactive forces and energy levels affecting the pseudo­twofold symmetry of the transmembrane domain of the protein and probably its catalytic function. These results support the involvement of ADCY3 in the pathology of the disease and point towards the requirement of defining protein function and evaluating the clinical significance of the detected variants.

Methylation status of hypothalamic Mkrn3 promoter across puberty.

Makorin RING finger protein 3 (MKRN3) is an important factor located on chromosome 15 in the imprinting region associated with Prader-Willi syndrome. Imprinted MKRN3 is expressed in hypothalamic regions essential for the onset of puberty and mutations in the gene have been found in patients with central precocious puberty. The pubertal process is largely controlled by epigenetic mechanisms that include, among other things, DNA methylation at CpG dinucleotides of puberty-related genes. In the present study, we investigated the methylation status of the Mkrn3 promoter in the hypothalamus of the female mouse before, during and after puberty. Initially, we mapped the 32 CpG dinucleotides in the promoter, the 5'UTR and the first 50 nucleotides of the coding region of the Mkrn3 gene. Moreover, we identified a short CpG island region (CpG islet) located within the promoter. Methylation analysis using bisulfite sequencing revealed that CpG dinucleotides were methylated regardless of developmental stage, with the lowest levels of methylation being found within the CpG islet region. In addition, the CpG islet region showed significantly lower methylation levels at the pre-pubertal stage when compared with the pubertal or post-pubertal stage. Finally, in silico analysis of transcription factor binding sites on the Mkrn3 CpG islet identified the recruitment of 29 transcriptional regulators of which 14 were transcriptional repressors. Our findings demonstrate the characterization and differential methylation of the CpG dinucleotides located in the Mkrn3 promoter that could influence the transcriptional activity in pre-pubertal compared to pubertal or post-pubertal period. Further studies are needed to clarify the possible mechanisms and effects of differential methylation of the Mkrn3 promoter.

Bone disease in thalassaemia major: recent advances in pathogenesis and clinical aspects.

Bone is a dynamic organ, constantly changing metabolically and being remodelled through the balanced activity of osteoclast and osteoblast on trabecular surfaces. Osteoporosis represents a continuum, in which multiple pathogenic mechanisms converge to cause loss of bone mass and deterioration of microarchitecture of skeletal structure. In thalassaemia major (TM), progressive 'aging' of bone starts in early childhood, through the gradual development of an imbalance between augmented osteoclastic resorption and insufficient osteoblastic bone formation. Chronic anemia, iron toxicity and endocrine complications, via a complex mechanism, lead to alterations in the RANK/RANKL/OPG system in favor of increased osteoclastic activity and enhanced osteoblastic dysfunction. Hypogonadotrophic hypogonadism and delayed puberty are the most common endocrine complications in patients with TM; they also contribute to osteopenia and osteoporosis, which is present in more than 50% of patients. There are gender differences not only in the prevalence but also in the severity of the osteoporosis syndrome. The anabolic effects of GH and IGF-1 on bone formation are important for the acquisition of bone mass, mainly during childhood and puberty. In TM, GH secretory dysfunction is common and contributes to osteopenia and osteoporosis, along with other endocrinopathies such as hypoparathyroidism and vitamin D deficiency, hypothyroidism and diabetes. Prevention is with no doubt the first step in the management of osteoporosis in TM, with the final goal of preventing bone loss and fractures. The management of patients with TM should start as early as birth in order to minimize the disease complications. Induction of puberty at a proper age with estrogens in girls and testosterone in boys and later treatment of hypogonadism with HRT are vital steps in the prevention of bone disease in TM. Biphosphonates, the well known medication for osteoporosis, have been tried in the treatment of TM-osteoporosis with promising outcomes. Since the origin of bone disease in TM is multifactorial and some of the underlying pathogenic mechanisms are still unclear, further research in this field is needed, which will allow the design of optimal therapeutic measures.

Microcalcifications without a thyroid nodule as the sole sign of papillary thyroid carcinoma.

SUMMARY: Our objective is to demonstrate the importance of considering microcalcifications even without evidence of nodules as a potential sign of malignancy. Current guidelines, such as those of the British Thyroid Association, acknowledge the clinical significance of microcalcifications only when found within nodules. In this case, they are considered a suspicious feature, classifying the nodules as U5 (i.e. high risk) where fine-needle aspiration biopsy (FNAB) is warranted, following the high likelihood of cancer in these nodules. In addition, there is a dearth of evidence of ultrasound scan (USS) detection of microcalcifications in the thyroid gland outside of nodules, along with their associated clinical implications. Yet, this clinical manifestation is not so infrequent considering that we do encounter patients in the clinic showing these findings upon ultrasound examination. Three patients who presented to our clinic with thyroid-related symptoms were shown to have areas of microcalcifications without a nodule upon sonographic evaluation of their thyroid gland. These incidentally detected hyperechoic foci were later confirmed to correspond to areas of papillary thyroid carcinoma (PTC) on histopathological examination of resected tissue following thyroidectomy. Four more cases were identified with sonographic evidence of microcalcifications without nodules and given their clinical and other sonographic characteristics were managed with active surveillance instead. LEARNING POINTS: Echogenic foci known as microcalcifications may be visible without apparent association to nodular structures. Microcalcifications without nodules may not be an infrequent finding. Microcalcifications are frequently indicative of malignancy within the thyroid gland even without a clearly delineated nodule. Empirically, the usual guidelines for the management of thyroid nodules can be applied to the management of microcalcifications not confined to a nodule, but such a finding per se should be classified as a 'high-risk' sign.

Pathogenic and Low-Frequency Variants in Children With Central Precocious Puberty.

Background: Central precocious puberty (CPP) due to premature activation of GnRH secretion results in early epiphyseal fusion and to a significant compromise in the achieved final adult height. Currently, few genetic determinants of children with CPP have been described. In this translational study, rare sequence variants in MKRN3, DLK1, KISS1, and KISS1R genes were investigated in patients with CPP. Methods: Fifty-four index girls and two index boys with CPP were first tested by Sanger sequencing for the MKRN3 gene. All children found negative (n = 44) for the MKRN3 gene were further investigated by whole exome sequencing (WES). In the latter analysis, the status of variants in genes known to be related with pubertal timing was compared with an in-house Cypriot control cohort (n = 43). The identified rare variants were initially examined by in silico computational algorithms and confirmed by Sanger sequencing. Additionally, a genetic network for the MKRN3 gene, mimicking a holistic regulatory depiction of the crosstalk between MKRN3 and other genes was designed. Results: Three previously described pathogenic MKRN3 variants located in the coding region of the gene were identified in 12 index girls with CPP. The most prevalent pathogenic MKRN3 variant p.Gly312Asp was exclusively found among the Cypriot CPP cohort, indicating a founder effect phenomenon. Seven other CPP girls harbored rare likely pathogenic upstream variants in the MKRN3. Among the 44 CPP patients submitted to WES, nine rare DLK1 variants were identified in 11 girls, two rare KISS1 variants in six girls, and two rare MAGEL2 variants in five girls. Interestingly, the frequent variant rs10407968 (p.Gly8Ter) of the KISS1R gene appeared to be less frequent in the cohort of patients with CPP. Conclusion: The results of the present study confirm the importance of the MKRN3-imprinted gene in genetics of CPP and its key role in pubertal timing. Overall, the results of the present study have emphasized the importance of an approach that aligns genetics and clinical aspects, which is necessary for the management and treatment of CPP.

GnRH Deficient Patients With Congenital Hypogonadotropic Hypogonadism: Novel Genetic Findings in ANOS1, RNF216, WDR11, FGFR1, CHD7, and POLR3A Genes in a Case Series and Review of the Literature.

Background: Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic disease caused by Gonadotropin-Releasing Hormone (GnRH) deficiency. So far a limited number of variants in several genes have been associated with the pathogenesis of the disease. In this original research and review manuscript the retrospective analysis of known variants in ANOS1 (KAL1), RNF216, WDR11, FGFR1, CHD7, and POLR3A genes is described, along with novel variants identified in patients with CHH by the present study. Methods: Seven GnRH deficient unrelated Cypriot patients underwent whole exome sequencing (WES) by Next Generation Sequencing (NGS). The identified novel variants were initially examined by in silico computational algorithms and structural analysis of their predicted pathogenicity at the protein level was confirmed. Results: In four non-related GnRH males, a novel X-linked pathogenic variant in ANOS1 gene, two novel autosomal dominant (AD) probably pathogenic variants in WDR11 and FGFR1 genes and one rare AD probably pathogenic variant in CHD7 gene were identified. A rare autosomal recessive (AR) variant in the SRA1 gene was identified in homozygosity in a female patient, whilst two other male patients were also, respectively, found to carry novel or previously reported rare pathogenic variants in more than one genes; FGFR1/POLR3A and SRA1/RNF216. Conclusion: This report embraces the description of novel and previously reported rare pathogenic variants in a series of genes known to be implicated in the biological development of CHH. Notably, patients with CHH can harbor pathogenic rare variants in more than one gene which raises the hypothesis of locus-locus interactions providing evidence for digenic inheritance. The identification of such aberrations by NGS can be very informative for the management and future planning of these patients.

Central Precocious Puberty Caused by Novel Mutations in the Promoter and 5'-UTR Region of the Imprinted MKRN3 Gene.

Background: Central Precocious Puberty (CPP) is clinically defined by the development of secondary sexual characteristics before the age of 8 years in girls and 9 years in boys. To date, mutations in the coding region of KISS1, KISS1R, PROKR2, DLK1, and MKRN3 genes have been reported as causative for CPP. This study investigated the presence of causative mutations in both the promoter and the 5'-UTR regions of the MKRN3 gene. Methods: Sanger DNA sequencing was used for screening the proximal promoter and 5'-UTR region of the MKRN3 gene in a group of 73 index girls with CPP. Mutations identified were cloned in luciferase reporter gene vectors and transiently transfected in GN11 cells in order to check for changes in the activity of the MKRN3 promoter. GN11 cells were previously checked for Mkrn3 expression using lentivirus mediated knock-down. In silico analysis was implemented for the detection of changes in the mRNA secondary structure of the mutated MKRN3 5'-UTR. Results: Three novel heterozygous mutations (-166, -865, -886 nt upstream to the transcription start site) located in the proximal promoter region of the MKRN3 gene were identified in six non-related girls with CPP. Four of these girls shared the -865 mutation, one the -166, and another one the -886. A 5'-UTR (+13 nt downstream to the transcription start site) novel mutation was also identified in a girl with similar clinical phenotype. Gene reporter assay evaluated the identified promoter mutations and demonstrated a significant reduction of MKRN3 promoter activity in transfected GN11 cells. In silico analysis for the mutated 5'-UTR predicted a significant change of the mRNA secondary structure. The minimum free energy (MFE) of the mutated 5'-UTR was higher when compared to the corresponding wild-type indicating less stable RNA secondary structure. Conclusion: Our findings demonstrated novel genetic alterations in the promoter and 5'-UTR regulatory regions of the MKRN3 gene. These changes add to another region to check for the etiology of CPP.

46,XY complete gonadal dysgenesis in a familial case with a rare mutation in the desert hedgehog (DHH) gene.

PURPOSE: Disorders of sex development (DSD) have been linked to gene defects that lead to gonadal dysgenesis. Herein, we aimed to identify the genetic cause of gonadal dysgenesis in a patient with primary amenorrhoea tracing it to a phenotypic female carrying a 46,XY karyotype of a consanguineous family. METHODS AND RESULTS: Whole exome sequencing (WES) was performed and revealed in homozygosity the rare and only once reported p.Arg164Pro missense mutation in exon 2 of the desert hedgehog (DHH) gene. Sanger sequencing was used to validate this candidate variant both in the patient, the parents, and two siblings. Both brother and sister of the index patient were found negative for the p.Arg164Pro mutation, while the consanguineous parents were found to carry the mutation in the heterozygous state. Neither the parents nor the unaffected siblings showed any reproductive malformations. CONCLUSIONS: Defects in the DHH gene have been reported as a very rare cause of DSD, and this report increases the number of 46,XY gonadal dysgenesis cases. Additionally, the present study highlights the importance of genetic validation of patients with DSD, since this is likely to alleviate the considerable psychological distress experienced by both the patient and the parents.

Hormonal dysregulation and bones in thalassaemia--an overview.

Bone disease (BDT) represents a prominent cause of morbidity in patients of both sexes with thalassaemia major (TM). The exact pathogenesis of BDT in TM is multifactorial, still unclear and complicated. Peak bone mass is achieved shortly after completion of puberty and normally remains stable until the third decade of life. After the age of 30 years, age related bone loss begins. Growth hormone (GH) and sex steroids have a crucial role in bone remodelling and therefore are important in helping to establish and maintain peak bone mass for both sexes. The anabolic effects of GH and IGF-1 in bone are important not only for the acquisition of bone mass during adolescence but also for the maintenance of skeletal architecture during adult life. GH deficiency is not a rare finding in adult patients with TM, thus contributing to the development of BDT. Furthermore, patients with TM are often hypogonadal, and therefore the lack of sex steroids in critical periods, such as puberty, contributes to the failure to achieve optimal peak bone mass and to maintain bone mass later in life. Sex steroids probably act by increasing the expression of RANKL by osteoblastic cells, and alterations in the RANK/RANKL/OPG system in favour of osteoclasts are characteristic in TM, where the ratio of sRANKL/OPG is increased. It is still not clear whether DEXA scan is the gold standard for determination of bone density in thalassaemics and if so, whether the WHO criteria for defining osteopenia and osteoporosis are relevant to patients with TM. The question therefore arises whether other methods should be adopted, since DEXA may often overestimate BDT in these patients. BDT in thalassaemia represents a unique clinical entity with a multifactorial aetiology and of complex mechanisms which need to be clarified. It is essential for us to understand the underlying mechanisms of bone destruction and the bony defect at the ultrastructural level in order to be able to design not only preventive strategies but also therapeutic measures.

Endocrine complications in patients with Thalassaemia Major.

Patients with multi-transfused thalassaemia major may develop severe endocrine complications due to iron overload. The anterior pituitary is particularly sensitive to iron overload which disrupts hormonal secretion resulting in hypogonadism, short stature , acquired hypothyroidism and hypoparathyroidism. Glucose intolerance and diabetes mellitus are also common in thalassaemic patients. The severity of the clinical manifestation and laboratory findings in thalassaemia largely depends on the genotype; thus homozygotes or compound heterozygotes for the mutations beta0 or beta+ depend for life on frequent transfusions. A multicenter study in Cyprus including 435 patients showed hypogonadotrophic hypogonadism in 32.5%, short stature in 35%, acquired hypothyroidism in 5.9%, hypoparathyroidism in 1.2% and diabetes mellitus in 9.4%. A slowing down of growth velocity and a reduced or absent pubertal growth spurt is observed in early adolescence leading to short adult height. Delayed or absent puberty and hypogonadism may result in fertility problems which affect enormously the life of thalassemics. Glucose intolerance in adolescence and diabetes mellitus later in life are also frequent complications mainly due to iron overload, chronic liver disease and genetic predisposition. Primary hypothyroidism and hypoparathyroidsm usually appear in the second decade of life; are related to iron overload and may be reversible at an early stage by intensive chelation. Osteopenia and osteoporosis due to a complicated pathogenesis represent prominent causes of morbidity in young adults of both genders with thalassaemia. Early recognition and prevention of the endocrine complications, by early and regular chelation therapy, is mandatory for the improvement of the quality of life and psychological outcome of these patients.

Variations in the 3'UTR of the CYP21A2 Gene in Heterozygous Females with Hyperandrogenaemia.

Heterozygosity for CYP21A2 mutations in females is possibly related to increased risk of developing clinical hyperandrogenism. The present study was designed to seek evidence on the phenotype-genotype correlation in female children, adolescents, and women with CYP21A2 mutations and variants in the 3'UTR region of the gene. Sixty-six patients out of the 169 were identified as carriers of CYP21A2 mutations. Higher values of stimulated 17 hydroxyprogesterone (17-OHP) levels were found in the carriers of the p.Val281Leu mutation compared to the carriers of other mutations (mean: 24.7 nmol/l versus 15.6 nmol/l). The haplotype of the ∗52C>T, ∗440C>T, and ∗443T>C in the 3'UTR was identical in all heterozygous patients with p.Val281Leu and the haplotype of the ∗12C>T and ∗52C>T was identical in all heterozygous patients with the p.Gln318∗. In conclusion, hyperandrogenaemic females are likely to bear heterozygous CYP21A2 mutations. Carriers of the mild p.Val281Leu mutation are at higher risk of developing hyperandrogenism than the carriers of more severe mutations. The identification of variants in the 3'UTR of CYP21A2 in combination with the heterozygous mutation may be associated with the mild form of nonclassic congenital adrenal hyperplasia and reveal the importance of analyzing the CYP21A2 untranslated regions for the appropriate management of this category of patients.

Diagnosis and management of Silver-Russell syndrome: first international consensus statement.

This Consensus Statement summarizes recommendations for clinical diagnosis, investigation and management of patients with Silver-Russell syndrome (SRS), an imprinting disorder that causes prenatal and postnatal growth retardation. Considerable overlap exists between the care of individuals born small for gestational age and those with SRS. However, many specific management issues exist and evidence from controlled trials remains limited. SRS is primarily a clinical diagnosis; however, molecular testing enables confirmation of the clinical diagnosis and defines the subtype. A 'normal' result from a molecular test does not exclude the diagnosis of SRS. The management of children with SRS requires an experienced, multidisciplinary approach. Specific issues include growth failure, severe feeding difficulties, gastrointestinal problems, hypoglycaemia, body asymmetry, scoliosis, motor and speech delay and psychosocial challenges. An early emphasis on adequate nutritional status is important, with awareness that rapid postnatal weight gain might lead to subsequent increased risk of metabolic disorders. The benefits of treating patients with SRS with growth hormone include improved body composition, motor development and appetite, reduced risk of hypoglycaemia and increased height. Clinicians should be aware of possible premature adrenarche, fairly early and rapid central puberty and insulin resistance. Treatment with gonadotropin-releasing hormone analogues can delay progression of central puberty and preserve adult height potential. Long-term follow up is essential to determine the natural history and optimal management in adulthood.