Bing-Li Liu

A splice site mutation combined with a novel missense mutation of LHCGR cause male pseudohermaphroditism.

Leydig cell hypoplasia (LCH) is a rare form of male pseudohermaphroditism caused by inactivating mutations in the luteinizing hormone receptor gene (LHCGR). The majority of LHCGR mutations are located in the coding sequence, resulting in impairment of either LH/CG binding or signal transduction. We report a Chinese family with two siblings (46, XY and 46, XX) carrying a missense mutation (c. 455 T>C, p. Ile152Thr) and a splice site mutation (c. 537‐3 C>A). Computational analysis of the missense mutation in the three‐dimensional structural model predicted it might influence the distribution of hydrogen bonds and intermolecular contacts between the hormone and receptor. Consistent with these findings, in vitro mutant analysis revealed a marked impairment of human chorionic gonadotropin binding and signal transduction. The splice‐acceptor mutation (c. 537‐3 C>A) resulted in abnormal splicing of LHCGR mRNA, skipping exon 7. This report expands the genotypic spectrum of LHCGR mutations, with relevant implications for the molecular analysis of this gene.

A refined study of FCRL genes from a genome-wide association study for Graves' disease.

To pinpoint the exact location of the etiological variant/s present at 1q21.1 harboring FCRL1-5 and CD5L genes, we carried out a refined association study in the entire FCRL region in 1,536 patients with Graves’ disease (GD) and 1,516 sex-matched controls by imputation analysis, logistic regression, and cis-eQTL analysis. Among 516 SNPs with P<0.05 in the initial GWAS scan, the strongest signals associated with GD and correlated to FCRL3 expression were located at a cluster of SNPs including rs7528684 and rs3761959. And the allele-specific effects for rs3761959 and rs7528684 on FCRL3 expression level revealed that the risk alleles A of rs3761959 and C of rs7528684 were correlated with the elevated expression level of FCRL3 whether in PBMCs or its subsets, especially in CD19+ B cells and CD8+ T subsets. Next, the combined analysis with 5,300 GD cases and 4,916 control individuals confirmed FCRL3 was a susceptibility gene of GD in Chinese Han populations, and rs3761959 and rs7528684 met the genome-wide association significance level (Pcombined = 2.27×10−12 and 7.11×10−13, respectively). Moreover, the haplotypes with the risk allele A of rs3761959 and risk allele C of rs7528684 were associated with GD risk. Finally, our epigenetic analysis suggested the disease-associated C allele of rs7528684 increased affinity for NF-KB transcription factor. Above data indicated that FCRL3 gene and its proxy SNP rs7528684 may be involved in the pathogenesis of GD by excessive inhibiting B cell receptor signaling and the impairment of suppressing function of Tregs.

Identification of steroid biosynthetic defects in genotype‐proven heterozygous individuals for 17α‐hydroxylase/17,20‐lyase deficiency

Objective  P450c17 deficiency (17α‐hydroxylase/17,20‐lyase deficiency, 17OHD) is a rare form of congenital adrenal hyperplasia caused by CYP17A1 gene mutations. The D487_F489 deletion in exon 8 and Y329fs in exon 6 are relatively frequent mutations of the CYP17A1 gene in China that completely abolish the enzyme activity of P450c17. However, little remains known about steroid biosynthetic functions in carriers with these mutations in a single allele of the CYP17A1 gene, who are assumed to have 50% P450c17 activity. We investigated adrenal steroidogenic function in genotype‐proven heterozygotes carrying such mutations in the CYP17A1 gene in vivo.

A unique exonic splicing mutation in the CYP17A1 gene as the cause for steroid 17α-hydroxylase deficiency

BACKGROUND 17α-Hydroxylase/17,20-lyase deficiency (17OHD) caused by a mutation in the CYP17A1 gene is characterized by hypertension, hypokalemia, and abnormal development of the genitalia. The majority of CYP17A1 mutations are located in the coding sequence, and several intronic splicing site mutations have been reported. OBJECTIVE A 2.5-year-old girl with 46,XY disordered sex development exhibited a nearly normal basal cortisol level and reduced sexual steroids. This study is aimed to explore the molecular basis and analyze its possible influence on the phenotype of the patient. METHODS AND RESULTS Mutation analysis revealed compound heterozygous CYP17A1 mutations, with c.985_987delinsAA in one allele and a synonymous substitution (c.1263G>A) in another allele. In vitro expression analysis of the allelic minigene showed that the novel nucleotide variation located in exon 8 induces a splicing signal, which results in an aberrant splicing of CYP17A1 mRNA and a missing portion of exon 8. The translation product includes the deletion of six or seven amino acids from residue position 415 without causing a frameshift. Consistent with the result of molecular modeling, functional studies in transiently transfected HEK-293T cells with the aberrantly spliced enzyme proteins showed that the deleted proteins completely abolished the enzyme activity. However, RT-PCR indicated the existence of a small fraction of normal, functionally intact enzyme, which may explain the partial masculinization of this patient. CONCLUSION This is the first description of an exonic splicing mutation in CYP17A1 relevant to the 17OHD phenotype. It also demonstrates the importance of studying synonymous change in such patients with less severe phenotype.

Identifying a novel mutation of CYP17A1 gene from five Chinese 17α-hydroxylase/17, 20-lyase deficiency patients

Mutations of CYP17A1 gene could cause complete or partial, combined or isolated 17α-hydroxylase/17,20-lyase enzyme deficiencies (17OHD). We intended to investigate the CYP17A1 mutation in five unrelated patients and analyze its possible influence on phenotype of an atypical 17OHD patient presented with micropenis, hypertension and intermittent hypokalemia. Steroid hormones were assayed in these patients. A novel missense mutation (c.1169C>G, p. Thr390Arg) located in exon 7 was detected in one of the patients. Homozygous c. 985_987delinsAA, p. Tyr329fs mutation was found in two patients, while compound heterozygous mutations (c. 985_987delinsAA, p. Tyr329fs/c. 932-939 del, p. Val311fs and c. 287G>A, p. Arg96Gln/c. 985_987delinsAA, p. Tyr329fs) were found in two other patients, respectively. Then, steric model analysis of CYP17A1 showed that the novel mutation T390R changed the local structure as well as the electrostatic potential of the nearby beta sheet. Finally, site-directed mutagenesis and in vitro expression were used to analyze the activity of novel mutant CYP17A1. It indicated the T390R mutant retained part of enzyme activity, which was consistent to the clinical features. In conclusion, we identified a novel missense mutation of CYP17A1 gene from a patient with micropenis, hypertension and intermittent hypokalemia, which varied from other four patients. It also expanded our understanding of genotype-phenotype correlation of the disease.

Functional study of an aberrant splicing variant of the human luteinizing hormone (LH) receptor.

The luteinizing hormone receptor (LHR) is a member of a subfamily of G protein-coupled receptors that is characterized by its alternative splicing. In a previous study, we identified a splice site mutation of intron 6 (IVS6-3C>A) in a patient suffering from Leydig cell hypoplasia, which leads to aberrant splicing of LHR mRNA. In vitro expression analysis confirmed that this mutation results in the skipping of exon 7 in the mature mRNA of the LHR gene. In this study, we determined the impact of IVS6-3C>A on the RNA secondary structure and function of LHR-Del7. The three-dimensional structure of the leucine-rich repeats in LHR was predicted by molecular modeling. Radioactive ligand-binding assays verified that LHR-Del7 has no binding affinity for hCG. Furthermore, we detected negligible cAMP production in cells transfected with LHR-Del7. Cells co-expressing LHR-WT and LHR-Del7 were able to generate cAMP in response to hCG, but there was no significant difference between cells transfected with LHR-WT/vector and LHR-WT/LHR-Del7, although the variant was able to localize to cell surface, similar to wild-type receptor. These results indicated that LHR-Del7 does not have a dominant negative effect on LHR-WT cell surface expression, and although the pathological splicing variant LHR-Del7 was able to localize to cell membranes it failed to bind hCG and had no effect on wild-type LHR.

A novel intronic mutation and a missense mutation of MEN1 identified in two Chinese families with multiple endocrine neoplasia type 1.

Background: Multiple endocrine neoplasia type 1 (MEN1) caused by MEN1 mutation is widely recognized. To date, 14 novel mutations were reported in Chinese and intronic mutations are getting more attention. Aim: To explore clinical features and MEN1 mutations in two Chinese families suffering from MEN1. Methods: Nineteen individuals (10 males and 9 females) from two unrelated families with MEN1 were studied. Mutations of MEN1 were analyzed by direct sequencing of PCR products. In vitro splicing analysis was also performed with minigenes containing both wildtype and novel mutant fragments. Through the RNAstructure program, we analyzed the secondary structure of the wild type MEN1 pre-mRNA and then introduced T>G mutation at +2 donor splice site of intron 7. Results: Clinical features of 3 patients in two families were described, and 5 individuals were proven to be carriers of MEN1 mutation without apparent symptoms. A novel splicing site mutation of the intron 7 (IVS7+2 T→G) was identified in the first family. In vitro analysis also verified this mutation caused the aberrant splicing of MEN1 mRNA. With the RNAstructure program, we could figure out that the global secondary structure as well as the number of stems and loops of pre-mRNA greatly changed after this mutation. The mutation c. 1227 C>A (C409X) was identified in another family, which also caused the truncation of menin. Conclusion: We reported a novel intronic mutation and a missense mutations in two Chinese families suffering from MEN1.

Identification of a novel mutation in CYP17A1 gene

17α-hydroxylase/17,20-lyase deficiency (17OHD) is a rare autosomal recessive genetic disease that is characterized by low-renin hypertension, hypokalemia, and abnormal development of the genitalia. Mutations in the CYP17A1 gene account for this disease. We aim to investigate the CYP17A1 mutation and analyze its possible influence on phenotype in a Chinese patient with 17OHD. Steroid hormones were assayed. The 8 exons of the CYP17A1 gene were amplified and directly sequenced. Wild-type and mutant CYP17A1 cDNA were cloned into pcDNA3.1 expression vectors and transfected into 293T cells. Finally, 17-hydroxylase and 17,20-lyase activity were detected by using progesterone and 17-hydroxypregnenolone as the substrates. A novel missense mutation c.716 G>A located in exon 4 that changed the amino acid from arginine to glutamine (R239Q) was discovered in the patient. Steric model analysis of CYP17A1 showed that R239Q changed the local structure and the electrostatic potential. Functional study indicated that the R239Q mutant caused the complete loss of both 17α-hydroxylase and 17,20-lyase activities. Our study expanded the CYP17A1 mutation spectrum. With a functional study, we confirmed that the novel mutation caused the complete loss of both 17α-hydroxylase and 17,20-lyase activities.

Treatment of acute multiorgan dysfunction occurring in congenital adrenal hyperplasia

A 23-year-old female was admitted to the hospital with a 2-day history of vomiting, diarrhea, and fever. The patient had a history of clitoral hypertrophy at birth, which was surgically corrected in her childhood. She presented with primary amenorrhea, and her chromosome karyotype was 46, XX. Approximately 12 months before admission, her steroid hormone profile showed elevated levels of testosterone (T 3.31 ng/ml, 0.15–0.51 ng/ml), dehydroepiandrosterone (DHEA 618.9 lg/dl, 19.00–391.00 lg/dl), progesterone (P 9.79 ng/ml, 0.2–0.9 ng/ml), and 17-hydroxyprogesterone (17OHP 10.99 ng/ml, 0.23–1.36 ng/ml), while the levels of follicle-stimulating hormone (FSH 5.83 mIU/ml, 21–104 mIU/ml) and luteinizing hormone (LH 3.58 mIU/ml, 0.9–58.6 mIU/ml) were decreased. A pelvic ultrasound indicated the presence of a uterus and ovaries. The patient was diagnosed with congenital adrenal hyperplasia (CAH) and 21-hydroxylase deficiency (21OHD). Then, she was treated with cortisone acetate (25 mg qd). Her levels of 17OHP (6.83 ng/ml) and T (0.15 ng/ml) decreased 6 months after treatment. Nine months after the treatment, she experienced menstruation and withdrew herself from the drug. At the time of her arrival at our hospital, a physical examination showed that she was hirsute, and her breasts were underdeveloped. Her heart rate was 100 bpm, and her blood pressure was undetectable. Blood tests indicated a WBC of 9 9 10/l (4–9.2 9 10/l), neutrophils 75.9 % (50–70 %), a PLT of 41 9 10/l (85–303 9 10/l), 6.25 mmol/l (3.5–5.1 mmol/l) potassium, and 134 mmol/l (135–147 mmol/l) sodium. She also had hypoglycemia (2.4 mmol/l, 3.9–6.1 mmol/l), and the levels of serum creatinine (Scr) (445 lmol/l, 53–115 lmol/l), amylase (653 U/l, 25–125 U/l), and TnI (2.5 ng/ml, 0–0.04 ng/ml) were increased. A blood gas analysis demonstrated a pH of 7.11 and a BE of -23.7 mmol/l. A stool occult blood test was positive. On the 2nd day after admission, she showed impaired liver function (GPT 4800 U/l, 1–65 U/l and GOT 14 750 U/l, 15–37 U/l) and decreased urine volume in combination with a gradually increased Scr. She was diagnosed with multiple organ dysfunction syndrome (MODS) and adrenal crisis. DNA was extracted from her peripheral leukocytes, and mutations were analyzed by sequencing the whole CYP21A2 gene. Following conventional treatment, she underwent hemodiafiltration (HDF) on D7 and D8. However, the Scr (D9 327 lmol/l; D13 123 lmol/l) and liver function (D6 GPT 533 U/l and GOT 242 U/l; D10 GPT 204 U/l and GOT 42 U/l) continued to decline (Table 1). Because the patient was in shock and had adrenal cortex dysfunction, she was also administered hydrocortisone (D2 to D6, 200 mg/d). The patient gradually recovered and was discharged from the hospital on D14. When the patient arrived at our hospital, she had gastrointestinal disorders, a normal body temperature and shock. Considering her history of CAH, an adrenal crisis was suspected. An infection and cortisone acetate withdrawal might have been the inducing factors. Therefore, it B. Han Y. Lu J. Qiao (&) Department of Endocrinology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China e-mail: qiaoj2001@126.com