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Design: Thyroid-stimulating hormone deficiency (TSHD) is a rare disease. It may be isolated, secondary to abnormalities of genes involved in TSH biosynthesis, or associated with other pituitary deficits or abnormalities of genes involved in pituitary ontogenesis. Several genes are involved in thyrotroph development and function. Objective: Our aim was to determine the genetic causes of TSHD, either isolated (ITSHD) or associated with somatotroph deficiency (TSHD-GHD), in the cohort of patients from the GENHYPOPIT network. Methods: Next-generation sequencing (NGS) analyses were performed as a panel of genes on a cohort of patients with non-syndromic ITSHD or TSHGHD. The variants were classified according to the American College of Medical Genetics classification reviewed by the NGS-Diag network and correlated with the phenotype. Class 3, 4, and 5 single-nucleotide variants were checked by Sanger sequencing and copy number variants by multiplex ligation-dependent probe amplification (MLPA). Results: A total of 64 index cases (22 ITSHD and 42 TSHD-GHD) were included in this cohort. A genetic cause was identified in 26.5% of patients, with 36.3% in the ITSHD group (variants in TSH$ and IGSF1) and 21.4% in TSHD-GHD (variants in IGSF1, TSH$, TRHR, GH1, POU1F1, and PROP1). Among the pathogenic and likely pathogenic variants identified, 42% were in IGSF1, including six not previously reported. Conclusion: Our results show that IGSF1 variants represent the most frequent aetiology of TSH deficiency. Despite a systematic NGS approach and the identification of new variants, most patients remain without a molecular diagnosis. Larger scale studies, such as exome or genome studies, should be considered in the future. [ABSTRACT FROM AUTHOR]

Objective: Congenital hyperinsulinism (HI) is the most common cause of persistent hypoglycemia in children. In addition to typical focal or diffuse HI, some cases with diazoxide-unresponsive congenital HI have atypical pancreatic histology termed Localized Islet Nuclear Enlargement (LINE) or mosaic HI, characterized by histologic features similar to diffuse HI, but confined to only a region of pancreas. Our objective was to characterize the phenotype and genotype of children with LINE-HI. Design: The phenotype and genotype features of 12 children with pancreatic histology consistent with LINE-HI were examined. Methods: We compiled clinical features of 12 children with LINE-HI and performed next-generation sequencing on specimens of pancreas from eight of these children to look for mosaic mutations in genes known to be associated with diazoxide-unresponsive HI (ABCC8, KCNJ11, and GCK). Results: Children with LINE-HI had lower birth weights and later ages of presentation compared to children with typical focal or diffuse HI. Partial pancreatectomy in LINE-HI cases resulted in euglycemia in 75% of cases; no cases have developed diabetes. Low-level mosaic mutations were identified in the pancreas of six cases with LINE-HI (three in ABCC8, three in GCK). Expression studies confirmed that all novel mutations were pathogenic. Conclusion: These results indicate that post-zygotic low-level mosaic mutations of known HI genes are responsible for some cases of LINE-HI that lack an identifiable germ-line mutation and that partial pancreatectomy may be curative for these cases. [ABSTRACT FROM AUTHOR]

Objective: The autoimmune polyendocrine syndrome type 1 (APS-1) is an autosomal recessive disorder characterised by immune dysregulation and autoimmune endocrine gland destruction. APS-1 is caused by biallelic mutations affecting the autoimmune regulator (AIRE) gene on chromosome 21q22.3, which facilitates immunological selftolerance. The objective was to investigate >300 probands with suspected APS-1 or isolated hypoparathyroidism for AIRE abnormalities. Methods: Probands were assessed by DNA sequence analysis. Novel variants were characterised using 3D modelling of the AIRE protein. Restriction enzyme and microsatellite analysis were used to investigate for uniparental isodisomy. Results: Biallelic AIRE mutations were identified in 35 probands with APS-1 and 5 proba nds with isolated hypoparathyroidism. These included a novel homozygous p.(His14Pro) mutation, predicted to disrupt the N-terminal caspase activation recruitment domain of the AIRE protein. Furthermore, an apparently homozygous AIRE mutation, p.Leu323fs, was identified in an APS-1 proband, who is the child of non-consanguineous asymptomatic parents. Microsatellite analysis revealed that the proband inherited two copies of the paternal mutant AIRE allele due to uniparental isodisomy. Hypoparathyroidism was the most common endocrine manifestation in AIRE mutation-positive probands and >45% of those harbouring AIRE mutations had at least two diseases out of the triad of candidiasis, hypoparathyroidism, and hypoadrenalism. In contrast, type 1 diabetes and hypothyroidism occurred more frequently in AIRE mutation-negative probands with suspected APS-1. Around 30% of AIRE mutation-negative probands with isolated hypoparathyroidism harboured mutations in other hypoparathyroid genes. Conclusions: This study of a large cohort referred for AIRE mutational analysis expands the spectrum of genetic abnormalities causing APS-1. [ABSTRACT FROM AUTHOR]

Background: The human INHA gene encodes the inhibin subunit alpha protein, which is common to both inhibin A and B. The functional importance of inhibins in male sex development, sexual function, and reproduction remain largely unknown. Objective: We report for the first time two male siblings with homozygous INHA mutations. Methods: The medical files were examined for clinical, biochemical, and imaging data. Genetic analysis was performed using next-generation and Sanger sequencing methods. Results: Two brothers complained of gynecomastia, testicular pain, and had a history of hypospadias. Biochemistry revealed low serum testosterone, high gonadotropin and anti-Mullerian hormone, and very low/undetectable inhibin concentrations, where available. Both patients had azoo spermia in the spermiogram. We have identified a homozygous 2 bp deletion (c.208_209delAG, R70Gfs*3) variant, which leads to a truncated INHA protein in both patients, and confirmed heterozygosity in the parents. The exter nal genital development, pubertal onset and progression, reproductive functions, serum gonadotropins, and sex hormones of mother and father, who were heterozygous carriers of the identified mutation, were normal. Conclusion: Homozygosity for INHA mutations causes decreased prenatal and postnatal testosterone production and infertility in males, while the heterozygous female and male ca rriers of INHA mutations do not have any abnormality in sex development and reproduction. [ABSTRACT FROM AUTHOR]

Objective: Mutations in the KATP channel genes, ABCC8 and KCNJ11, are the most common cause of congenital hyperinsulinism. The diagnosis of KATP-hyperinsulinism is important for the clinical management of the condition. We aimed to determine the clinical features that help to identify KATP-hyperinsulinism at diagnosis. Design: We studied 761 individuals with KATP-hyperinsulinism and 862 probands with hyperinsulinism of unknown aetiology diagnosed before 6 months of age. All were referred as part of routine clinical care. Methods: We compared the clinical features of KATP-hyperinsulinism and unknown hyperinsulinism cases. We performed logistic regression and receiver operator characteristic (ROC) analysis to identify the features that predict I KATP-hyperinsulinism. Results: Higher birth weight, diazoxide unresponsiveness and diagnosis in the first week of life were independently associated with KATP-hyperinsulinism (adjusted odds ratio: 4.5 (95% CI: 3.4-5.9), 0.09 (0.06-0.13) and 3.3 (2.0-5.0) respectively). Birth weight and diazoxide unresponsiveness were additive and highly discriminatory for identifying KATP-hyperinsulinism (ROC area under the curve for birth weight 0.80, diazoxide responsiveness 0.77, and together 0.88, 95% CI: 0.85-0.90). In this study, 86% born large for gestation and 78% born appropriate for gestation and who did not respond to diazoxide treatment had KATP-hyperinsulinism. In contrast, of those individuals born small for gestation, none who were diazoxide responsive and only 4% of those who were diazoxide unresponsive had KATP-hyperinsulinism. Conclusions: Individuals with hyperinsulinism born appropriate or large for gestation and unresponsive to diazoxide treatment are most likely to have an ABCC8 or KCNJ11 mutation. These patients should be prioritised for genetic testing of KATP channel genes. [ABSTRACT FROM AUTHOR]

Context: 17α-Hydroxylase/17,20-lyase deficiency (17OHD) caused by mutations in the CYP17A1 gene is a rare form of congenital adrenal hyperplasia typically characterised by cortisol deficiency, mineralocorticoid excess and sex steroid deficiency. Objective: To examine the phenotypic spectrum of 17OHD by clinical and biochemical assessment and corresponding in silico and in vitro functional analysis. Design: Case series. Patients and results: We assessed eight patients with 17OHD, including four with extreme 17OHD phenotypes: two siblings presented with failure to thrive in early infancy and two with isolated sex steroid deficiency and normal cortisol reserve. Diagnosis was established by mass spectrometr y-based urinary steroid profiling and confirmed by genetic CYP17A1 analysis, revealing homozygous and compound heterozygous seque nce variants. We found novel (p.Gly111Val, p.Ala398Glu, p.Ile371Thr) and previously described sequence variants (p.Pro409Leu, p.Arg347His, p.Gly436Arg, p.Phe53/54del, p.Tyr60IlefsLys88X). In vitro functional studies employing an overexpression system in HEK293 cells showed that 17,20-lyase activity was invariably decreased while mutant 17α-hydroxylase activity retained up to 14% of WT activity in the two patients with intact cortis ol reserve. A ratio of urinary corticosterone over cortisol metabolites reflective of 17α-hydroxylase activity correlated well with clinical phenotype s everity. Conclusion: Our findings illustrate the broad phenotypic spectrum of 17OHD. Isolated sex steroid deficiency with normal stimulated cortisol has not been reported before. Attenu ation of 17α-hydroxylase activity is readily detected by urinary steroid profiling and predicts phenotype severity. [ABSTRACT FROM AUTHOR]

Objective: Primary aldosteronism (PA) is the most common form of secondar y and curable hypertension. Different germline and somatic mutations are found in aldosterone-produci ng adenoma (APA) and familial forms of the disease, while the causes of bilateral adrenal hyperplasia (BAH) remain largely unknown. Adrenalectomy is the recommended treatment for patients with APA; however, 6% of patients are no t cured and show persistent PA after surgery suggesting BAH. The objective of this study was to analyze clin ical data of patients with APA without biochemical success after adrenalectomy as well as the histological and gen etic characteristics of their adrenal glands. Design and methods: Clinical data of 12 patients with partial and absent biochemic al cure were compared to those from 39 PA patients with hormonal cure after surgery. Histologi cal, morphological, and genetic characterization of the adrenals was carried out by CYP11B2 and CYP11B1 immunostaining and by CYP11B2-guided NGS. Results: Patients with absent hormonal cure displayed a longer duration of arterial hypertension and lower lateralization index of aldosterone production. In ten patients, APAs expressing CYP11B2 were identified. No difference in histological and morphological characteristics was observed between patients with or without a hormonal cure. Somatic mutations in APA driver genes were identified in all CYP 11B2 positive APAs; CACNA1D mutations were the most frequent genetic abnormality. Conclusions: Patients with partial and absent biochemical cure were diagnos ed later and exhibited a lower lateralization index of aldosterone production, suggesting asymmetric aldosterone production in the context of BAH. Somatic mutations in adrenal glands from those patients indicat e common mechanisms underlying BAH and APA. [ABSTRACT FROM AUTHOR]

Background: Loss-of-function germline MEN1 gene mutations account for 75-95% of patients with multiple endocrine neoplasia type 1 (MEN1). It has been postulated that mutations in non-coding regions of MEN1 might occur in some of the remaining patients; however, this hypothesis has not yet been fully investigated. Objective: To sequence for the entire MEN1 including promoter, exons and introns in a large MEN1 cohort and determine the mutation profile. Methods and patients: A target next-generation sequencing (tNGS) assay comprising 7.2 kb of the full MEN1 was developed to investigate germline mutations in 76 unrelated MEN1 probands (49 familial, 27 sporadic). tNGS results were validated by Sanger sequencing (SS), and multiplex ligation-dependent probe amplification (MLPA) assay was applied when no mutations were identifiable by both tNGS and SS. Results: Germline MEN1 variants were verified in coding region and splicing sites of 57/76 patients (74%) by both tNGS and SS (100% reproducibility). Thirty-eight different pathogenic or likely pathogenic variants were identified, including 13 new and six recurrent variants. Three large deletions were detected by MLPA only. No mutation was detected in 16 patients. In untranslated, regulatory or in deep intronic MEN1 regions of the 76 MEN1 cases, no point or short indel pathogenic variants were found in untranslated, although 33 benign/likely benign and three new VUS variants were detected. Conclusions: Our study documents that point or short indel mutations in non-coding regions of MEN1 are very rare events. Also, tNGS proved to be a highly effective technology for routine genetic MEN1 testing. [ABSTRACT FROM AUTHOR]

Twenty years ago, we described kindred of 105 individuals with isolated GH deficiency (IGHD) in Itabaianinha County, in northeast Brazil, carrying a homozygous mutation in the GH-releasing hormone receptor gene. These subjects exhibit markedly reduced GH responsiveness to stimulatory tests and anterior pituitary hypoplasia. Serum concentrations of IGF-I, IGF binding protein type 3 and the acid-labile subunit are markedly reduced, with a lesser reduction of IGF-II. The most striking physical findings of these IGHD individuals are the proportionate short stature, doll facies, high-pitched voice and visceral obesity with reduced fat-free mass. There is neither microphallus, nor neonatal hypoglycemia. Puberty is delayed, menopause anticipated, but fertility is preserved in both genders. The reduction in bone sizes is not even, with mean standard deviation scores for height of -7.2, total maxillary length of -6.5, total facial height of -4.3 and cephalic perimeter of -2.7. In addition, the non-osseous growth is not uniform, preserving some organs, like pancreas, liver, kidney, brain and eyes and compromising others such as thyroid, heart, uterus and spleen. These subjects present higher prevalence of dizziness, mild high-tones sensorineural hearing loss, reduction of vascular retinal branching points, increase of optic disk, genu valgum and increased systolic blood pressure. Biochemically, they have high low density lipoprotein cholesterol and C-reactive protein levels, but maintain increased insulin sensitivity and do not show premature atherosclerosis. Finally, they have normal immune function and normal longevity. This review details the findings and summarizes 20 years of clinical research carried out in this unique population. [ABSTRACT FROM AUTHOR]

Objective: Acid-labile subunit (ALS) deficiency (ACLSD), caused by homozygous or compound heterozygous IGFALS mutations, is associated with moderate short stature, delayed puberty, low serum IGF-I and ALS and extremely low serum IGFBP-3. Its effect on birth weight, head circumference, bone mineral density (BMD), serum IGF-II and IGFBP-2 is uncertain, as well as the phenotype of heterozygous carriers of IGFALS mutations (partial ACLSD). Design: From all available members of five Turkish families, carrying three mutations in exon 2 of IGFALS (c.1462G > A, p.Asp488Asn (families A, B, E); c.251A > G, p.Asn84Ser (families C and E) and c.1477del, p.Arg493fs (family D)), clinical, laboratory and BMD data were collected. Methods: Auxological and biochemical findings were expressed as SDS for age and gender. Ternary complex formation in serum was investigated by size-exclusion chromatography. BMD using DXA bone densitometry was adjusted for height and age (Ha-BMD z-score). Results: In ACLSD (n = 24), mean ± s.d. height SDS (-2.7 ± 1.2), head circumference SDS (-2.3 ± 0.5) and body mass index (BMI) (-0.6 ± 1.0 SDS) were lower than those in partial ACLSD (n = 26, P ≤ 0.01) and birth weight SDS (n = 7) tended to be lower (-2.2 ± 1.1 vs -0.6 ± 0.3 in partial ACLSD (P = 0.07)). Serum IGF-I was -3.7 ± 1.4 vs -1.0 ± 1.0, IGF-II: -5.6 ± 0.7 vs -1.3 ± 0.7, ALS: <-4.4 ± 1.2 vs -2.1 ± 0.9 and IGFBP-3: -9.0 ± 1.9 vs -1.6 ± 0.8 SDS respectively (P < 0.001). Ha-BMD z-score was similar and normal in both groups. Conclusions: To the known phenotype of ACLSD (i.e. short stature, reduced serum levels of IGF-I and ALS, extremely low serum IGFBP-3 and disturbed ternary complex formation), we add reduced birth weight, head circumference and serum IGF-II. [ABSTRACT FROM AUTHOR]

Background: Homozygous inactivating mutations of the calcium-sensing receptor (CaSR) lead to neonatal severe hyperparathyroidism (NSHPT), whereas heterozygous inactivating mutations result in familial hypocalciuric hypercalcemia (FHH). It is unknown why in some cases heterozygous CaSR mutations cause neonatal hyperparathyroidism (NHPT) clinically similar to NSHPT but with only moderately elevated serum calcium. Methods: A literature survey was conducted to identify patients with heterozygous CaSR mutations and NHPT. The common NHPT CaSR mutants R185Q and R227L were compared with 1 5 mutants causing only FHH in the heterozygous state. We studied in vitro calcium signaling including the functional consequences of co-expression of mutant and wild-type (wt) CaSR, patients' phenotype, age of disease manifestation and mode of inheritance. Results: All inactivating CaSR mutants impaired calcium signaling of wt-CaSR regardless of the patients' clinical phenotype. The absolute intracellular calcium signaling response to physiologic extracellular calcium concentrations in vitro showed a high correlation with patients' serum calcium concentrations in vivo, which is similar in NHPT and FHH patients with the same genotype. Pedigrees of FHH families revealed that paternal inheritance per se does not necessarily lead to NHPT but may only cause FHH. Conclusions: There is a significant correlation between in vitro functional impairment of the CaSR at physiologic calcium concentrations and the severity of alterations in calcium homeostasis in patients. Whether a particular genotype leads to NHPT or FHH appears to depend on additional predisposing genetic or environmental factors. An individual therapeutic approach appears to be warranted for NHPT patients. [ABSTRACT FROM AUTHOR]

Aim: We previously reported that biallelic mutations in dual oxidase 2 (DUOX2) cause transient hypothyroidism. Since then, many cases with DUOX2 mutations have been reported. However, the clinical features and prognosis of individuals with DUOX2 defects have not been clarified. Objective: We investigated the prognosis of patients with congenital hypothyroidism (CH) due to DUOX2 mutations. Patients: Twenty-five patients were identified by a neonatal screening program and included seven familial cases. Their serum TSH values ranged from 18.9 to 734.6 mU/l. Twenty-two of the patients had low serum free thyroxine (fT4) levels (0.17-1.1 ng/dl). Twenty-four of the patients were treated with L-thyroxine. Methods: We analyzed the DUOX2, thyroid peroxidase, Na+/I- symporter, and dual oxidase maturation factor 2 genes of these 25 patients by PCR-amplified direct sequencing. An additional 11 genes were analyzed in 11 of the 25 patients using next-generation sequencing. Results: All patients had biallelic DUOX2 mutations, and seven novel alleles were detected. Fourteen of the patients were able to discontinue replacement therapy, and seven were receiving reduced L-thyroxine doses. Normalization of thyroglobulin lagged several years behind the completion of treatment. Two patients showed permanent hypothyroidism. Except for one case of a learning disability, growth and psychomotor development were normal. Conclusion: The prognosis of Japanese patients with DUOX2 defects was usually transient CH. Delayed improvement of thyroglobulin indicates that these patients have subclinical hypothyroidism. Hypothyroidism did not recur in patients during the study period (up to 18 years old). [ABSTRACT FROM AUTHOR]

Background: Bilateral macronodular adrenal hyperplasia (BMAH) is a rare cause of Cushing's syndrome (CS) and its familial clustering has been described previously. Recent studies identified that ARMC5 mutations occur frequently in BMAH, but the relation between ARMC5 mutation and the expression of aberrant G-protein-coupled receptor has not been examined in detail yet. Methods:We studied a large French-Canadian family with BMAH and sub-clinical or overt CS. Screening was performed using the 1-mg dexamethasone suppression test (DST) in 28 family members. Screening for aberrant regulation of cortisol by various hormone receptors were examined in vivo in nine individuals. Sequencing of the coding regions of ARMC5 gene was carried out. Results: Morning ambulating cortisol post 1 mg DST were >50 nmol/l in 5/8 members in generation II (57-68 years old), 9/22 in generation III (26-46 years old). Adrenal size was enlarged at different degrees. All affected patients increased cortisol following upright posture, insulin-induced hypoglycemia and/or isoproterenol infusion. β-blockers led to the reduction of cortisol secretion in all patients with the exception of two who had adrenalectomies because of β-blockers intolerance. We identified a heterozygous germline variant in the ARMC5 gene c.327_328insC, (p.Ala110Argfs*9) in nine individuals with clinical or subclinical CS, in four out of six individuals with abnormal suppression to dexamethasone at initial investigation and one out of six individuals with current normal clinical screening tests. Conclusions: Systematic screening of members of the same family with hereditary BMAH allows the diagnosis of unsuspected subclinical CS associated with early BMAH. The relation between the causative ARMC5 mutation and the reproducible pattern of aberrant b-adrenergic and V1-vasopressin receptors identified in this family remains to be elucidated. [ABSTRACT FROM AUTHOR]

Context and objective: Idiopathic central precocious puberty (iCPP) is defined as early activation of the hypothalamic- pituitary-gonadal axis in the absence of identifiable central lesions. Mutations of the makorin RING finger 3 (MKRN3) gene are associated with iCPP. We aimed to assess the frequency of MKRN3 mutations in iCPP and to compare the phenotypes of patients with and without MKRN3 mutations. Design: An observational study was carried out on patients recruited at pediatric hospitals in France and Italy. Forty-six index CPP cases were screened for mutations in the MKRN3 coding sequence: 28 index cases of familial cases and 18 cases did not report any familial history of CPP. The endocrine phenotype was compared between MKRN3 mutated and non-mutated patients. Results: MKRN3 mutations were identified in one sporadic and 13 familial cases. We identified five new heterozygous missense mutations predicted to be deleterious for protein function and two frameshift mutations, one new and the other recurrent, predicted to result in truncated proteins. Age at puberty onset varied very little among patients with MKRN3 mutations and puberty occurred earlier in these patients than in those without MKRN3 mutations (6.0 years (5.4-6.0) vs 7.0 years (6.0-7.0), P=0.01). Conclusions: MKRN3 mutations are common in familial iCPP. MKRN3 is one of the gatekeepers of the postnatal activation of the gonadotropic axis. [ABSTRACT FROM AUTHOR]

Context:3b-hydroxysteroid dehydrogenase deficiency (3bHSD) is a rare disorder of sexual development and steroidogenesis. There are two isozymes of 3bHSD, 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. Objective: To describe the molecular genetics, the steroid biochemistry, the (immuno-)histochemistry and the clinical implications of a loss-of-function HSD3B2 mutation. Methods: Biochemical, genetic and immunohistochemical investigations on human biomaterials. Results: 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. Conclusions: 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. [ABSTRACT FROM AUTHOR]

Background: Thyroid cancer incidence has dramatically increased worldwide over the last two decades. The rise is mostly due to an increased detection of small papillary thyroid carcinomas (PTCs) (≤20 mm), predominantly microPTC (≤ 10 mm). Although small tumors generally have an excellent outcome, a considerable percentage may have a more aggressive disease and worse prognosis. The clinical challenge is to preoperatively identify those tumors that are more likely to recur. Aim: To improve risk stratification and patient management, we sought to determine the prognostic value of BRAF V600E, NRAS or RET/PTC mutations in patients with PTC measuring <20 mm, mainly microPTC. Methods: The prevalence of RET/PTC fusion genes was examined by quantitative RT-PCR. BRAF V600E and NRAS Q61 mutations were determined by PCR sequencing. To further elucidate why some small PTC are less responsive to radioactive iodine treatment therapy, we explored if these genetic alterations may modulate the expression of iodine metabolism genes (NIS, TPO, TG, TSHR and PDS) and correlated with clinico-pathological findings that are predictors of recurrence. Results: This study shows that tumors measuring ≤20 mm exhibited higher prevalence of BRAF V600E mutation, which correlated with aggressive histopathological parameters, higher risk of recurrence, and lower expression of NIS and TPO. Although this correlation was not found when microPTC were evaluated, we show that tumors measuring 7-10 mm, which were positive for BRAF mutation, presented more aggressive features and lower expression of NIS and TPO. Conclusion: We believe that our find ings will help to decide the realistic usefulness of BRAF V600E mutation as a preoperative marker of poor prognosis in small PTC, primarily in microPTC. [ABSTRACT FROM AUTHOR]

Objective: Primary macronodular adrenal hyperplasia (PMAH) is a rare type of Cushing's syndrome (CS) that results in increased cortisol production and bilateral enlargement of the adrenal glands. Recent work showed that the disease may be caused by germline and somatic mutations in the ARMC5 gene, a likely tumor suppressor gene (TSG). We investigated 20 different adrenal nodules from one patient with PMAH for ARMC5 somatic sequence changes. Design: All of the nodules were obtained from a single patient who underwent bilateral adrenalectomy. DNA was extracted by standard protocol and the ARMC5 sequence was determined by the Sanger method. Results: Sixteen of 20 adrenocortical nodules harbored, in addition to what appeared to be the germline mutation, a second somatic variant. The p.Trp476* sequence change was present in all 20 nodules, as well as in normal tissue from the adrenal capsule, identifying it as the germline defect; each of the 16 other variants were found in different nodules: six were frame shift, four were missense, three were nonsense, and one was a splice site variation. Allelic losses were confirmed in two of the nodules. Conclusion: This is the most genetic variance of the ARMC5 gene ever described in a single patient with PMAH: each of 16 adrenocortical nodules had a second new, 'private,' and - in most cases - completely inactivating ARMC5 defect, in addition to the germline mutation. The data support the notion that ARMC5 is a TSG that needs a second, somatic hit, to mediate tumorigenesis leading to polyclonal nodularity; however, the driver of this extensive genetic variance of the second ARMC5 allele in adrenocortical tissue in the context of a germline defect and PMAH remains a mystery. [ABSTRACT FROM AUTHOR]

Objective: Mutations of the fibroblast growth factor receptor 3 (FGFR3) cause various forms of short stature, of which the least severe phenotype is hypochondroplasia, mainly characterized by disproportionate short stature. Testing for an FGFR3 mutation is currently not part of routine diagnostic testing in children with short stature without disproportion. Design: A three-generation family A with dominantly transmitted proportionate short stature was studied by whole-exome sequencing to identify the causal gene mutation. Functional studies and protein modeling studies were performed to confirm the pathogenicity of the mutation found in FGFR3. We performed Sanger sequencing in a second family B with dominant proportionate short stature and identified a rare variant in FGFR3. Methods:Exome sequencing and/or Sanger sequencing was performed, followed by functional studies using transfection of the mutant FGFR3 into cultured cells; homology modeling was used to construct a three-dimensional model of the two FGFR3 variants. Results:A novel p.M528I mutation in FGFR3 was detected in family A, which segregates with short stature and proved to be activating in vitro. In family B, a rare variant (p.F384L) was found in FGFR3, which did not segregate with short stature and showed normal functionality in vitro compared with WT. Conclusions:Proportionate short stature can be caused by a mutation in FGFR3. Sequencing of this gene can be considered in patients with short stature, especially when there is an autosomal dominant pattern of inheritance. However, functional studies and segregation studies should be performed before concluding that a variant is pathogenic. [ABSTRACT FROM AUTHOR]

Context: Within the last two decades, heterozygous loss-of-function PAX8 mutations have been reported in patients with a wide degree of thyroid gland dysfunction and growth despite the presence of identical mutations. Objectives: To search for PAX8 mutations in a cohort of patients with congenital hypothyroidism (CH) and various types of thyroid gland defects. Design: A cross-sectional study was conducted in a cohort of patients. Setting: The French neonatal screening program was used for recruiting patients. Patients: A total of 118 patients with CH, including 45 with familial and 73 with sporadic diseases, were included in this study. The thyroid gland was normal in 23 patients had hypoplasia, 25 had hemithyroid agenesis, 21 had athyreosis, and 21 had ectopy. Results: We found four different PAX8 mutations (p.R31C, p.R31H, p.R108X, and p.I47T) in ten patients (six patients with CH and four family members), two with sporadic and eight with familial diseases. Imaging studies performed in the index cases showed ectopic thyroid gland (n = 2), hypoplasia (n = 2), eutopic lobar asymmetry (n = 1), and eutopic gland compatible with dyshormonogenesis (n = 1). The previously reported p.R31C and the novel p.I47T PAX8 mutations are devoid of activity. Conclusion: Four different PAX8 mutations were detected in six index patients with CH (ten total subjects). The p.R31C, p.R31H, and p.R108X mutations have been reported. The novel p.I47T PAX8 mutation presented loss of function leading to CH. Thyroid ectopy was observed in two cases of PAX8 (p.R31H) mutation, a finding that has not been reported previously. We observed a high inter-individual and intra-familial variability of the phenotype in PAX8 mutations, underlining that population genetic studies for CH should include patients with various clinical presentations. [ABSTRACT FROM AUTHOR]

Objective: To date, no evidence of robust genotype-phenotype correlation or disease modifiers for multiple endocrine neoplasia type 1 (MEN1) syndrome has been described, leaving the highly variable clinical presentation of patients unaccounted for. Design: As the CDKN1B (p27) gene causes MEN4 syndrome and it is transcriptionally regulated by the product of the MEN1 gene (menin), we sought to analyze whether p27 influences the phenotype of MEN1-mutated patients. The cohort consisted of 100 patients carrying germline MEN1 gene mutations and 855 population-matched control individuals. Methods: Genotyping of the coding p27 c.326TOG (V109G) variant was performed by sequencing and restriction site digestion, and the genotypes were associated with clinical parameters by calculating odds ratios (ORs) and their 95% CIs using logistic regression. Results: There were significant differences in p27 V109G allele frequencies between controls and MEN1-mutated patients (ORZ2.55, PZ0.019, CIZ1.013-5.76). Among patients who are R30 years old carrying truncating MEN1 mutations, the T allele was strongly associated with susceptibility to tumors in multiple glands (three to four glands affected vs one to two glands affected; ORZ18.33; PZ0.002, CIZ2.88-16.41). This finding remained significant after the Bonferroni's multiple testing correction, indicating a robust association. No correlations were observed with the development of MEN1-related tumors such as hyperparathyroidism, pituitary adenomas, and enteropancreatic and adrenocortical tumors. Conclusions: Our study suggests that the p27 tumor suppressor gene acts as a disease modifier for the MEN1 syndrome associated with MEN1 germline mutations. If confirmed in independent patient cohorts, this finding could facilitate the management of this clinically complex disease. [ABSTRACT FROM AUTHOR]

Background: Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive inherited endocrine diseases. Steroid 11b-hydroxylase (P450c11) deficiency (11OHD) is the second most common form of CAH. Aim: The aim of the study was to study the functional consequences of three novel CYP11B1 gene mutations (p.His125Thrfs*8, p.Leu463_Leu464dup and p.Ser150Leu) detected in patients suffering from 11OHD and to correlate this data with the clinical phenotype. Methods: Functional analyses were done by using a HEK293 cell in vitro expression system comparing WT with mutant P450c11 activity. Mutant proteins were examined in silico to study their effect on the three-dimensional structure of the protein. Results: Two mutations (p.His125Thrfs*8 and p.Leu463_Leu464dup) detected in patients with classic 11OHD showed a complete loss of P450c11 activity. The mutation (p.Ser150Leu) detected in a patient with non-classic 11OHD showed partial functional impairment with 19% of WT activity. Conclusion: Functional mutation analysis enables the correlation of novel CYP11B1 mutations to the classic and non-classic 11OHD phenotype respectively. Mutations causing a non-classic phenotype show typically partial impairment due to reduced maximum reaction velocity comparable with non-classic mutations in 21-hydroxylase deficiency. The increasing number of mutations associated with non-classic 11OHD illustrate that this disease should be considered as diagnosis in patients with otherwise unexplained hyperandrogenism. [ABSTRACT FROM AUTHOR]

Objective: The p.V600E BRAF and RAS mutations are found in 30-80% of differentiated thyroid carcinoma (DTC). BRAF mutation has been associated with poor prognosis. This study investigated the role of molecular studies in preoperative diagnosis of DTC and the association of p.V600E mutation with prognostic factors. Design: Prospective study. Methods: A total of 202 patients with cytological diagnosis of Bethesda III-VI underwent preoperative molecular studies and subsequent thyroidectomy. p.V600E and RAS mutations were studied in the cytology smears, using real-time PCR genotyping technique. The BRAF mutation (BRAF+ or BRAF-) was correlated with histological and clinical findings. Results: Molecular study of 172 nodules with Bethesda III-V cytology improved negative predictive value and accuracy of Bethesda III and IV diagnosis. BRAF mutation was present in 65% of 94 DTC and p.Q61R NRAS in one. Except for age, BRAF+ and BRAF- did not differ in sex, tumor size, histological subtype, multifocality, vascular invasion, extrathyroidal extension, or prognostic staging. Among papillary carcinomas, lymph node (LN) metastasis was diagnosed in 23% BRAF+ and 37% BRAF-. Distant metastasis occurred in four BRAF-. Recurrent or persistent disease was more frequent in BRAFK (26.7 vs 3.3% BRAF+, P=0.002) along follow-up of 29.8±10 months. BRAFC patients without LN metastasis by pre-operative evaluation submitted to thyroidectomy with central neck dissection (CND) had more frequent LN metastasis (45 vs 5% no CND, P=0.002), but no difference in clinical outcome was observed. Conclusions: Pre-operative identification of BRAF mutation improved cytological diagnosis of DTC, but it was not associated with poor prognostic factors. Prophylactic CND did not guarantee better outcome in BRAF+ patients. [ABSTRACT FROM AUTHOR]

Objective: Although the succinate dehydrogenase (SDH)-related tumor spectrum has been recently expanded, there are only rare reports of non-pheochromocytoma/paraganglioma tumors in SDHx-mutated patients. Therefore, questions still remain unresolved concerning the aforementioned tumors with regard to their pathogenesis, clinicopathological phenotype, and even causal relatedness to SDHx mutations. Absence of SDHB expression in tumors derived from tissues susceptible to SDH deficiency is not fully elucidated. Design and methods: Three unrelated SDHD patients, two with pituitary adenoma (PA) and one with papillary thyroid carcinoma (PTC), and three SDHB patients affected by renal cell carcinomas (RCCs) were identified from four European centers. SDHA/SDHB immunohistochemistry (IHC), SDHx mutation analysis, and loss of heterozygosity analysis of the involved SDHx gene were performed on all tumors. A cohort of 348 tumors of unknown SDHx mutational status, including renal tumors, PTCs, PAs, neuroblastic tumors, seminomas, and adenomatoid tumors, was investigated by SDHB IHC. Results: Of the six index patients, all RCCs and one PA displayed SDHB immunonegativity in contrast to the other PA and PTC. All immunonegative tumors demonstrated loss of the WT allele, indicating bi-allelic inactivation of the germline mutated gene. Of 348 tumors, one clear cell RCC exhibited partial loss of SDHB expression. Conclusions: These findings strengthen the etiological association of SDHx genes with pituitary neoplasia and provide evidence against a link between PTC and SDHx mutations. Somatic deletions seem to constitute the second hit in SDHB-related renal neoplasia, while SDHx alterations do not appear to be primary drivers in sporadic tumorigenesis from tissues affected by SDH deficiency. [ABSTRACT FROM AUTHOR]

Objective: Adrenal vein sampling (AVS) is a technically demanding procedure required for the identification of suitable candidates for unilateral adrenalectomy in primary aldosteronism. Recently, somatic KCNJ5 KC-channel mutations in aldosterone-producing adenoma (APA) patients have been shown to influence steroid gradients during AVS. These and other recently identified genetic modifiers (ATP1A1 and ATP2B3) might affect the final diagnosis and treatment of the affected patients. Design: Fifty-nine patients with APAs who had undergone successful AVS (adrenal vein cortisol:peripheral cortisol ratio R2) and had undergone a mutation analysis of their tumor tissue were studied. The mutation status of the APAs was as follows: 19 KCNJ5 mutations, eight ATPase mutations (five ATP1A1 and three ATP2B3), and 32 patients with none of these mutations. Methods: The lateralization index (ratio of aldosterone:cortisol on the side of the adenoma to aldosterone to cortisol on the contralateral side) and the contralateral suppression index (ratio of aldosterone:cortisol on the contralateral side to aldosterone to cortisol in the periphery) were calculated for the KCNJ5-mutated, ATPase-mutated, and the KCNJ5/ATPase mutation-negative APA patients. Results: The lateralization indices of the ATPase mutation carriers had a median of 19.9 compared with a median of 16.0 in the KCNJ5 mutation carriers and that of 20.5 in the KCNJ5/ATPase mutation-negative patients. The contralateral suppression indices of the ATPase-mutated patients had a median of 0.1 compared with a median of 0.4 in the KCNJ5 mutation carriers and that of 0.2 in the KCNJ5/ATPase mutation-negative patients. The differences between the genetic groups were not statistically significant. Conclusions: We did not find evidence for a clinically important impact of mutation status on steroid gradients during AVS. [ABSTRACT FROM AUTHOR]

Objective: Due to a strong genotype-phenotype correlation, the timing of prophylactic thyroidectomy in rearranged during transfection (RET) gene mutation carriers is usually dictated by genetic analysis. Subjects and methods: We report a nationwide retrospective study of the clinical data of 77 French patients from 19 families with a mutation in codon 790 of the RET proto-oncogene. Results: The average age at diagnosis was 35.6 yearsG20.5. Thirty-nine patients were women. Fiftyfive patients underwent operations for the treatment of medullary thyroid carcinoma (MTC) at the mean age of 38 years (4-82 years). The mean follow-up duration was 89 months. TNM staging was as follows: T0NxMx in 19, TxNxMx in 1, T1NxMx in 22, T1N1Mx in 8, T2N1Mx in 1 and T3N1Mx in four patients. In the T1/x-Nx group, 96% were considered cured after surgery. In the N1 group (n=13), six patients had multifocal disease and five patients were cured. Age and gender were not significant predictors of remission. Twenty-two patients did not undergo an operation (age 1.5-78 years); among them, 11 patients had a mean basal calcitonin (CT) level of 9.8 pg/ml (2-24) after 53 months of follow-up. One patient had been operated on for phaeochromocytoma (PHEO), and their CT level remained normal for 262 months. Conclusions: This study confirms that RET 790 mutation is associated with a non-aggressive form of multiple endocrine neoplasia type 2, as 28% of the patients were followed up without thyroidectomy, 25% had been thyroidectomised with no tumour being detected and even patients with MTC had slowevolving disease. Moreover, only one patient had PHEO, and no-one had primary hyperparathyroidism. [ABSTRACT FROM AUTHOR]

Objective: Loss-of-function calcium-sensing receptor (CAR) mutations cause elevated parathyroid hormone (PTH) secretion and hypercalcaemia. Although full Car deletion is possible in mice, most human CAR mutations result from a single amino acid substitution that maintains partial function. However, here, we report a case of neonatal severe hyperparathyroidism (NSHPT) in which the truncated CaR lacks any transmembrane domain (CaRR392X), in effect a full CAR 'knockout'. Case report: The infant (daughter of distant cousins) presented with hypercalcaemia (5.5-6 mmol/l corrected calcium (2.15-2.65)) and elevated PTH concentrations (650-950 pmol/l (12-81)) together with skeletal demineralisation. NSHPT was confirmed by CAR gene sequencing (homozygous c.1174C-to- T mutation) requiring total parathyroidectomy during which only two glands were located and removed, resulting in normalisation of her serum PTH/calcium levels. Design and methods: The R392X stop codon was inserted into human CAR and the resulting mutant (CaRR392X) expressed transiently in HEK-293 cells. Results: CaRR392X expressed as a 54 kDa dimeric glycoprotein that was undetectable in conditioned medium or in the patient's urine. The membrane localisation observed for wild-type CaR in parathyroid gland and transfected HEK-293 cells was absent from the proband's parathyroid gland and from CaRR392X-transfected cells. Expression of the mutant was localised to endoplasmic reticulum consistent with its lack of functional activity. Conclusions: Intriguingly, the patient remained normocalcaemic throughout childhood (2.5 mM corrected calcium, 11 pg/ml PTH (10-71), age 8 years) but exhibited mild asymptomatic hypocalcaemia at age 10 years, now treated with 1-hydroxycholecalciferol and Ca2+ supplementation. Despite representing a virtual CAR knockout, the patient displays no obvious pathologies beyond her calcium homeostatic dysfunction. [ABSTRACT FROM AUTHOR]

Objective: In the field of fine-needle aspiration cytology, the category of suspicious for malignancy (SM) thyroid lesions, that bears 55-85% risk of malignant histology, is a challenging topic in which morphology alone is not always able to make a correct diagnosis. Recently, immunocytochemistry (ICC) has been referred to as helpful in differentiating low- and high-malignant risk lesions and BRAF activating mutations have been identified in a significant amount of papillary thyroid carcinomas (PTC). The introduction of the liquid-based cytology (LBC) may simplify the application of these techniques to thyroid cytology . Design: Our aim is to evaluate the diagnostic and prognostic role of both ICC and BRAF mutation for the SM category on LBC. Methods: From October 2010 through June 2011, 113 LBC cytological cases (including 37 SM and 76 PTC) underwent surgery . All cases were studied for BRAF mutation and ICC. Results: ICC resulted positive in 26 (86.6%) histologically malignant SM with 15 of which (40.5%) expressing a BRAF mutation. Overall, 63 cases showed a BRAF mutation resulting in PTC. Concerning the prognostic role of BRAF mutation for the two categories, we reported a significant correlation with multifocality, nodal involvement and extra-capsular invasion (P < 0.0001). Conclusions: Special techniques such as ICC and molecular markers might be successfully carried out on LBC-processed material. For both categories, ICC is more sensitive whereas BRAF analysis is an interesting support due to its high specificity adding a prognostic value in both SM and PTCs. [ABSTRACT FROM AUTHOR]

Background: Pseudohypoaldosteronism type 1 (PHA1) is a monogenic disease caused by mutations in the genes encoding the human mineralocorticoid receptor (MR) or the a (SCNN1A), β (SCNN1B) or γ (SCNN1G) subunit of the epithelial Na+ channel (ENaC). While autosomal dominant mutation of the MR cause renal PHA1, autosomal recessive mutations of the ENaC lead to systemic PHA1. In the latter, affected children suffer from neonatal onset of multi-organ salt loss and often exhibit cystic fibrosis-like pulmonary symptoms. Objective: We searched for underlying mutations in seven unrelated children with systemic PHA1, all offsprings of healthy consanguineous parents. Methods and results: Amplification of the SCNN1A gene and sequencing of all 13 coding exons unraveled mutations in all of our patients. We found five novel homozygous mutations (c.587_588insC in two patients, c.1342_1343insTACA, c.742delG, c.189C>A, c.1361-2A>G) and one known mutation (c.1474C>T) leading to truncation of the aENaC protein. All parents were asymptomatic heterozygous carriers of the respective mutations, confirming the autosomal recessive mode of inheritance. Five out of seven patients exhibited pulmonary symptoms in the neonatal period. Conclusion: The a subunit is essential for ENaC function and mutations truncating the pore-forming part of the protein leading to systemic PHA1. Based on current knowledge, the pulmonary phenotype cannot be satisfactorily predicted. [ABSTRACT FROM AUTHOR]

Objective: Most cases of familial isolated pituitary adenomas with mutated aryl hydrocarbon receptorinteracting protein (AIP:HGNC:358) gene develop somatotropinomas. They are characterised by an aggressive clinical phenotype including early age at diagnosis, large tumours and frequent invasiveness. There is little information on AIP gene mutations' prevalence in isolated somatotropinomas characterised by poor response to somatostatin analogue treatment. The aim of this study was to investigate the prevalence of AIP mutations in non-familial cases of somatotropinomas with poor response to conventional treatment. Design and methods: Fifty patients with acromegaly (22 males/28 females, age 51±18 years) and 60 controls were included in this study performed at eight University Hospitals in Spain. None had family history of pituitary adenomas or other endocrine tumors. All patients failed to respond to conventional treatment including surgery and somatostatin analogues. Some patients received adjuvant radiotherapy and most cases required pegvisomant (PEG) treatment for normalisation of IGF1. AIP analysis was performed in DNA extracted from peripheral leucocytes, using standardised PCR protocol in which the coding regions of exons 1, 2, 3, 4, 5 and 6 were amplified. Possible deletions/duplications were studied using multiplex ligation-dependent probe amplification. Results: Sequence changes of potential different significance that could be considered as mutations or variations of unknown significance (VUS) of the AIP gene were found in four patients (8%). In two cases, two different mutations previously described were found: p.Arg9Gln and p.Phe269Phe. Two other VUS were also found: c.787+24C>T in intron 5 and c.100-18C>T in intron 1. Age at diagnosis ranged from 21 to 50 years old, and in all patients, the tumor was a macroadenoma depicting IGF1 normalisation under PEG treatment. Conclusions: AIP germline mutations show a low, but non-negligible, prevalence in non-familial acromegaly patients with tumors resistant to treatment with somatostatin analogues. [ABSTRACT FROM AUTHOR]

Objective: The genetic mutations causing the constitutive activation of MEK/ERK have been regarded as an initiating factor in papillary thyroid carcinoma (PTC). The ERK-specific dual specificity phosphatase 6 (DUSP6) is part of the ERK-dependent transcriptional output. Therefore, the coordinated regulation of the activities of ERK kinases and DUSP6 may need to be reestablished to make new balances in PTC. Methods: To investigate the role of DUSP6 in the regulation of ERK1/2 (MAPK3/1)-dependent transcription, 42 benign neoplasms and 167 PTCs were retrospectively analyzed by immunohistochemistry with dideoxy sequencing to detect BRAFV600E mutation. Results: The expressions of total ERK1/2, DUSP6, c-Fos (FOS), c-Myc (MYC), cyclin D1, and PCNAwere markedly increased in PTC compared with those in benign neoplasms. However, phospho-ERK1/2 was detected in only eight (4.8%) cases out of 167 PTC samples. Unexpectedly, the staining intensity and nuclear localization of ERK1/2 were not affected by the presence or absence of the BRAFV600E mutation. However, the expressions of c-Fos and PCNA were elevated in BRAFV600E-positive PTC compared with those in BRAFV600E-negative PTC. Interestingly, the higher staining intensities of DUSP6 were associated with the level of total ERK1/2 expression (PZ0.04) and with high-risk biological features such as age (PZ0.05), tumor size (PZ0.01), and extrathyroidal extension (linear by linear association, PZ0.02). In addition, DUSP6 silencing significantly decreased the cell viability and migration rate of FRO cells. Conclusions: The coordinated upregulation of total ERK1/2 and its phosphatase, DUSP6, is related to bare detection of phospho-ERK1/2 in PTC regardless of BRAFV600E mutation status. A link between DUSP6 expression and high-risk features of PTC suggested that DUSP6 is an important independent factor affecting the signaling pathways in established PTC. [ABSTRACT FROM AUTHOR]

Objective: Familial hypocalciuric hypercalcemia (FHH) syndrome is a rare benign condition, inherited as an autosomal dominant trait, in which inactivating mutations of the calcium-sensing receptor (CASR) gene affects the body's ability to regulate calcium homeostasis. Its outcome is featured by increased levels of serum calcium, moderate hypophosphatemia, and inadequately normal or elevated circulating parathyroid hormone levels. Affected patients are mostly asymptomatic and do not benefit from surgical resection of their mildly enlarged parathyroids. Design: We evaluated for hypercalcemia an Italian family that was identified via a young adult male proband referred to our center for parathyroidectomy. Methods: The patients and the family members were evaluated both biochemically and genetically as suspected FHH subjects. An in vitro functional study was performed by site-directed mutagenesis, and CASR activity was monitored by measuring intracellular calcium ([Ca2+]i Results: The patient had a novel germline heterozygous CASR mutation (c.361_364GATT; p.D121del/fsX122). The mutation caused a premature stop codon at codon 122, exiting a truncated protein. The biochemical phenotype of all family members carrying the heterozygous deletion was concordant with classic FHH syndro Conclusions: Our findings confirm the role of CASR gene mutational analysis to offer a valuable addition for the recognition of FHH in hypercalcemic patients not yet characterized for a positive familial history of hypercalcemia, the only condition that identifies CASR gene mutations in hypercalcemia. [ABSTRACT FROM AUTHOR]

Objective: The established causes of GH insensitivity include defects of the GH receptor and STAT5B. The latter condition is also characterized by severe immunodeficiency. A recent case with short stature, GH resistance, and immunodeficiency due to an I&kgr;B mutation suggests that the NF-&kgr;B pathway may interact with STAT5B signaling. Design: Here, we present a case of a short child with several congenital anomalies as well as GH insensitivity and mild immunodeficiency associated with a mosaic de novo duplication of chromosome 17q21-25, suggesting that overexpression of one of the duplicated genes may be implicated in GH resistance. Methods and results: In vitro studies on blood lymphocytes showed disturbed signaling of the CD28 pathway, involving NF-&kgr;B and related proteins. Functional studies on cultured skin fibroblasts revealed that NF-&kgr;B activation, PI3K activity, and STAT5 phosphorylation in response to GH were suppressed, while the sensitivity to GH in terms of MAPK phosphorylation was increased. An in silico analysis of the duplicated genes showed that MAP3K3 and PRKCA are associated with the NF-&kgr;B pathway. Baseline MAP3K3 expression in T-cell blasts (TCBs) was normal, but PRKCA expression in TCBs and fibroblasts was significantly higher than that in control cells. Conclusions: We conclude that the 17q21-25 duplication is associated with GH insensitivity and disturbed STAT5B, PI3K, and NF-&kgr;B signaling, possibly due to PRKCA mRNA overexpression. [ABSTRACT FROM AUTHOR]

Objective: Genetic disorders of calcium metabolism arise in a familial or sporadic setting. The calciumsensing receptor (CASR) plays a key role in maintaining calcium homeostasis and study of the CASR gene can be clinically useful in determining etiology and appropriate therapeutic approaches. We report two cases of novel CASR gene mutations that illustrate the varying clinical presentations and discuss these in terms of the current understanding of CASR function. Patients and methods: A 16-year-old patient had mild hypercalcemia associated with low-normal urinary calcium excretion and normal-to-high parathyroid hormone (PTH) levels. Because of negative family history, familial hypocalciuric hypercalcemia was originally excluded. The second patient was a 54-year-old man with symptomatic hypocalcemia, hyperphosphatemia, low PTH, and mild hypercalciuria. Familial investigation revealed the same phenotype in the patient's sister. The coding region of the CASR gene was sequenced in both probands and their available first-degree relatives Results: The first patient had a novel heterozygous inactivating CASR mutation in exon 4, which predicted a p.A423K change; genetic analysis was negative in the parents. The second patient had a novel heterozygous activating CASR mutation in exon 6, which predicted a p.E556K change; the affected sister of the proband was also positive. Conclusions:We reported two novel heterozygous mutations of the CASR gene, an inactivating mutation in exon 4 and the first activating mutation reported to date in exon 6. These cases illustrate the importance of genetic testing of CASR gene to aid correct diagnosis and to assist in clinical management. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To comprehensively phenotype parents identified with nonclassic congenital adrenal hyperplasia (NCCAH) by family genetic studies, termed here as cryptic NCCAH and to define the incidence of cryptic NCCAH in the parents of a large cohort of patients with 21-hydroxylase deficiency. DESIGN: Genotyping was performed on 249 parents of 145 unrelated congenital adrenal hyperplasia (CAH) patients. Parents with two CYP21A2 mutations underwent extensive evaluation. RESULTS: Of the 249 parents, ten (4%; seven females and three males) were identified as having cryptic NCCAH. The majority was of ethnicities previously reported to have a higher incidence of NCCAH. Cosyntropin stimulation performed in eight parents provided biochemical confirmation (17-hydroxyprogesterone range 56–364 nmol/l) and cortisol response was ≤500 nmol/l in three parents (38%). Of the seven women (27–54 years) with cryptic NCCAH, four had prior infertility, two reported irregular menses, two had treatment for hirsutism, one had androgenic alopecia. Men were asymptomatic. All cryptic NCCAH parents reported normal puberty and had normal height. Adrenal hypertrophy and a small adrenal myelolipoma were observed in two parents; testicular adrenal rest tissue was not found. CONCLUSIONS: Parents diagnosed with NCCAH by genetic testing are mostly asymptomatic. Temporary female infertility and suboptimal cortisol response were commonly observed. Ongoing glucocorticoid therapy is not indicated in adults with CAH identified by family genotype studies unless symptomatic, but glucocorticoid stress coverage should be considered in select cases. Parents of a child with CAH have a 1:25 risk of having NCCAH; if the mother of a child with CAH has infertility, evaluation for NCCAH is indicated. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Congenital hyperinsulinism (CHI) is characterized by persistent hypoglycemia due to the inappropriate insulin secretion. Inactivating mutations in the ABCC8 and KCNJ11 genes, which encode the sulfonylurea receptor 1 and Kir6.2 subunits of the ATP-sensitive K+(KATP) channel in pancreatic β-cell, are the most common cause of CHI. We studied the genetic etiology and phenotypes of CHI in Korean patients. METHODS: ABCC8 and KCNJ11 mutational analysis was performed in 17 patients with CHI. Medical records were retrospectively reviewed to identify phenotypes. RESULTS: Mutations (12 ABCC8 and three KCNJ11) were identified in 82% (14/17) of patients. Of these, nine ABCC8 mutations (E100X, W430X, c.1630+1G>C, D813N, Q923X, E1087_A1094delinsDKSDT, Q1134H, H1135W, and E1209Rfs) and one KCNJ11 mutation (W91X) were novel. Of the 14 patients, four had confirming recessively inherited CHI. The remaining ten patients had single heterozygous mutations. The majority (12/17) of patients were medically responsive. Of the five diazoxide-responsive patients, four had an ABCC8 mutation. The five patients unresponsive to medical management and one diazoxide-responsive patient underwent pancreatectomy and had diffuse histology. Of the operated six patients, two had recessively inherited mutations; three patients had a single heterozygous mutation (one maternally and two paternally inherited); and one patient had no identifiable KATPchannel mutation. CONCLUSIONS: This is the first study to report genotype and phenotype correlations among Korean patients with CHI. Mutations in ABCC8 and KCNJ11 are the most common causes of CHI in Korean patients. Similar to other studies, there is marked genetic heterogeneity and no clear genotype–phenotype correlation. [ABSTRACT FROM AUTHOR]

OBJECTIVE: In children with congenital hyperinsulinism (CHI), KATPchannel genes (ABCC8 and KCNJ11) can be screened rapidly for potential pathogenic mutations. We aimed to assess the contribution of rapid genetic testing to the clinical management of CHI. DESIGN: Follow-up observational study at two CHI referral hospitals. METHODS: Clinical outcomes such as subtotal pancreatectomy, 18F-Dopa positron emission tomography–computed tomography (PET–CT) scanning, stability on medical treatment and remission were assessed in a cohort of 101 children with CHI. RESULTS: In total, 32 (32%) children had pathogenic mutations in KATPchannel genes (27 in ABCC8 and five in KCNJ11), of which 11 (34%) were novel. In those negative at initial screening, other mutations (GLUD1, GCK, and HNF4A) were identified in three children. Those with homozygous/compound heterozygous ABCC8/KCNJ11 mutations were more likely to require a subtotal pancreatectomy CHI (7/10, 70%). Those with paternal heterozygous mutations were investigated with 18F-Dopa PET–CT scanning and 7/13 (54%) had a focal lesionectomy, whereas four (31%) required subtotal pancreatectomy for diffuse CHI. Those with maternal heterozygous mutations were most likely to achieve remission (5/5, 100%). In 66 with no identified mutation, 43 (65%) achieved remission, 22 (33%) were stable on medical treatment and only one child required a subtotal pancreatectomy. CONCLUSIONS: Rapid genetic analysis is important in the management pathway of CHI; it provides aetiological confirmation of the diagnosis, indicates the likely need for a subtotal pancreatectomy and identifies those who require 18F-Dopa PET–CT scanning. In the absence of a mutation, reassurance of a favourable outcome can be given early in the course of CHI. [ABSTRACT FROM AUTHOR]

Three syndromes affecting the thyroid gland are described in the literature separately: familial nonautoimmune hyperthyroidism, sporadic congenital nonautoimmune hyperthyroidism, and autonomous adenomas. Recent studies have shown that these three syndromes are caused by similar activating mutations of the TSH receptor gene (TSHR), and that the consequences of these mutations on the physiology and gene expression of the thyroid are qualitatively, but not quantitatively, similar. The three syndromes and two suggested unrecognized variants are in fact facets of the same disease, genetic hyperthyroidism due to TSHR mutations, the expression of which depends on the intensity of activation, its timing, and on the number of affected cells. [ABSTRACT FROM AUTHOR]

CONTEXT: Somatic mutations at residue R132 of isocitrate dehydrogenase 1 (IDH1) were recently discovered in gliomas and leukaemia at a high frequency. IDH1 is a metabolic gene, and the R132 mutations create a new enzymatic activity. OBJECTIVES: To determine whether IDH1 had somatically acquired mutations in thyroid carcinomas. DESIGN: Exons 4 and 6 of IDH1 were sequenced in a large panel of thyroid tumours (n=138) and compared with the patients normal DNA (n=26). We also correlated IDH1 mutations with clinical–pathological data and BRAF and RAS mutational status. RESULTS: We identified four novel and two previously described non-synonymous variants in thyroid carcinomas, which were absent in benign tumours and paired normal thyroid. Although IDH1 variants occurred at higher frequency in follicular thyroid carcinomas, follicular variant of papillary thyroid carcinoma (PTC) and undifferentiated thyroid carcinomas than the observed variants in classical PTC (15/72 vs 3/37), it was not significant (P=0.1). Sequence alignment across several species shows that all IDH1 genetic alterations occurred at evolutionarily conserved residues located within the active site, and therefore, are likely to affect protein function. Unlike other tumours, IDH1 and BRAF or RAS mutations are not mutually exclusive. There was no association between IDH1 mutational status and clinical characteristics. CONCLUSION: IDH1-acquired genetic alterations are highly prevalent in thyroid carcinomas (16%). Our findings not only extend our understanding of the molecular mechanism underlying pathogenesis of thyroid tumours, but also emphasize the biological differences between tumour types. Those tumours with IDH1 mutations might benefit from therapies that exploit this alteration. [ABSTRACT FROM AUTHOR]

The article reports on a study to investigate the prevalence of hepatocyte nuclear factor 4 alpha (HNF4A) mutations in a large cohort of patients with diazoxide responsive hyperinsulinemic hypoglycemia (HH). The study included 220 patients with HH responsive to diazoxide, who underwent sequencing of the ABCC8, KCNJ11, GCK, GLUD1, and/or HNF4A genes and were diagnosed with hyperinsulinism (HI). The study reveals that HNF4A mutations are one of the most common cause of diazoxide responsive HH.

The polyglandular autoimmune syndromes (PAS) comprise a wide spectrum of autoimmune disorders and are divided into a very rare juvenile (PAS type I) and a relatively common adult type with (PAS II) or without adrenal failure (PAS III). First clinical manifestation of PAS I usually occurs in childhood, whereas PAS II mostly occurs during the third and fourth decades. PAS I is caused by mutations in the autoimmune regulatory (AIRE) gene on chromosome 21 and is inherited in an autosomal recessive manner. Mutations in the AIRE gene result in defect proteins which cause autoimmune destruction of target organs by disturbing the immunological tolerance of the patients. Genetic testing may identify patients with PAS I, but not those with PAS II/III. For PAS II/III, susceptibility genes are known which increase the risk for developing autoimmune disorders, but must not be causative. These are certain HLA genes, the cytotoxic T lymphocyte antigen gene, and the protein tyrosine phosphatase nonreceptor type 22 gene on chromosomes 6, 2 and 1 respectively. Actual diagnosis of PAS involves serological measurement of organ-specific autoantibodies and subsequent functional testing. Management of patients with PAS including their family relatives is best performed in centres with special expertise in autoimmune endocrine disorders. [ABSTRACT FROM AUTHOR]

In hypophosphatemic rickets, there are both inherited and acquired forms, where X-linked dominant hypophosphatemic rickets (XLH) is the most prevalent genetic form and caused by mutations in the phosphate-regulating endopeptidase (PHEX) gene. XLH is associated with growth retardation and bone deformities. The renal tubular cells have an important role in calcium and phosphate metabolism, where the 1α-hydroxylase enzyme metabolizes the conversion of 25 (OH)-vitamin D to potent 1,25 (OH)2-vitamin D, whereas the sodium–phosphate transporter controls tubular phosphate reabsorption. The pathophysiological defect in XLH is speculated to cause an increase in a circulating phosphate regulating hormone termed phosphatonin (fibroblast growth factor 23 is the primary phosphatonin candidate), which leads to inhibition of 1α-hydroxylase, and simultaneously to inhibition of the sodium–phosphate transporter domain NPT2c leading to parathyroid hormone-independent phosphaturia. Hence, current treatment of XLH is 1,25 (OH)2-vitamin D or the vitamin D analog alfacalcidol and elementary phosphorus. Unfortunately, patients with XLH may develop nephrocalcinosis, secondary or tertiary hyperparathyroidism, and in some situations also hypertension and cardiovascular abnormalities. We describe a patient with XLH caused by a novel missense mutation in the PHEX gene, who on treatment with alfacalcidol and oral phosphate had normal growth and minimal bone deformities, but who subsequently developed moderate nephrocalcinosis, significant hyperparathyroidism, hypercalcemia, renal failure, and hypertension. We also report the use of the calcimimetic drug cinacalcet in the successful treatment of hypercalcemia and hyperparathyroidism. [ABSTRACT FROM AUTHOR]

INTRODUCTION: Multiple endocrine neoplasia type 2A (MEN2A) is an autosomal dominant hereditary disorder, associated with a cluster of germline gain-of-function mutations of the RET proto-oncogene (RET), mainly in exons 10–15. The G533C mutation in exon 8 of the RET is rare and has been mainly related to the familial medullary thyroid carcinoma. PATIENTS-METHODS: We describe the RET G533C mutation in exon 8 of the RET in two unrelated female index patients, with MEN2A phenotype, consisting of pheochromocytoma which was the presenting feature and medullary thyroid carcinoma. In addition, 12 family members were also studied. DNA extraction, PCR, and sequencing of RET was performed in exons 7–19 and 21, following standard procedures. RESULTS: The mutation was found in both index patients and in 6 out of 12 family members (50%). Three of them were biochemically affected with histologically proven medullary thyroid carcinoma in two of them while there are no certain clues regarding the other three members as they declined further evaluation. CONCLUSION: Patients with MEN2A should be also searched in exon 8 while positive carriers of this mutation should be screened annually for pheochromocytoma or other components of the syndrome. [ABSTRACT FROM AUTHOR]

INTRODUCTION: Familial hypocalciuric hypercalcemia (FHH) is a lifelong, benign, inherited condition caused by inactivating mutations in the calcium-sensing receptor (CASR) gene. Both FHH and primary hyperparathyroidism (PHPT) are characterized by elevated P-calcium, normal or elevated plasma-parathyroid hormone (P-PTH), and typically normal renal function. In PHPT, vitamin D metabolism is typically characterized by low plasma levels of 25-hydroxyvitamin D (25OHD), and high plasma levels of 1,25-dihydroxyvitamin D (1,25(OH)2D). In FHH, the vitamin D metabolism is not very well known. OBJECTIVE: To compare and evaluate plasma 25OHD, 1,25(OH)2D, and PTH in FHH and PHPT. DESIGN: Cross-sectional study. MATERIALS: About 66 FHH patients with mutations in the CASR gene, 147 patients with surgically verified PHPT, and 46 controls matched to FHH patients according to age (±5 years), sex, and season. All patients had a P-creatinine <140 µmol/l. METHODS: We measured P-calcium, P-Ca2+, P-albumin, P-creatinine, P-phosphate, P-magnesium, and P-PTH by standard laboratory methods. P-25OHD and P-1,25(OH)2D were measured by RIA or enzyme immunoassay. In FHH, all protein-coding exons in the CASR gene were sequenced and aligned to GenBank reference sequence NM_000388.2. RESULTS: PHPT patients had higher body mass index (2p<0.01), together with higher P-PTH (2p<0.01) and P-1,25(OH)2D (2p<0.01) compared with FHH patients. The groups had similar levels of P-Ca2+and of P-25OHD. The phenotypic expression of the CASR mutations (as determined by the degree of hypercalcemia) did not influence the levels of P-1,25(OH)2D. CONCLUSION: Even though P-calcium and P-25OHD were comparable, P-1,25(OH)2D and P-PTH differed between FHH and PHPT. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Anaplastic thyroid cancer (ATC) is often unoperable and chemotherapy and radiotherapy are the main treatments. Until now ‘primary ATC cell cultures’ (ANA) have been developed from surgical biopsies. The possibility to obtain ANA from fine-needle aspiration (FNA-ANA) and to test their sensitivity to different drugs could increase the effectiveness of treatments and avoid unnecessary surgical procedures. DESIGN: To obtain FNA-ANA from six ATC patients before undergoing surgery and to evaluate the chemosensitivity of FNA-ANA to chemotherapeutic agents and thiazolidinediones (TZD). METHODS AND RESULTS: FNA-ANA from the six ATC patients were cultured in RPMI 1640 and propagated in DMEM. Chemosensitivity was evaluated by inhibiting the proliferation with increasing concentrations of five different chemotherapeutic agents (bleomycin, cisplatin, gemcitabine, etoposide, and carboplatin) or TZD (rosiglitazone). Chemotherapeutic agents significantly inhibited (P<0.0001) FNA-ANA proliferation, such as TZD (P<0.001); etoposide was the most effective in reducing cell growth. Another ANA culture for each patient was obtained from a biopsy specimen; the results for the chemosensitivity tests were similar to those obtained with FNA-ANA. The V600EBRAF mutation was observed in two ATC patients; the inhibition of proliferation by drugs was similar in tumors with or without V600EBRAF mutation. CONCLUSIONS: Our study demonstrates 1) the possibility to obtain FNA-ANA, and opens the way to the use of FNA-ANA to test the chemosensitivity to different drugs (chemotherapeutic agents or TZD; and possibly the radiosensitivity) in each patient, avoiding unnecessary surgical procedures and the administration of inactive chemotherapeutics; and 2) that etoposide is highly effective in reducing ATC cell growth in vitro. [ABSTRACT FROM AUTHOR]

CONTEXT: Noonan syndrome (NS) is characterized by short stature, typical facial dysmorphology and congenital heart defects. Short-term effect of GH therapy in NS is beneficial, reports on the effect on adult height are scarce. OBJECTIVE: To determine the effect of long-term GH therapy in children with NS. DESIGN: Twenty-nine children with NS were treated with GH until final height was reached. SETTING: Hospital endocrinology departments. PATIENTS: Children with the clinical diagnosis of NS, with mean age at the start of therapy of 11.0 years, 22 out of 27 tested children had a mutation in the protein tyrosine phosphatase, non-receptor-type 11 gene (PTPN11 gene). INTERVENTIONS: GH was administered subcutaneously at 0.05 mg/kg per day until growth velocity was 1 cm/6 months. MAIN OUTCOME MEASURE: Linear growth (height) was measured at 3-month intervals in the first year and at 6-month intervals thereafter until final height. RESULTS: At the start of treatment, median height SDS (H-SDS) was –2.8 (–4.1 to –1.8) and 0.0 (–1.4 to +1.2), based on national and Noonan standards respectively. GH therapy lasted for 3.0–10.3 years (median, 6.4), producing mean gains in H-SDS of +1.3 (+0.2 to +2.7) and +1.3 (–0.6 to +2.4), based on national and Noonan standards respectively. In 22 children with a mutation in PTPN11 mean gain in H-SDS for National standards was +1.3, not different from the mean gain in the five children without a mutation in PTPN11+1.3 (P=0.98). CONCLUSION: Long-term GH treatment in NS leads to attainment of adult height within the normal range in most patients. [ABSTRACT FROM AUTHOR]

OBJECTIVE: The existence of genotype–phenotype correlation in multiple endocrine neoplasia type 1 (MEN1) is controversial. Two founder mutations of the MEN1 gene in Northern Finland gave us an opportunity to compare clinical features among heterozygotes of different mutations. Design and methods: Study cohort included 82 MEN1 heterozygotes who were tested for MEN1 during the years 1982–2001. Medical records were reviewed for manifestations of MEN1, other tumours and cause of death by the end of August 2003. Logistic regression analysis was used in evaluating the impact of age, gender and mutational status of affected heterozygotes on the likelihood of developing manifestations of MEN1. RESULTS: Founder mutations 1466del12 and 1657insC were found in 39 and 29 individuals, and D418N, G156R and R527X mutations in 9, 3 and 2 individuals respectively. Except for pituitary adenoma and nonfunctional pancreatic tumour (NFPT), age was a risk factor for all the disease manifestations. For NFPT, frameshift/nonsense mutations (1657insC, R527X) gave an odds ratio (OR) of 3.26 (95% confidence intervals (CI), 1.27–8.33; P = 0.014) compared with in-frame/missense mutations (1466del12, D418N, G156R); including the founder mutation carriers (n = 68) only, the 1657insC mutation gave an OR of 3.56 (CI, 1.29–9.83; P = 0.015). For gastrinoma, in-frame/missense mutations predicted the risk with an OR of 6.77 (CI, 1.31–35.0; P = 0.022), and in the founder mutations group the 1466del12 mutation gave an OR of 15.09 (CI, 1.73–131.9, P = 0.014). CONCLUSIONS: In this study population, NFPT was more common in the frameshift/nonsense or 1657insC mutation carriers, whereas gastrinoma was more common in the in-frame/missense or 1466del12 mutation carriers. [ABSTRACT FROM AUTHOR]

Background: Phytosterolaemia (sitosterolaemia) is a rare autosomal recessive condition caused by mutations on the ABCG5 and ABCG8 gut transporter proteins. This leads to accumulation of plantderived cholesterol-like molecules in blood and tissues. Case: We describe a family of Bangladesh origin, where three siblings (two males and one female) have homozygous mutations for phytosterolaemia, and exhibit short stature and adrenal failure with the female having ovarian failure. Findings: The index case (18-year-old female) and her sibling (16 years) have adrenal insufficiency with hyperpigmentation and raised levels of ACTH, at 367 and 690 ng/l respectively. The youngest child at 7 years has normal adrenal function. In addition, the index case has ovarian failure and sibling 2 has partial growth hormone deficiency. Conclusion: Although short stature is a recognised phenomenon, no previous association has been made between phytosterolaemia and other endocrine abnormalities. We postulate that the elevated plant sterol levels in phytosterolaemia may interfere with endocrine hormone synthesis; in particular, we present evidence that adrenal cholesterol metabolism may be preferentially affected, accounting for the adrenal insufficiency. [ABSTRACT FROM AUTHOR]

Background: Idiopathic short stature (ISS) includes a range of conditions. Some are caused by defects in the GH-IGF-I axis. ISS is an approved indication for GH therapy in the USA and a similar approval in Europe may be imminent. Genetic analysis for single-gene defects has made enormous contributions to understanding the physiology of growth regulation. Can this type of investigation help in predicting growth responses to GH or IGF-I therapy? Methods: The rationale for choice of GHor IGF-I therapy inISS is reviewed. Many ISS patientshave lowIGFI, but most can generate IGF-I levels in response to short-term GH administration. Some GH resistance seems to be present. Mutation analysis in several cohorts of GHIS and ISS patients is reviewed. Results: Low IGF-I levels suggest either unrecognised GH deficiency or GH resistance. In classical GHIS patients, there was a positive relationship between IGFBP-3 levels and height SDS. No relationship exists between mutations and phenotype. There is awide variability of phenotype in patients carrying identical mutations. HeterozygousGHreceptor (GHR)mutationswere present in!5%of ISS patients and their role in causing growth defects is questionable. Exceptions are dominant negative mutations that have been shown to disturb growth. Conclusions: Analysis for single-gene defects does not give sensitive predictions of phenotype and cannot predict responses to GHor IGF-I therapy. Endocrine abnormalities have closer correlationswith phenotype and may thus be a better guide to therapeutic responsiveness. [ABSTRACT FROM AUTHOR]