Functional And Structural Analysis Of Four Novel Mutations Of CYP21A2 Gene And Genotype-phenotype Correlation In 72 Chinese Patients With 21-hydroxylase Deficiency

Congenital adrenal hyperplasia(CAH)is a group of autosomal recessive disorders.The worldwide prevalence of the classical CAH ranges from 1:10,000 to 1:20,000 births.While the incidence of CAH in China is merely 1 in 28000,which is much lower than the other populations.Steroid 21-hydroxylase deficiency(210HD)is the most common enzymatic defect that accounts for about 95%of the congenital adrenal hyperplasia(CAH).Phenotypically,CAH is consist of the classic salt-wasting(SW),the classic simple virilizing(SV)and the nonclassical(NC)forms.The phenotype is closely associated with the genotype.The 21-hydroxylase gene(CYP21A2)is located on chromosome 6(6p21.3),adjacent to its nonfunctional pseudogene(CYP21P).The CYP21A2 and CYP21P genes consist of 10 exons and sharing a high homology about 98%.The transfer of genetic material from the CYP21P pseudogene into the CYP21A2 gene accounts for about 70%-75%of CYP21A2 disease-causing mutations.Although most patients carry CYP21P-derived mutations,an increasing number of naturally occurring mutations independently from the pseudogene has been found in last years,which is of grant importance in the study of CYP21 protein.Our analysis of genotype-phenotype correlations in a Chinese cohort provides a framework for clinicians to predict the general phenotype of CAH for a given genotype.Moreover,we evaluated the functional role of 4 novel mutants(p.L199X,p.E321 del,p.H393Q and p.L459_P464 del)in detail,including in vitro and in silico analysis,which can enrich the human genomic database and provide novel insights into the structure-function relationship of CYP21 and other P450 enzymes.Part I.Clinical data collection and mutation analysis of the patientsObjectivesTo analyze the clinical features of 72 patients with 21-OHD,and to study the mutational characters of these patients.Methods1.From 2012-2017,a total of 72 unrelated patients who were treated in Shandong Provincial Hospital and diagnosed as having CAH were enrolled to investigate the clinical,biochemical,and genetic characteristics of this disorder.Symptoms and laboratory findings of all the members were collected.2.Genomic DNA was extracted from peripheral blood leukocytes sampled from all the patients.Specific amplification of CYP21A2 by PCR was performed,followed by sequencing of the entire CYP21A2 gene.The resulting sequences were compared to the corresponding wild-type sequences of CYP21A2 using the AutoAssembler software.3.The distribution and frequency of the mutations were studied.4.Homologous analysis was made for the novel mutations,Multiple amino acid alignment of CYP21 orthologs from the Uniprot database including sequences from species in the following order:human(P08686),pig(P15540),bovine(P00191),dog(Q8WNW0),cat(Q2LA60),rat(Q64562)and mouse(P03940).Results1.The patients consisted of 37 males and 35 females.The average diagnosed age was 3.5±7.6 yr(range 0-29).Among them,47 patients(65.3%)were diagnosed as SW phenotype,11(15.3%)were SV type and 14(19.4%)were NC type.2.Hyperpigmentation was the most common symptom(62.5%).Other common symptoms were clitoromegaly in 37.5%and advanced penile growth in 13.9%of the patients.56 patients were infants when diagnosed,among them,milk regurgitation(24 in 56,42.9%)and no weight increase(7 in 56,12.5%)were the most common symptoms.3.The mean value of FSH(2.52+12.51 IU/L)and LH(1.69±2.52 IU/L)were lower than normal value:5-40 IU/L and 5-25 IU/L.To determine the predicting value of serum FSH,LH and PRL levels in disease severity,we constructed ROC curves and calculated the area under the curve(AUC).The ROC curves suggested that the AUC value of FSH for the prediction of the severe SW phenotype was up to 0.862(CI(95%):0.735-0.943,P<0.001),with an estimated sensitivity and specificity of 75.00%and 100.00%,respectively.While the AUC value of LH for predicting the SW phenotype was 0.669(CI(95%):0.522-0.796,P=0.0364),with an estimated sensitivity and specificity of 87.50%and 44.44%,respectively.4.The most frequently mutations in the present research were I2G(32.6%),Large lesion(20.6%),I173N(17%)and Q319X(7.1%).Novel mutations were detected on 2.8%of alleles(4 of 141).5.The mutate amino acid residues were highly homologous in the CYP21 proteins of mammals.Conclusions1.SW is the most common phenotype of all the 72 21-OHD patients.The most common symptoms is hyperpigmentation among all the patients and milk regurgitation among infant patients.2.CYP21A2 mutational analysis was performed in 72 patients with 21-OHD.The overall detection rate of mutation was 97.9%(141/144).The most frequently mutations in the present research were I2G,which is similar to the previous study in other populations.Part ?.The genotype-phenotype correlation study in 72 Chinese patients with 21-hydroxylase deficiencyObjectivesTo figure out the relation of genotype and phenotypeMethods1.The 72 patients were divided into four groups according to their genotypes.2.Analyze the association between the genotype and phenotype among the four groups.Results1.36 genotypes were found in the 72 patients.Patients were divided into four groups according to their genotype.The categorization based on the established in vitro residual enzyme activity and on the assumption that the mildest mutation determines the phenotype in compound heterozygotes:group null 12(7 males,5 females),group A 26(19 males,7 females),group B 16(3 males,13 females),and group C 11(4 males,7 females).The rest were patients with novel mutations and one mutation(4males,3 females).2.In group null,all 12 patients(100%)had the classic SW form as expected.In group A,23 of the 26 patients(88.5%)had the anticipated SW form,and only 1 patient(3.8%)was diagnosed with the SV form and 2(7.7%)were diagnosed with the NC form.There were 5(31.3%),6(37.5%)and 5(31.3%)patients of 16 patients in group B exhibited SW,SV and NC phenotypes,and 2(20%),4(40%),4(40%)of 10 patients in group C,respectively.3.The mean value of FSH(2.52±2.51 IU/L)and LH(1.69±2.52 IU/L)were lower than normal value:5-40 IU/L and 5-25 IU/L.To determine the predicting value of serum FSH and LH levels in disease severity,we constructed ROC curves and calculated the area under the curve(AUC).The value of FSH and LH for the prediction of the severe S W phenotype was up to 0.862 and 0.669,respectively.ConclusionsWe found a high correlation between genotype and phenotype in the severe groups(group null,A)of our 72 patients.All the patients(100%)in group null had the expected SW phenotype.And 88.5%of the patients in group A had the SW phenotype as anticipated.Our genotype-phenotype correlation was poor for group B and C.In group B,5(31.3%)patients had the predicted SV form In group C,only 4(36.4%)of the 11 patients had the predicted NC form.Meanwhile,we demonstrated that serum FSH level may serve as a potential marker for the prediction of disease severity.Part III.In vitro and in silico analysis of the four novel mutations(L199X,E321del,H393Q and L459_P464 del)of CYP21A2 geneObjectivesTo evaluate the functional role of 4 novel mutants(p.L199X,p.E321del,p.H393Q and p.L459_P464 del)in detail,including in vitro and in silico analysis,which can enrich the human genomic database and provide novel insights into the structure-function relationship of CYP21 and other P450 enzymes.Methods1.The human full-length CYP21A2 cDNA was cloned into the pcDNA 3.1 vector,resulting in the pcDNA3.1-CYP21A2-WT construct.Four novel mutations were introduced by site-directed mutagenesis using the corresponding mutagenesis primers.The plasmids were named according to the designed mutations pcDNA3.1-CYP21A2-L199X,E321del,H393Q,L459_P464del,I173N,V282L,P454S and pcDNA3-CYP21A2-P483S.In addition to these mutations,four control mutations were used for validation:1173N,V282L,P454S and P483S with about 4.4%,18%,24-38%,and 54-61%residual 21-hydroxylase activity.2.Activity of 21-hydroxylase in intact COS-7 cells was determined 48h after transfection.The cells were incubated for 1 h at 37? with 500 ?1 DMEM medium containing 0.5 ?Ci 3H-labeled substrate(P or 170HP),2 ?mol/L unlabeled substrate,and 8 mmol/L nicotinamide adenine dinucleotide phosphate reduced(NADPH).After incubation,steroids were extracted from the culture medium,evaporated to dryness,and dissolved in methanol.The steroids were separated by Thin Layer Chromatography(TLC)and the radioactivity was measured by liquid scintillation counting.21-Hydroxylase activity was expressed as a percentage of conversion grade,taking the activity of cells expressing the CYP21A2-WT protein as 100%after correction for total protein and for the activity of cells transfected with the empty pcDNA3.1 plasmid.3.The effect of novel mutations(the deletion of residues 199-485,residue 321,residues 459-464,and mutation His393GIn)on the structure of the CYP21 protein was evaluated by studying the 4Y8W structure of human cytochrome P450 21A2 using PyMoL 1.7.6.4.Multiple amino acid sequences of CYP21 orthologs from species including human(P08686),pig(P15540),bovine(P00191),dog(Q8WNW0),cat(Q2LA60),rat(Q64562)and mouse(P03940)were obtained from the Uniprot database.Results1.The activity of the normal protein(WT)was defined as 100%,conversion of P and 170HP for p.I173N,p.V282L,p.P454S and p.P483S were 9.5±2.4%/9.0±0.6%,41.3± 5.2%/44.3±2.8%.48.0 ± 2.4%/55.9±2.3%and 81.3±3.0%/81±2.0%,respectively.2.The L199X,E321del,H393Q and L459-464del mutants demonstrated a reduced CYP21 activity of 0.9±0.73%,2.4±0.72%,2.2±0.57%and 3.5± 1.33%compared with the wild-type activity for the conversion of P to deoxycorticosterone.While for the conversion for 170HP to 11-deoxycortisol,the aforementioned mutations presented 1.1 ±0.4%,3.0±1.6%,2.5±0.6%and 3.6±1.1%CYP21 activity,respectively.3.The deletion of the residues at the position]99-485 leaded to loss of electrostatic interactions with the substrate progesterone and heme,while the deletion of glutamic acid at position 321 disturbed the interaction with Leu-345.In addition,a change to glutamine at position 393 influenced the interaction with Arg-355.The remove of the residues at the positions 459-464 resulted in loss of electrostatic interactions with the residues His-310 and Gln-475.4.The residue 321,393 and residues 459-464 were highly conserved in CYP21 of mammals.Conclusions1.Consistent with previous research,conversion of P and 170HP for p.I173N,p.V282L,p.P454S and p.P483S were gradually increased,which demonstrated that the design and performance of the assays are reliable.2.CYP21 proteins containing p.L199X,p.E321del,p.H393Q and p.L459-P464del mutations showed severe loss of enzyme function,the enzymatic residual activity for all the isolated mutations was lower than 5%for both two substrates.Especially for the mutation L199X,which showed a conversion rate of lower than 1%for P to deoxycorticosterone.3.The in silico structure analysis showed that all the four novel mutations destroyed the 3D structure of the protein,which supports the results of the in vitro assays.4.Combined with the salt wasting symptom and early diagnosed age found in the four patients presented the novel mutations,we deduced that all the four novel mutations could cause a severe decline of the enzyme activity and lead to the SW type.