Adaptation of the electron transport chain improves the biocatalytic efficiency of progesterone 17α hydroxylation / 生物工程学报

17α hydroxylase is a key enzyme for the conversion of progesterone to prepare various progestational drug intermediates. To improve the specific hydroxylation capability of this enzyme in steroid biocatalysis, the CYP260A1 derived from cellulose-mucilaginous bacteria Sorangium cellulosum Soce56 and the Fpr and bovine adrenal-derived Adx4-108 derived from Escherichia coli str. K-12 were used to construct a new electron transfer system for the conversion of progesterone. Selective mutation of CYP260A1 resulted in a mutant S276I with significantly enhanced 17α hydroxylase activity, and the yield of 17α-OH progesterone reached 58% after optimization of the catalytic system in vitro. In addition, the effect of phosphorylation of the ferredoxin Adx4-108 on 17α hydroxyl activity was evaluated using a targeted mutation technique, and the results showed that the mutation Adx4-108T69E transferred electrons to S276I more efficiently, which further enhanced the catalytic specificity in the C17 position of progesterone, and the yield of 17α-OH progesterone was eventually increased to 74%. This study provides a new option for the production of 17α-OH progesterone by specific transformation of bacterial-derived 17α hydroxylase, and lays a theoretical foundation for the industrial production of progesterone analogs using biotransformation method.

Clinical characteristics of 5 patients with 17α-hydroxylase deficiency and LC-MS/MS method for the measurement of steroid hormones / 中华内分泌代谢杂志

Objective:To analyze clinical characteristics of 17α-hydroxylase deficiency, and to facilitate the understanding and management of the disease.Methods:A retrospective analysis of the clinical characteristics and biochemical results of 5 cases with 17α-hydroxylase deficiency diagnosed and treated from 2018 to 2020.Results:All 5 patients were female as social gender, and reached adulthood upon first clinic visit to our department and got diagnosed. All 5 cases had hypertension, hypokalemia, bilateral adrenal hyperplasia or adenoma, osteoporosis, and typical hormone changes related to steroid synthesis.Conclusion:Steroid hormone tests with liquid chromatography tandem mass spectrometry(LC-MS/MS) enable early diagnosis of 17α-hydroxylase deficiency, assessment of the type and degree of enzyme deficiency, and choice of treatment. For such patients, it is necessary to give appropriate anti-osteoporosis therapy.

Clinical characteristics and CYP17A1 gene mutation analysis in patients with 17α-hydroxylase/17, 20-lyase deficiency and testicular tumor / 中华内科杂志

The 17α-hydroxylase/17, 20-lyase deficiency (17-OHD) is a rare disease. The clinical characteristics and gene mutation of 2 late-diagnosed 17-OHD patients with testicular tumor admitted to our hospital from March 2018 to February 2019 were analyzed retrospectively. The two 17-OHD patients were female (46, XY). Laparoscopic abdominal exploration found undeveloped testicles in grey-yellow or grey-red in the groin and iliac fossa. The testicles were removed and showed malignancy in pathology study. Sequencing of the CYP17A1 gene identified c.1247G>A/c.1427T>C and c.985_987delTACinsAA/c.1306G>A complex heterozygous mutations. Taking together, the possibility of 17-OHD should be considered in patients with hypertension, hypokalemia, adrenal adenomatoid hyperplasia together with 46, XY gonadal dysplasia, so as to make early diagnosis and treatment, and avoid dysplastic testicular turning to malignancy.

Study on six patients with 17α-hydroxylase/17,20-lyase deficiency / 中华内分泌代谢杂志

Objective To improve the understanding of 17α-hydroxylase/17, 20-lyase deficiency ( 17OHD ) disease. Methods The clinical data of six patients suffering from 17OHD were analyzed retrospectively. Results Two patients with complete combined defect had typical clinical presentation, including absence of secondary sexual characteristics, primary amenorrhea, hypertension, hypokalamia, lower gonadal hormone levels, as well as elevated corticotropin and progesterone levels. TAC329AA homozygous mutation,IVS1+2T＞C, and c.775 776delAT complex heterozygous mutation were found in 2 cases. Four cases of partial combined defect showed high progesterone, lower gonadal hormones and dehydroepiandrosterone-sulfate levels. Three females (46, XX) showed spontaneous menstrual and primary infertility, and two of them got successful pregnancy with assisted reproductive technology. TAC329AA heterozygous mutation was found in those 4 cases. Conclusions TAC329AA mutations are common in 17OHD, and heterozygous or homozygous mutation of TAC329AA may be the genetic and molecular basis for determining whether these patients present as partial or complete defect. The elevated plasma progesterone in non-pregnancy combined with lower gonadal hormones and dehydroepiandrosterone-sulfate is the main character of patients with partial 17OHD. Less severe patients may be able to get successful pregnancy with assisted reproductive technology.

Siblings with congenital adrenal cortex hyperplasia (17-α-hydroxylase deficiency) and literature review / 临床儿科杂志

Objective To report clinical characteristics and genetic results of two sisters suffered from congenital adrenal cortex hyperplasia (17-α-hydroxylase deficiency), and relevant literatures were reviewed. Methods Clinical manifestation and laboratory examination data of two sister cases of 17-α-hydroxylase deficiency enrolled in Capital Institute of Pediatrics in March 2016 were analyzed. Sanger sequencing and MLPA for CYP17A1 genes were performed and the parents' genes were also verified. Results The two patients were four years and 10 years old, both suffered from hypokalemia after infections, and hypergonadotrophin gonad hypofunction. One case was with slightly high blood pressure. Laboratory test results showed potassium fluctuation tendency in 1.9~4.0 mmol/L, 17-OHP and DHEA was decreased. Enhanced CT showed different degree of adrenal gland enlargement. Chromosome examination of the older sister is 46, XY. Both sisters demonstrated heterozygous mutation of CYP17A1 gene. The molecular genetic analysis suggested a c.985_987delTACinsAA from father and a deletion spanning exons 1-7 of the CYP17A1 gene from mother. Conclusion 17-α-hydroxylase enzyme deficiency can be diagnosed before adolescence. Clinical hypokalemia with unknown reason and high blood pressure may indicate the disease. The diagnosis can be confirmed with gene sequencing of CYP17A1.

A cross-sectional study to assess any possible linkage of C/T polymorphism in CYP17A1 gene with insulin resistance in non-obese women with polycystic ovarian syndrome.

Background & objectives: Insulin resistance (IR) is a major confounding factor in polycystic ovarian syndrome (PCOS) irrespective of obesity. Its exact mechanism remains elusive till now. C/T polymorphism in the -34 promoter region of the CYP17 gene is inconsistently attributed to elucidate the mechanism of IR and its link to hyperandrogenemia in obese PCOS patients. In the present study we aimed to evaluate any association of this polymorphism with IR in non-obese women with PCOS. Methods: Polymorphism study was performed by restriction fragment length polymorphism (RFLP) analysis of the Msp A1 digest of the PCR product of the target gene in 75 PCOS cases against 73 age and BMI matched control women. Serum testosterone, BMI and HOMA-IR (homeostatic model of assessment-insulin resistance) were analyzed by standard techniques. A realistic cut-off value for the HOMA-IR was obtained through receiver operating characteristic (ROC) curve for exploring any possible link between IR and T/C polymorphism in the case group. Results: Significant increases in serum testosterone and HOMA-IR values were observed among the case group (P<0.001) without any significant elevation in BMI and FBG compared to controls. Cut-off value for IR in the PCOS patients was 1.40 against a maximum sensitivity of 0.83 and a minimum false positivity of 0.13. The analysis revealed an inconclusive link between the C/T polymorphic distribution and insulin resistant case subjects. Interpretation & conclusions: The results showed that CYP17A1 gene was not conclusively linked to either IR or its associated increased androgen secretion in non-obese women with PCOS. We propose that an increased sensitivity of insulin on the ovarian cells may be the predominant reason for the clinical effects and symptoms of androgen excess observed in non-obese PCOS patients in our region.

Male pseudohermaphroditism and gene mutation analysis:one case report / 中华内分泌代谢杂志

[Summary] The clinical and genetic characteristics of a patient with male pseudohermaphroditism, being considered as an isolated 17, 20-lyase deficiency case, were analyzed. The social gender of the patient aged 30-year-old was female. The patient presented with 46, XY karyotype, unclosed epiphysis, after perineal block resection, hypergonadotropic hypogonadism, while the production of mineralocorticoids and glucocorticoids hormone was intact. A503V heterozygous mutation in exon 13 and a deletion in intron 11 of POR gene were detected. The gene mutations may lead to the occurrence of the isolated 17,20-lyase deficiency.

Rare type of congenital adrenal hyperplasia / 中华实用儿科临床杂志

Congenital adrenal hyperplasia (CAH) is an autosomal recessive genetic disease due to glucocorticoid biosynthesis enzyme deficiency,in addition to the common types such as 21-hydroxylase,11 β-hydroxylase deficiency,also include rare types such as 3 β-hydroxysteroid dehydrogenase deficiency,17α-hydroxylase deficiency and congenital lipoid adrenal hyperplasia (CLAH).These rare types of CAH have multiple clinical manifestations,which are easily missed or misdiagnosed.This article focus on molecular genetics,pathophysiology,clinical manifestations and treatment principles of above 3 rare types of CAH.

Adrenal myelolipoma with congenital 17a-hydroxylase deficiency: a pedigree report and review of the literature / 中华内分泌代谢杂志

Objective To analyse the clinical and pathological characteristics of congenital adrenal hyperplasia (CAH) with adrenal myelolipoma (AML),and to improve the awareness of CAH.Methods The P450c17a gene in a pedigree with CAH and the clinical and pathological characteristics of three sisters of the pedigree with CAH and adrenal myelolipoma were studied;and the pertinent literatures reported abroad were analyzed and summarized.Results (1) A heterozygous point mutation 1178 base A to T in exon 6,and a heterozygous deletion mutation 1 457-1 465 bases TCGACTCTT of exon 8 were detected in P450c17 (CYP17) gene of three sisters of the pedigree with CAH.(2) The clinical characteristics of three sisters were hypertension,hypokalemia,primary amenorrhea,and gonadal dysgenesis.Without regular treatment after the diagnosis of CAH,the three sisters experienced many years of elevated serum ACTH levels,and developed AML.Conclusion The long-term high ACTH level plays an important role in the development of AML,although the exact mechanism of CAH with adrenal myelolipoma remains still unclear.CAH patients require long-term regular treatment.

Approach to the patient with 17α-hydroxylase/17, 20-lyase deficiency / 中华内分泌代谢杂志

17α-hydroxylase/17,20-lyase deficiency (17OHD) is a rare cause of congenital adrenal hyperplasia.The patient predominantly presents with low-renin hypertension,hypokalemia,lack of secondary sexual development,and in women with primary amenorrhea,in male with pseudohermaphroditism.We herewith analyse the clinical features of a case of 17OHD diagnosed by gene sequencing.And the etiology,clinical manifestations,genetic features,diagnosis and treatment for 17OHD were reviewed.

An unique exonic splicing mutation in the CYPI7AI gene as the cause of steroid 17α-hydroxylase deficiency / 中华内分泌代谢杂志

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.

A clinical and genetic analysis of a pedigree with two 46,XY patients suffering from 17α-hydroxylase deficiency / 中华内科杂志

Objective To investigate the molecular defects of CYPl7A1 gene in a pedigree with two 46,XY patients suffering from 17α-hydroxylase deficiency (17-OHD) and explore the steroid biosynthetic difference in carriers of 17-OHD before and after adrenocorticotrophic hormone (ACTH) test.Methods Clinical data and hormone profiles were collected from the members of the pedigree.CYPl7A1 genotyping was performed in the patients and family members with PCR-direct sequencing.A short ACTH test was evaluated in some cases.Results The CYP17 genes of the patients were proved to hold a homozygous mutation with a base deletion and a base transversion (TAC/AA) in exon 6,which produced a missense mutation of Tyr→ Lvs at codon 329 and changed the open reading frame following this codon.The hormone response of the carriers after ACTH stimulation was abnormal between the patients and normal controls.Conclusion 17-OHD in this family was caused by CYP17A1 mutation (TAC329AA):some hormonal response to ACTH stimulation Was abnormal in carriers.