A Case of Pseudohypoparathyroidism Misdiagnosed as Idiopathic Epilepsy for 5 Years: Clinical Analysis and Follow-up Outcomes.

We report a 15-year-old Chinese girl who presented with intermittent seizure episodes and had been misdiagnosed as having idiopathic epilepsy 5 years previously. Laboratory testing revealed hypocalcemia, hyperphosphatemia, and a high parathyroid hormone (PTH) concentration. She was subsequently shown to have pseudohypoparathyroidism type Ib (PHPIb) based on the results of methylation analysis of the GNAS gene, which showed a loss of methylation of the differentially methylated regions (DMR) of GNAS-AS1, GNAS-XL, and GNAS-A/B; and a gain of methylation of the DMR of the GNAS-NESP55 region. We adjusted the patient's medication by prescribing calcium and calcitriol supplements, and gradually reduced the doses of antiepileptic drugs, until they had been completely discontinued. As a result, the patient did not experience any further seizures or epileptiform symptoms; and had normal plasma calcium, phosphorus, and 25-hydroxyvitamin D concentrations and 24-hour urinary calcium excretion. In addition, her PTH concentration gradually normalized over 12 months, and no urinary stones were found on ultrasonographic examination. In conclusion, the clinical presentation of PHP is complex, and the condition is often misdiagnosed. The diagnosis and follow-up of the present patient have provide valuable insights that should contribute to informed clinical decision-making and the implementation of appropriate treatment strategies.

Clinical and genetic characteristics for 4 patients with Type Ib pseudohypoparathyroidism. / 4例Ibåž‹å‡æ€§ç”²çŠ¶æ—è ºåŠŸèƒ½å‡é€€ç—‡çš„ä¸´åºŠå’Œé—ä¼ å­¦ç‰¹å¾.

Pseudohypoparayhyroidism (PHP) is a rare autosomal dominant or recessive genetic disorder characterized by low calcium, high phosphorus, and target organ resistance to parathyroid. The clinical characteristics and genetic features in 4 patients with Type Ib PHP in the Third Xiangya Hospital, Central South University, have been reviewed. All 4 patients had low calcium, high phosphorus, and parathyroid resistance. Among them, 2 patients had slightly elevated thyroid stimulating hormone and mild features of Albright's hereditary osteodystrophy, and one patient had hypokalemia. No guanine nucleotide-binding protein alpha-stimulating activity polypeptide 1 (GNAS) and gene variant associated with hypokalemia were identified using the whole exome sequencing. The results of the methylation-specific multiple ligation-dependent probe amplification showed that there were abnormal methylation of the upstream differentially methylated regions of GNAS in the 4 patients. There were phenotype overlap among the various subtypes of PHP. Detection of GNAS gene methylation in patients with clinical suspicion of Type Ib PHP is helpful for the diagnosis and treatment of PHP.

Clinical and genetic analysis of pseudohypoparathyroidism complicated by hypokalemia: a case report and review of the literature.

BACKGROUND: Pseudohypoparathyroidism (PHP) encompasses a highly heterogenous group of disorders, characterized by parathyroid hormone (PTH) resistance caused by mutations in the GNAS gene or other upstream targets. Here, we investigate the characteristics of a female patient diagnosed with PHP complicated with hypokalemia, and her family members. CASE PRESENTATION AND GENE ANALYSIS: A 27-year-old female patient occasionally exhibited asymptomatic hypocalcemia and hypokalemia during her pregnancy 1 year ago. Seven months after delivery, she experienced tetany and dysphonia with diarrhea. Tetany symptoms were relieved after intravenous calcium gluconate supplementation and she was then transferred to our Hospital. Laboratory assessments of the patient revealed hypokalemia, hypocalcemia and hyperphosphatemia despite elevated PTH levels. CT scanning of the brain revealed globus pallidus calcification. Possible mutations in GNAS and hypokalemia related genes were identified using WES, exon copies of STX16 were analized by MLPA and the methylation status of GNAS in three differential methylated regions (DMRs) was analyzed by methylation-specific polymerase chain reaction, followed by confirmation with gene sequencing. The patient was clinically diagnosed with PHP-1b. Loss of methylation in the A/B region and hypermethylation in the NESP55 region were detected. No other mutations in GNAS or hypokalemia related genes and no deletions of STX16 exons were detected. A negative family history and abnormal DMRs in GNAS led to a diagnosis of sporadic PHP-1b of the patient. CONCLUSIONS: Hypokalemia is a rare disorder associated with PHP-1b. Analysis of genetic and epigenetic mutations can aid in the diagnosis and accurate subtyping of PHP.

A novel GNAS mutation in pseudohypoparathyroidism type 1a in a Chinese man presented with recurrent seizure: a case report.

BACKGROUND: Pseudohypoparathyroidism is a rare genetic disease characterized by hypocalcaemia and hyperphosphataemia due to the defect to the guanine nucleotide-binding protein alpha subunit (GNAS) gene. Patients with pseudoparathyroidism type 1a and 1c could manifest Albright's hereditary osteodystrophy and multiple hormone resistance including gonadotropin and thyroid stimulating hormone. CASE PRESENTATION: Here we report a Chinese man who presented with fatigue, recurrent seizure and Albright's hereditary osteodystrophy. His genetic study revealed a heterozygote mutation in the GNAS gene [NM_000516.4(GNAS): c2787_2788del (p.Val930AspfsTer12)]. After calcium and calcitriol supplement, his seizures achieved partially remission. CONCLUSIONS: We report a case of PHP1a or 1c with a novel frameshift mutation in GNAS gene in a patient presenting with AHO, as well as TSH and partial gonadotropin resistance. This mutation in this case has not been reported in literature and adds to the spectrum of genetic mutations related to PHP.

Mutational Mosaics of Cell-Free DNA from Pancreatic Cyst Fluids.

BACKGROUND: Pancreatic cyst fluids (PCFs) enriched in tumor-derived DNA are a potential source of new biomarkers. The study aimed to analyze germinal variants and mutational profiles of cell-free (cf)DNA shed into the cavity of pancreatic cysts. METHODS: The study cohort consisted of 71 patients who underwent endoscopic ultrasound fine-needle aspiration of PCF. Five malignant cysts, 19 intraductal papillary mucinous neoplasms (IPMNs), 11 mucinous cystic neoplasms (MCNs), eight serous cystic neoplasms (SCNs), and 28 pseudocysts were identified. The sequencing of 409 genes included in Comprehensive Cancer Panel was performed using Ion Proton System. The mutation rate of the KRAS and GNAS canonical loci was additionally determined using digital PCR. RESULTS: The number of mutations detected with NGS varied from 0 to 22 per gene, and genes with the most mutations were: TP53, KRAS, PIK3CA, GNAS, ADGRA2, and APC. The frequencies of the majority of mutations did not differ between non-malignant cystic neoplasms and pseudocysts. NGS detected KRAS mutations in malignant cysts (60%), IPMNs (32%), MCNs (64%), SCNs (13%), and pseudocysts (14%), with GNAS mutations in 20%, 26%, 27%, 13%, and 21% of samples, respectively. Digital PCR-based testing increased KRAS (68%) and GNAS (52%) mutations detection level in IPMNs, but not other cyst types. CONCLUSIONS: We demonstrate relatively high rates of somatic mutations of cancer-related genes, including KRAS and GNAS, in cfDNA isolated from PCFs irrespectively of the pancreatic cyst type. Further studies on molecular mechanisms of pancreatic cysts malignant transformation in relation to their mutational profiles are required.

Renal Tubular Dysfunction Fully Accounts for Plasma Biochemical Abnormalities in Type 1A Pseudohypoparathyroidism.

Context: Type 1A pseudohypoparathyroidism (PHP-1A) is characterized by target organ resistance to PTH. Patients can present with various dysmorphic features; however, renal failure has not been classically described. Case Description: A female patient came to our attention at the age of 7 years with characteristic signs of PTH resistance (i.e., hypocalcemia, hyperphosphatemia, and high serum PTH levels). She also presented with hypothyroidism, early-onset obesity, short metacarpal bones, and multiple subcutaneous ossifications, leading to a clinical diagnosis of pseudohypoparathyroidism. In addition to her genetic condition, she had bilateral renal hypodysplasia that was slowly progressing to end-stage kidney disease. She received a kidney transplant at the age of 16 years and, after transplantation, experienced rapidly normalized calcium, phosphate, and PTH levels, allowing f withdrawal of vitamin D supplementation. Conclusions: To the best of our knowledge, ours is the first report of a patient with PHP-1A undergoing kidney transplantation. Normalization of biochemical parameters after the procedure demonstrated that renal tubular resistance to PTH is sufficient to explain the calcium/phosphate abnormalities observed in PHP-1A.

Analysis of the influence of the T393C polymorphism of the GNAS gene on the clinical expression of primary hyperparathyroidism.

BACKGROUND: The receptor of parathyroid hormone and parathyroid hormone-related-protein (PTH/PTHrp) is located in the cell membrane of target tissues - kidney and osteoblasts. It is a G protein-coupled-receptor whose Gsα subunit is encoded by the GNAS gene. Our aim was to study whether the single nucleotide polymorphism (SNP) T393C of the GNAS gene is associated with renal stones, bone mineral density (BMD), or bone remodelling markers in primary hyperparathyroidism (PHPT). METHODS: An analysis was made of clinical and biochemical parameters and densitometric values in three areas and their relationship with the T393C SNP of the GNAS gene in 261 patients with primary hyperparathyroidism and in 328 healthy controls. Genotyping was performed using the Custom Taqman® SNP Genotyping assay. RESULTS: The genotype frequencies of GNAS T/C 393 were similar in the control and PHPT groups. No association was found between genotypes and clinical expression of PHPT (renal stones and bone fractures). A nonstatistically significant trend was seen to lower BMD in the lumbar spine, femoral neck, and total hip in both PHPT and control C homozygote subjects. CONCLUSION: Genetic susceptibility to PHPT related to the GNAS T393C polymorphism or a major influence in its development and clinical expression were found. A C allele-related susceptibility to lower BMD in trabecular bone in both PHPT and control subjects is not sufficient to suggest a more severe clinical expression of PHPT. This trend may be considered as a basis for further studies with larger sample sizes and complementary functional evaluation.

Effect of electroacupuncture on gene expression in calcium signaling pathway in hippocampal cells in mice with cerebral ischemia reperfusion.

OBJECTIVE: To observe the regulation of electroacupuncture on gene expression at calcium signaling pathways in mice with cerebral ischemia reperfusion. METHODS: Sixty male, inbred Kunming mice were randomly assigned to three groups: repeated cerebral ischemia reperfusion group (RG, n = 24), sham-operated group (SG, n = 12), and electroacupuncture group (EG, n = 24). Mice in RG and EG groups were modeled by repeated cerebral ischemia reperfusion surgery, and EG mice were treated with electroacupuncture for 30 min after recovery from anesthesia. Changes in gene expression profile of mice hippocampi were analyzed by global expression profile microarray. Genes that were up-regulated or down-regulated greater than 1.5 folds were considered to be biologically meaningful. Real-time quantitative polymerase chain reaction (q-PCR) method was used to verify the expression of selected genes based on the algorithm [2^ (ΔΔCt)]. RESULTS: Compared with SG mice, 242 genes showed different in expressions in RG mice: 107 down-regulated and 135 up-regulated. Compared with RG mice, 609 genes showed a difference of expression in EG mice: 315 down-regulated and 375 up-regulated. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated two pathways: calcium signaling and long-term potentiation in which 11 differentially expressed genes selected. Six of the 11 genes in the calcium signaling pathway were verified after real-time q-PCR testing. CONCLUSION: Electroacupuncture treatment of cerebral ischemia reperfusion appears to regulate Atp2a2, Cacna1e, Camk2a, Gnas, Grm1, Rapgef3 genes in the calcium signaling pathway.

Toward the molecular dissection of peritoneal pseudomyxoma.

BACKGROUND: Outcome of pseudomyxoma peritonei (PMP) after cytoreductive surgery (CRS) and hypertermic intraperitoneal chemotherapy (HIPEC) is heterogeneous even after adjusting for clinico-pathological prognostic variables. The identification of additional prognostic or even predictive biomarkers is an unmet clinical need. PATIENTS AND METHODS: Forty patients with mucinous appendiceal tumors and PMP were clinically eligible and had evaluable tumor samples obtained after CRS and HIPEC. We carried out next-generations sequencing (NGS) of 50 gene's hotspot regions contained in the Hotspot Cancer Panel v2 using the Ion Torrent Personal Genome Machine platform (Life Technologies). RESULTS: KRAS and GNAS mutations were found in 72% and 52%, and their allelic frequency was below 10% in 55% and 43% of samples, respectively. KRAS and GNAS mutations were associated with worse progression-free survival (PFS) at univariate analysis (P = 0.006 and 0.011, respectively). At multivariate analysis, only KRAS mutations were independently associated with PFS (P = 0.012); GNAS mutations were not-being significantly associated with other poor prognostic features such as incomplete cytoreduction or KRAS mutations. Validation of results was carried out in an independent bi-institutional cohort of 25 patients and the prognostic effect of KRAS mutations was again confirmed in the multivariate model (P = 0.029). NGS approach allowed the discovery of other potentially druggable mutations such as those in PI3K, AKT, LKB1, FGFR3 and PDGFRA. CONCLUSIONS: Given the homogeneity of this series and the sensitivity of NGS in this low-cellularity tumor, we demonstrated for the first time a poor prognostic role of KRAS mutations.

12(S)-HETrE, a 12-Lipoxygenase Oxylipin of Dihomo-γ-Linolenic Acid, Inhibits Thrombosis via Gαs Signaling in Platelets.

OBJECTIVE: Dietary supplementation with polyunsaturated fatty acids has been widely used for primary and secondary prevention of cardiovascular disease in individuals at risk; however, the cardioprotective benefits of polyunsaturated fatty acids remain controversial because of lack of mechanistic and in vivo evidence. We present direct evidence that an omega-6 polyunsaturated fatty acid, dihomo-γ-linolenic acid (DGLA), exhibits in vivo cardioprotection through 12-lipoxygenase (12-LOX) oxidation of DGLA to its reduced oxidized lipid form, 12(S)-hydroxy-8Z,10E,14Z-eicosatrienoic acid (12(S)-HETrE), inhibiting platelet activation and thrombosis. APPROACH AND RESULTS: DGLA inhibited ex vivo platelet aggregation and Rap1 activation in wild-type mice, but not in mice lacking 12-LOX expression (12-LOX(-/-)). Similarly, wild-type mice treated with DGLA were able to reduce thrombus growth (platelet and fibrin accumulation) after laser-induced injury of the arteriole of the cremaster muscle, but not 12-LOX(-/-) mice, supporting a 12-LOX requirement for mediating the inhibitory effects of DGLA on platelet-mediated thrombus formation. Platelet activation and thrombus formation were also suppressed when directly treated with 12(S)-HETrE. Importantly, 2 hemostatic models, tail bleeding and arteriole rupture of the cremaster muscle, showed no alteration in hemostasis after 12(S)-HETrE treatment. Finally, the mechanism for 12(S)-HETrE protection was shown to be mediated via a Gαs-linked G-protein-coupled receptor pathway in human platelets. CONCLUSIONS: This study provides the direct evidence that an omega-6 polyunsaturated fatty acid, DGLA, inhibits injury-induced thrombosis through its 12-LOX oxylipin, 12(S)-HETrE, which strongly supports the potential cardioprotective benefits of DGLA supplementation through its regulation of platelet function. Furthermore, this is the first evidence of a 12-LOX oxylipin regulating platelet function in a Gs α subunit-linked G-protein-coupled receptor-dependent manner.

Reductions in hypothalamic Gfap expression, glial cells and α-tanycytes in lean and hypermetabolic Gnasxl-deficient mice.

BACKGROUND: Neuronal and glial differentiation in the murine hypothalamus is not complete at birth, but continues over the first two weeks postnatally. Nutritional status and Leptin deficiency can influence the maturation of neuronal projections and glial patterns, and hypothalamic gliosis occurs in mouse models of obesity. Gnasxl constitutes an alternative transcript of the genomically imprinted Gnas locus and encodes a variant of the signalling protein Gαs, termed XLαs, which is expressed in defined areas of the hypothalamus. Gnasxl-deficient mice show postnatal growth retardation and undernutrition, while surviving adults remain lean and hypermetabolic with increased sympathetic nervous system (SNS) activity. Effects of this knock-out on the hypothalamic neural network have not yet been investigated. RESULTS: RNAseq analysis for gene expression changes in hypothalami of Gnasxl-deficient mice indicated Glial fibrillary acid protein (Gfap) expression to be significantly down-regulated in adult samples. Histological analysis confirmed a reduction in Gfap-positive glial cell numbers specifically in the hypothalamus. This reduction was observed in adult tissue samples, whereas no difference was found in hypothalami of postnatal stages, indicating an adaptation in adult Gnasxl-deficient mice to their earlier growth phenotype and hypermetabolism. Especially noticeable was a loss of many Gfap-positive α-tanycytes and their processes, which form part of the ependymal layer that lines the medial and dorsal regions of the 3(rd) ventricle, while ß-tanycytes along the median eminence (ME) and infundibular recesses appeared unaffected. This was accompanied by local reductions in Vimentin and Nestin expression. Hypothalamic RNA levels of glial solute transporters were unchanged, indicating a potential compensatory up-regulation in the remaining astrocytes and tanycytes. CONCLUSION: Gnasxl deficiency does not directly affect glial development in the hypothalamus, since it is expressed in neurons, and Gfap-positive astrocytes and tanycytes appear normal during early postnatal stages. The loss of Gfap-expressing cells in adult hypothalami appears to be a consequence of the postnatal undernutrition, hypoglycaemia and continued hypermetabolism and leanness of Gnasxl-deficient mice, which contrasts with gliosis observed in obese mouse models. Since α-tanycytes also function as adult neural progenitor cells, these findings might indicate further developmental abnormalities in hypothalamic formations of Gnasxl-deficient mice, potentially including neuronal composition and projections.

Cinacalcet as adjunctive therapy in pseudohypoparathyroidism type 1b.

BACKGROUND: In patients with pseudohypoparathyroidism type 1b (PHP1b) due to a tissue-specific imprinting defect in the G-protein α-subunit, skeletal disorders can arise from the bones being sensitive to parathyroid hormone (PTH) while the kidneys remain resistant to this hormone. CASE-DIAGNOSIS/TREATMENT: We report a 4.8-year-old girl with PHP1b who presented with an abnormal gait, severe skeletal changes and elevated levels of serum PTH (2844 pg/ml), phosphate (7.2 mg/dl) and bone turnover markers. Traditional treatment with calcium and calcitriol failed to suppress PTH secretion, which was still elevated at 2877 pg/ml after 14 months of therapy, nor did it correct the other clinical, biochemical and radiographic abnormalities. The addition of cinacalcet to the treatment regimen over the subsequent 32 months resulted in normalization of serum PTH (58 ng/ml), phosphate (4.9 mg/dl) and bone turnover markers, and resolution of the radiographic changes, with no adverse effects noted. CONCLUSIONS: Due to its ease of administration, we recommend the addition of cinacalcet into the armamentarium of medications available to treat children with PHP1b.

Virilizing sclerosing-stromal tumor of the ovary in a young woman with McCune Albright syndrome: clinical, pathological, and immunohistochemical studies.

CONTEXT: McCune-Albright syndrome (MAS) is characterized by polyostotic fibrous dysplasia, café-au-lait skin pigmentations, and gonadotropin-independent sexual precocious puberty, resulting from a somatic postzygotic activating mutation of the GNAS1 gene. SETTING: We report a virilizing sclerosing-stromal tumor of the ovary in a young female with MAS. PATIENT: She presented polyostotic fibrous dysplasia of the left upper and lower limbs and a café-au-lait skin spot in the posterior area of the neck. She had a history of precocious puberty, diagnosed at the age of 6 years and treated with cyproterone acetate until the age of 10 years; then she developed central puberty with severe oligomenorrhea. At the age of 23 years, she was hospitalized for a virilization syndrome including hirsutism, acne, deepening of the voice, amenorrhea, and clitoromegaly. Serum levels of T were dramatically increased (1293 ng/dl; normal range, 10-80). The abdominal computed tomography scan revealed a solid mass located on the left ovary. INTERVENTION: An ovariectomy was performed, and histological examination revealed a sclerosing-stromal tumor with pseudolobular pattern. RESULTS: Immunohistochemical studies revealed that the tumor cells expressed all steroidogenic enzymes involved in androgen synthesis. Molecular analysis revealed that ovarian tumor cells harbored the Arg 201 activating mutation in the GNAS1 gene. After surgery, T levels returned to normal, the patient retrieved a normal gonadal function, and she was able to become pregnant. CONCLUSION: This observation extends the clinical spectrum of ovarian pathology of women with MAS. However, the mechanisms causing this ovarian tumor remain unclear, even if the gsp oncogene has been implicated in the pathogenesis of some gonadal tumors.

Neuromuscular symptoms in a patient with familial pseudohypoparathyroidism type Ib diagnosed by methylation-specific multiplex ligation-dependent probe amplification.

Pseudohypoparathyroidism type Ib (PHP-Ib) is a rare genetic disorder characterized by hypocalcemia and hyperphosphatemia due to imprinting defects in the maternally derived GNAS allele. Patients with PHP-Ib are usually identified by tetany, convulsions, and/or muscle cramps, whereas a substantial fraction of patients remain asymptomatic and are identified by familial studies. Although previous studies on patients with primary hypoparathyroidism have indicated that hypocalcemia can be associated with various neuromuscular abnormalities, such clinical features have been rarely described in patients with PHP-Ib. Here, we report a 12-year-old male patient with familial PHP-Ib and unique neuromuscular symptoms. The patient presented with general fatigue, steppage gait, and myalgia. Physical examinations revealed muscular weakness and atrophies in the lower legs, a shortening of the bilateral Achilles' tendons and absence of deep tendon reflexes. Laboratory tests showed hypocalcemia, hyperphosphatemia, elevated serum intact PTH level, and impaired responses of urinary phosphate and cyclic AMP in an Ellsworth-Howard test, in addition to an elevated serum creatine kinase level. Clinical features of the patient were significantly improved after 1 month of treatment with alfacalcidol and calcium. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and subsequent PCR analyses identified a methylation defect at exon A/B of GNAS and a microdeletion involving exons 4-6 of the GNAS neighboring gene STX16 in the patient and in his asymptomatic brother. The results suggest that various neuromuscular features probably associated with hypocalcemia can be the first symptoms of PHP-Ib, and that MS-MLPA serves as a powerful tool for screening of GNAS abnormalities in patients with atypical manifestations.

Intrahepatic expression of genes related to metabotropic receptors in chronic hepatitis.

AIM: To screen for genes related to metabotropic receptors that might be involved in the development of chronic hepatitis. METHODS: Assessment of 20 genes associated with metabotropic receptors was performed in liver specimens obtained by punch biopsy from 12 patients with autoimmune and chronic hepatitis type B and C. For this purpose, a microarray with low integrity grade and with oligonucleotide DNA probes complementary to target transcripts was used. Evaluation of gene expression was performed in relation to transcript level, correlation between samples and grouping of clinical parameters used in chronic hepatitis assessment. Clinical markers of chronic hepatitis included alanine and aspartate aminotransferase, γ-glutamyltranspeptidase, alkaline phosphatase and cholinesterase activity, levels of iron ions, total cholesterol, triglycerides, albumin, glucose, hemoglobin, platelets, histological analysis of inflammatory and necrotic status, fibrosis according to METAVIR score, steatosis, as well as anthropometric body mass index, waist/hip index, percentage of adipose tissue and liver size in ultrasound examination. Gender, age, concomitant diseases and drugs were also taken into account. Validation of oligonucleotide microarray gene expression results was done with the use of quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The highest (0.002 < P < 0.046) expression among genes encoding main components of metabotropic receptor pathways, such as the α subunit of G-coupled protein, phosphoinositol-dependent protein kinase or arrestin was comparable to that of angiotensinogen synthesized in the liver. Carcinogenesis suppressor genes, such as chemokine ligand 4, transcription factor early growth response protein 1 and lysophosphatidic acid receptor, were characterized by the lowest expression (0.002 < P < 0.046), while the factor potentially triggering hepatic cancer, transcription factor JUN-B, had a 20-fold higher expression. The correlation between expression of genes of protein kinases PDPK1, phosphoinositide 3-kinase and protein kinase A (Spearman's coefficient range: 0.762-0.769) confirmed a functional link between these enzymes. Gender (P = 0.0046) and inflammation severity, measured by alanine aminotransferase activity (P = 0.035), were characterized by diverse metabotropic receptor gene expression patterns. The Pearson's coefficient ranging from -0.35 to 0.99 from the results of qRT-PCR and microarray indicated that qRT-PCR had certain limitations as a validation tool for oligonucleotide microarray studies. CONCLUSION: A microarray-based analysis of hepatocyte metabotropic G-protein-related gene expression can reveal the molecular basis of chronic hepatitis.

Postnatal changes in the expression pattern of the imprinted signalling protein XLαs underlie the changing phenotype of deficient mice.

The alternatively spliced trimeric G-protein subunit XLαs, which is involved in cAMP signalling, is encoded by the Gnasxl transcript of the imprinted Gnas locus. XLαs deficient mice show neonatal feeding problems, leanness, inertia and a high mortality rate. Mutants that survive to weaning age develop into healthy and fertile adults, which remain lean despite elevated food intake. The adult metabolic phenotype can be attributed to increased energy expenditure, which appears to be caused by elevated sympathetic nervous system activity. To better understand the changing phenotype of Gnasxl deficient mice, we compared XLαs expression in neonatal versus adult tissues, analysed its co-localisation with neural markers and characterised changes in the nutrient-sensing mTOR1-S6K pathway in the hypothalamus. Using a newly generated conditional Gnasxl lacZ gene trap line and immunohistochemistry we identified various types of muscle, including smooth muscle cells of blood vessels, as the major peripheral sites of expression in neonates. Expression in all muscle tissues was silenced in adults. While Gnasxl expression in the central nervous system was also developmentally silenced in some midbrain nuclei, it was upregulated in the preoptic area, the medial amygdala, several hypothalamic nuclei (e.g. arcuate, dorsomedial, lateral and paraventricular nuclei) and the nucleus of the solitary tract. Furthermore, expression was detected in the ventral medulla as well as in motoneurons and a subset of sympathetic preganglionic neurons of the spinal cord. In the arcuate nucleus of Gnasxl-deficient mice we found reduced activity of the nutrient sensing mTOR1-S6K signalling pathway, which concurs with their metabolic status. The expression in these brain regions and the hypermetabolic phenotype of adult Gnasxl-deficient mice imply an inhibitory function of XLαs in energy expenditure and sympathetic outflow. By contrast, the neonatal phenotype of mutant mice appears to be due to a transient role of XLαs in muscle tissues.

Acquired plate-like osteoma cutis.

Plate-like osteoma cutis is a rare disorder that has been historically classified as a congenital syndrome. It has a possible relationship to a mutation in the gene (GNAS1) that encodes the α-subunit of the stimulatory G protein, which regulates adenyl cyclase activity. We report a case of extensive plaque-like masses on the scalp and face with no abnormalities in calcium or phosphate metabolism and no preceding inflammatory cutaneous conditions. With less than ten reported cases, to our knowledge, this is one the few cases of acquired plate-like osteoma cutis described in the literature.

Long-term follow-up of a pseudohypoparathyroidism type 1A patient with missense mutation (Pro115Ser) in exon 5.

Pseudohypoparathyroidism (PHP) refers to end-organ resistance that primarily impairs the renal actions of parathyroid hormone (PTH). The patients with PHP type Ia (PHP-Ia), one of the 4 types of PHP, show resistance to other peptide hormones as well as clinical features of Albright hereditary osteodystrophy (AHO), a constellation of short stature, obesity, brachydactyly, ectopic ossifications, and/or mental retardation. Here we report clinical follow-up for a long-term period in a PHP-Ia case who had a missense mutation leading to the substitution of proline by serine (Prol115Ser) in exon 5 which has been reported previously in only two patients. An 11-year-old boy applied for hand spasm to our hospital. On physical examination, he had short stature, round-shaped face and brachydactly. Laboratory evaluation revealed PTH and TSH resistance. Molecular genetic analysis of the GNAS gene revealed a P115S substitution. The patient was followed up for 13 years. Normocalcaemia was achieved with reduced doses of calcitriol (0.25 µg/day) and calcium supplements (40 mg/kg/day). Daily requirement for levothyroxine supplementation was still high (2.3 µg/kg) to achieve euthyroidism. His pubertal development was Tanner stage V and he has no gonadotropin resistance. To our knowledge, this is the first report concerning long-term follow-up of this rare mutation. We believe that despite the genetic heterogeneity of AHO, phenotype/genotype correlations of this kind of rare mutations may help to understand progress of the disease.

Secretogranin II binds to secretogranin III and forms secretory granules with orexin, neuropeptide Y, and POMC.

Functional variations in the secretogranin III (SCG3) gene are associated with susceptibility to obesity. SCG3 forms secretory granules with orexin, melanin-concentrating hormone (MCH), neuropeptide Y (NPY), and POMC in the hypothalamus. In this study, we screened proteins for SCG3-binding activity and identified secretogranin II (SCG2) using a yeast two-hybrid system. Immunoprecipitation revealed that SCG2 interacts with SCG3. In situ hybridization and immunohistochemistry indicated that SCG2 was highly expressed in the lateral hypothalamic area, paraventricular nucleus, and arcuate nucleus of the hypothalamus. Double-labeling immunohistochemical analysis demonstrated that SCG2 was expressed in orexin-, MCH-, NPY-, and POMC-expressing neurons. SCG2 was also coexpressed with SCG3. Upon introduction into neuroblastoma cells, SCG2 was expressed in the cytosol and formed granule-like structures with SCG3, orexin, NPY, or POMC. SCG3 bound to POMC; however, it did not bind to orexin, MCH, or NPY. By contrast, SCG2 formed aggregates with orexin, MCH, NPY, and POMC. SCG2 may act as a hormone carrier for orexin, MCH, NPY, and POMC by binding with SCG3, which targets proteins to the secretory granules. SCG2 mRNA levels increased along with those of SCG3, orexin, MCH, and NPY after a 24-h fast, suggesting that the SCG2/SCG3 system may respond in an adaptive manner to acute body weight changes. However, this SCG2/SCG3 system appears to be unresponsive to chronic body weight changes, such as diet-induced obesity or obesity in ob/ob mice. We suggest that SCG2, as well as SCG3, may be a potential regulator of food intake based on its capacity to accumulate appetite-related hormones into secretory granules.