Objective To investigate a Chinese pedigree suffering from Leydig cell hypoplasia ( LCH) based on clinical data and genetic diagnosis. Methods The patient was diagnosed by means of clinical data, hormone profiles, and human chorionic gonadotropin ( hCC) test. The luteinizing hormone/chorionic gonadotropin receptor(LHCGR) gene of the patient and family members was amplified and sequenced. Results The patient presented with male pseudohermaphroditism, low level of testosterone, which did not respond to hCG. Genetic analysis of the LHCGR revealed two novel mutations: a missense mutation located in exon 5, resulting in Ile replaced by Thr in the extracellular domain; and a splice site mutation in the 3' terminal of intron 6( IVS6-3 C→A). Proband's sister (46, XX) who lacked clinical manifestations showed the identical genotype with the patient. Conclusions A mutation in the consensus sequence of 3' splice site, in addition to a missense mutation (Ile 152Thr)in the extracellular ligand-binding domain is the cause of inactivation of the LHCGR gene in patient with Leydig cell hypoplasia.

To investigate the clinical features, genetic diagnosis, and treatment of a patient with Prader-Willi syndrome(PWS). For a case with clinically suspected PWS, methylation specific PCR(MSPCR)amplification was applied to CpG islands of SNRPN(exon α)gene locus in the 15q11-q13. Furthermore, the diagnosis was comfirmed by the method of bisulfite sequencing PCR(BSPCR). Metabolic status before and after the operation of sleeve gastrectomy were compared. Absence of amplification of paternal allele on chromosome 15q11-q13 was detected in the case by MSPCR, different from the normal control. Results of BSPCR further proved a full methylation of CpG islands in the SNRPN gene locus. Four months after sleeve gastrectomy, systemic metabolic status and ventricular function were improved. MSPCR and BSPCR were both consistent with genetic diagnosis of PWS. Weight loss surgery is expected to be a major therapy of this disease.

Objective To analyze CYP17A1 gene mutation in a patient with 46,XY disordered sex development and to explore the possible influence on the phenotype of the patient.Methods Eight exons of CYP17AI gene in the patient and her parents were amplified and directly sequenced.In order to construct Mini-gene system,PCR fragments containing wildtype and mutant splicing sites were inserted in expression vector,and then transfected into cells.RT-PCR was used to observe the influence of splicing site mutation.Wildtype and aberrant splicing CYP17A1 cDNA expression plasmids were constructed and transfected into cells respectively,and CYP17A1 enzyme activity was tested in vitro.Results Mutation analysis revealed compound heterozygous CYP17A1 mutations,with Y329fs in one allele and a synonymous substitution( c.1263G＞A:GCG＞GCA) in another allele.In vitro analysis showed that the synonymous substitution induced a novel splicing site,which resulted in aberrant splicing of CYP17A1 mRNA and lacked six or seven amino acids after 415 in splicing product.In vitro transfection and enzyme activity experiment showed that the aberrant splicing product abolished the enzyme activity completely.However,this mutation did not completely influence splicing.The patient also had a part of normal splicing product,which was a coincidence to the phenotype of the patient.Conclusion This is the first description of an exonic splicing mutation in CYP17A1 relevant to the 17ot-hydroxylase deficiency phenotype.The functional study of the aberrant splicing variant has been initiated.

Objective To investigate the adrenal steroidogenic function in genotype-proven heterozygotes carrying mutations in CYP17A1 gene in vivo. Methods Eight patients and 14 family members from 5 families with 17-hydroxylase/17,20-lyase deficiency (17OHD) were recruited. The mutations of the CYP17A1 gene in these individuals were screened by direct sequencing of PCR products. The hormonal response to ACTH was evaluated in the 14 genotype-proven carriers and 45 age- and sex-matched normal subjects. Results Three mutations were found in 5 unrelated families. 14 carriers with CYP17A1 mutation were identified, including 7 heterozygotes with D487_F489del, 6 with Y329fs, and 1 for H373L. Compared to the normal subjects, the carriers exhibited lower basal and ACTH-stimulated cortisol levels, but higher ACTH-stimulated corticosterone level. The ratios of corticosterone to cortisol in the genotype-proven heterozygotes were higher than those of normal individuals at baseline and following ACTH-stimulation. Similarly, progesterone level and ratios of progesterone to 17-hydroxyprogesterone in the male heterozygotes were also higher than that of normal individuals before and after stimulation. No significant differences were observed in the hormone levels between two genotypes (D487_F489del vs Y329fs). Conclusions Genotype-proven carriers of 17OHD without apparent clinical symptoms exhibit decreased enzyme activity,analogous to mildly impaired adrenal 21-hydroxylase activity in the carriers of CYP21 A2 gene mutation.

Previous studies suggest that NGAL (neutro phil gelatinase-associated lipocalin) is involved in the transformation and development of esophageal carcinoma. Alteration of NGAL expression can trigger the change of cellular morphology in esophageal carcinoma cells. However, the mechanisms remain unclear. To get a better understanding of NGAL function in esophageal carcinoma, NGAL protein was expressed in methylotrophic yeast, Pichia pastoris, and purified by chromatography. EC1.71 cells expressed high levels of NGALR (NGAL receptor) and EC109 cells expressed low levels of NGALR were used as cells model. The trafficking and the possible function of NGAL protein were then analyzed in the esophageal carcinoma cells. The results showed that 5-FAM-labeled recombinant NGAL protein could internalize into the EC1.71 and EC109 cells. Furthermore, the internalized NGAL protein could induce the alteration of cellular morphology, resulting in generation of autophagosome, transcriptional up-regulation of genes associated with autophagy and increase of phospho-ERK1/2 (p-ERK1/2). Interestingly, the treatment with the NGAL protein did not affect the intracellular iron level. These data indicate that induced autophagy by exogenous NGAL protein is a mechanism that internalized NGAL plays important roles in esophageal carcinoma cells, independent with NGAL-mediated iron transport process, while ERK1/2 signal pathway is involved in activation of autophagy by exogenous NGAL protein.

Objective To analyze the present situation of glucose metabolism and the characteristics of blood glucose fluctuation in in-hospital type l diabetic patients (T1DM). Methods One hundred and forty-three hospitalized cases of T1DM patients from November 2012 to November 2016 were retrospectively analyzed.The percentage of adult-onset T1DM patients was 76.22%(109/143)and none adult-onset was 23.78%(34/143). The following data were collected: general information, the indexes of glucose metabolism and islet function.Seventy-two-hour continuous glucose monitoring(CGM) was carried on 40 patients as a subgroup.Results The average age was(40.29 ± 16.79)years.The onset age of diabetes was(33.57 ± 17.18)years.The disease duration was 4.0(1.0,10.0)years.The body mass index(BMI)was(20.68 ± 2.95)kg/m2.The fasting blood glucose(FBG)was(12.02 ± 5.40)mmol/L.The HbA1c was(9.80 ± 2.65)%.The fasting C-peptide was 0.08(0.01,0.38)nmol/L.The 2-hour postprandial C-peptide (C-P 2 h) was 0.12(0.01, 0.70) nmol/L. The anti-glutamic acid decarboxylase antibody was 12.08(8.16,20.56)μg/L.The islet-cell antibody was 4.85(2.66,12.07)μg/L.By using multivariate linear regression analysis, HbA1c were negatively related to the duration and BMI of T1DM. CGM: the mean blood glucose was (10.34 ± 2.97) mmol/L. The standard deviation of blood glucose was (2.89 ± 1.07) mmol/L. The mean amplitude glycemic excursions was (7.10 ± 3.09) mmol/L. The incidence of hypoglycemia was 10.00% (≤ 2.8 mmol/L) and 32.50% (≤ 3.9 mmol/L). Conclusions Adult-onset T1DM patients account for more than two-thirds. In-hospital T1DM patients have poor control of blood glucose, and they show the clinical characteristics of high blood glucose fluctuation and more hypoglycemia.