Clinical And Molecular Genetic Studies Of 17-hydroxylase/17,20-lyase Deficiency

Background17a-hydroxylase/17,20-lyase deficiency（17-OHD）is a rare form of congenital adrenal hyperplasia diseases.Mutations within the CYP17A1 gene is the molecular pathological basis of partial or complete loss of 17α-hydroxylase enzyme activity and 17,20-lyase enzyme activity.The CYP17A1 gene mutation was first reported by Yanase T in 1988.To date,more than 140 mutations in CYP17A1 have been identified in the Human Gene Mutation Database（http://www.hgmd.org/）.Lack of adrenal 17a-hydroxylase activity leads to low cortisol production,compensatory hyper-secretion of corticotrophin and overproduction of the 17-deoxysteroids 11-deoxycorticosterone（DOC）and corticosteroid.Excess DOC and corticosterone cause hypertension and hypokalemia in 17-OHD.Concomitant lack of gonadal and adrenal 17,20-lyase activity precludes sex steroid production.Male（46,XY）patients leads to male pseudo-hermaphroditism,usually grows with female external genitalia and female characteristics.Female（46,XX）patients have normal female genitalia at birth but absent sexual development at the time of puberty.In clinical practice,except complete 17-OHD,partial 17-OHD and isolated 17,20-lyase deficiency（ILD）are also present.17-OHD is a very rare endocrine disease,which can be easily missed or mis-diagnose in clinical practice.There are few reports about the long-term treatment and prognosis of patients with 17-OHD.Molecular genetic studies of Chinese 17-OHD are not yet sufficient.ObjectiveThe aim of this study is to improve the diagnosis and treatment of 17-OHD through the retrospective clinical analysis of 8 Chinese patients（5 patients were from the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University and 3 patients were from Qilu Hospital of Shandong University）from 2000 to 2015.In this study,genetic analysis were also examined in all 8 patients with 17-OHD in order to identify the mutations of CYP17A1 gene.To understand the further characteristics of CYP17A1 gene mutations in Chinese 17-OHD patients,we compared the difference of CYP17A1 gene mutations with other races.In the present study,we have also demonstrated the relationship between genotype and phenotype in 17-OHD patients.We try to expend the clinical characteristics of 17-OHD and CYP17A1 mutation speculum.SubjectsWe here reported eight Chinese patients who were diagnosed with17-OHD,along with a treatment and a long-term follow-up.Five patients were from the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University from 2000 to 2015.The other three patients were from Qilu Hospital of Shandong University during the period of 1983 to 2015.All patients were hospitalized patients from 6 different families.The social sex and phenotype of all eight patients were female（six were 46,XX;two were 46,XY）.Methods1.Clinical analysisA retrospective analysis of eight Chinese patients with 17-OHD was made according to clinical data（medical history,physical examination,family history,marriage history）,biochemistry（serum potassium,serum sodium,blood chloride,blood glucose,blood urea nitrogen,serum creatinine,24 hours urinary potassium,etc）,hormonal（serum cortisol,corticotrophin,testosterone,estradiol,luteinizing hormone,follicle stimulating hormone,progesterone,aldosterone,plasma renin activity,etc）,chromosome karyotype analysis,radiological characteristics（electrocardiogram,pelvic ultrasonography,abdominal ultrasonography,echocardiography,adrenal computed tomography,bone mineral density,etc）,long-term treatment（glucocorticoid therapy,primitive glandectomy,complementary sex hormone therapy,etc）,follow-up（blood pressure,serum potassium,adrenal imaging changes,bone mineral density）and prognosis.2.Molecular genetic studiesTotal genomic DNA of the patients was extracted from peripheral blood leukocytes.The eight coding exons and the flanking non-coding region sequences of the CYP17A1 gene were amplified into 1-8 pieces by polymerase chain reaction（PCR）.The PCR products were then purified and sequenced in a Genetic Analyzer.The sequencing results were compared with the reference sequence of CYP17A1 gene（NG₀07955.1）provided by GeneBank.Novel mutation of CYP17A1 gene affecting the protein features is predicted through online gene bioformatic prediction program.Results1.Clinical analysisEight patients with 17-OHD were from six different families.Among them,7 cases of complete 17-OHD patients and 1 case of partial 17-OHD patients.The newly diagnosed age varied from 15 to 26 years old.Most patients（7/8）had varying degrees of hypertension before treatment.All patients with 17-OHD suffered from varying degrees of hypokalemia before treatment.Seven patients with complete 17-OHD presented with primary amenorrhea.1 case of partial 17-OHD patients presented with secondary amenorrhea and recurrent ovarian cysts.All 17-OHD patients had a low body mass index before treatment.Serum cortisol was significantly declined in all 17-OHD patients,and serum ACTH levels were elevated.Serum testosterone levels were significantly lower than the normal adult male level in 2 cases of chromosome 46,XY patients,serum estradiol levels were significantly lower than the normal adult female level in 6 cases of chromosome 46,XX patients.Serum luteinizing hormone and follicle stimulating hormone were significantly elevated in all 17-OHD patients.We also observed that some patients were presented with markedly low bone mineral density（BMD）before therapy.The majority of the 17-OHD patient had normal or nearly normal blood pressure,and normal serum potassium level after treated with low dose dexamethasone（DEX）.Adrenal masses were discovered in 2 patients before treatment.In addition,4 patients developed low BMD or osteoporosis and 3 patients developed recurrent infections during long-term follow-up.2.Molecular genetic studiesThe mutations of CYP17A1 gene were analyzed by sequencing of PCR products in 8 cases of 17-OHD patients and some relatives.The results showed that all 17-OHD patients had CYP17A1 gene mutation.Moreover,a total of seven kinds of mutations were detected,2 missense mutations（L266V,P409R）,1 non-sense mutation,（Y329X）1 deletion mutation（D487_F489 del）,2 frame-shift mutation（Y329fs,V311fs）and 1 non-coding region mutation（-34T>C）.The majority of them（5/8）had homozygous mutations.Among them,P1-1 and P1-2 had D487_F489 del homozygous mutations,P5-1 and P5-2 had-34T>C homozygous mutation,P7 had Y329X homozygous mutation.Some patients（3/8）had complex heterozygous mutations,for example;P3 had both L266V and-34T>C mutations.P4 had both D487_F489 del and Y329fs mutations,P8 had both V311fs and P409R mutations.L266V was a novel mutation.Online gene bioformatic prediction program predicted that L266V could affect the protein features both in the secondary structure and the tertiary structure.Furthermore,varying forms of mutations identified in codon 329 confirmed the instability of codon 329 in our study.Conclusion1.All eight 17-OHD patients were diagnosed after the age of 15.So,this study can help to establish clear understanding about the disease and make early diagnosis of patients with 17-OHD.2.Minority of 17-OHD patients had adrenal mass before treatment.Some adrenal mass size can be significantly decrease after the glucocorticoids treatment.We recommended to monitor the adrenal mass size in 17-OHD patients with adrenal mass after dexamethasone therapy,in order to avoid unnecessary surgery.3.BMD was significantly reduced in some 17-OHD patients.In addition,partial 17-OHD patients developed low BMD or osteoporosis during long-term dexamethasone treatment.Clinicians should pay attention to bone health problems in 17-OHD patients.4.Infections were also common in patients with 17-OHD on long-term steroid treatment.Therefore,minimum dose of dexamethasone was used in 17-OHD patients as far as possible to avoid long-term complications of glucocorticoid.5.All eight 17-OHD patients had CYP17A1 gene mutation,a total of seven kinds of mutations were detected.The majority of whom（5/8）have homozygous mutations,whereas some patients（3/8）had complex heterozygous mutations.6.One novel mutation,L266V,was found in Patient 3.Online gene bioformatic prediction program predicted that L266V could affect the protein features both in the secondary structure and the tertiary structure.7.In addition,various forms of mutations identified in codon 329 in our study’confirmed the instability of codon 329.8.Seven patients with completely 17-OHD carried gene mutations which induced the deficiency of the entire activity of P450c17 enzyme,corresponding with their clinical features.One patient with partial 17-OHD carried a compound heterozygous mutation V311fs/P409R.We speculated that these 2 mutations lie far away from the active site of the P450c17 enzyme,therefore they conserve part activities of the P450c17 enzyme.Further functional conformational studies need to conduct on our hypothesis.