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A Novel Semiquantitative Polymerase Chain Reaction/Enzyme Digestion-Based Method for Detection of Large Scale Deletions/Conversions of the CYP21 Gene and Mutation Screening in Turkish Families with 21-Hydroxylase Deficiency

Department of Pediatrics, Divisions of Medical Genetics (T.T.) and Pediatric Endocrinology (F.B., H.G.), Istanbul Medical Faculty, and Divisions of Medical Genetics (O.U., M.Y.-A., H.K., B.W.) and Auxology (N.S.), Child Health Institute, Istanbul University, 34390 Istanbul, Turkey; and Department of Pediatrics, Weill Medical College, Cornell University (J.Q.W., D.X.S., R.C.W., M.I.N.), New York, New York 10029

Address all correspondence and requests for reprints to: Dr. Turgut Tukel, Department of Human Genetics, Mount Sinai School of Medicine, 1425 Madison Avenue, Box 1498, Room 14-52, New York, New York 10029. E-mail: turgut.tukel{at}mssm.edu. Or to: Dr. Bernd Wollnik, Institute of Child Health, Division of Medical Genetics, Istanbul University, Millet Cad., Capa, 34390 Istanbul, Turkey. E-mail: .

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
21-Hydroxylase deficiency is a recessively inherited disorder resulting from mutations in the CYP21 gene. The CYP21 gene is located along with the CYP21P pseudogene in the human leukocyte antigen major histocompatibility complex region on chromosome 6. Molecular diagnosis is difficult due to the 98% similarity of CYP21 and CYP21P genes and the fact that almost all frequently reported mutations reside on the pseudogene. Allele-specific PCR for the 8 most frequently reported point mutations was performed in 31 Turkish families with at least a single 21-hydroxylase-deficient individual. The allele frequencies of the point mutations were as follows: P30L, 0%; IVS2 (AS,A/C-G,-13), 22.5%; G1108nt, 3.2%; I172N, 11.4%; exon 6 cluster (I236N, V237E, M239K), 3.2%; V281L, 0%; Q318X, 8%; and R356W, 9.6%. Large deletions and gene conversions were detected by Southern blot analysis, and the allele frequencies were 9.6% and 22.5%, respectively. Sequence analysis of the gene, performed on patients with only 1 mutated allele, revealed 2 missense mutations (R339H and P435S). A novel semiquantitative PCR/enzyme digestion-based method for the detection of large scale deletions/conversions of the gene was developed for routine diagnostic purposes, and its accuracy was shown by comparison with the results of Southern blot analysis.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
CONGENITAL ADRENAL HYPERPLASIA (CAH) is a common autosomal recessive disorder that is most often caused by steroid 21-hydroxylase deficiency. The deficiency of this enzyme leads to various degrees of impaired cortisol and aldosterone synthesis accompanied by increased androgen biosynthesis. Depending on the residual activity of the enzyme, the clinical phenotype is divided into three forms: the salt-wasting (SW) and simple virilizing (SV) forms, also known as the classical forms, and the nonclassical (NC) form. The worldwide frequency of the classical forms is 1:14,000 live births. In the majority of cases, classical 21-hydroxylase deficiency causes salt wasting, which can be life threatening if untreated, particularly in the first weeks after birth. Classical 21-hydroxylase deficiency is the most common cause of ambiguous genitalia in females. It represents one of the few disorders for which prenatal diagnosis and treatment are feasible and effective to prevent birth defects (1, 2, 3, 4).

Steroid 21-hydroxylase is a microsomal enzyme responsible for the conversion of progesterone and 17-hydroxyprogesterone to 11-deoxycorticosterone and 11- deoxycortisol, respectively. The gene encoding the human 21-hydroxylase enzyme (CYP21) and a pseudogene (CYP21P) are located within the human leukocyte antigen class III gene region on 6p21.3, approximately 30 kb apart, in 3' positions of each of the two genes encoding the fourth component of complement, C4A and C4B (Fig. 1). The pseudogene is 98% and 96% homologous to the active gene in exons and introns, respectively. Both genes are 3.1 kb in size and are encoded by 10 exons (5, 6). Most of the deleterious point mutations reside on the pseudogene and are transferred to the active gene by gene conversions causing either complete or partial inactivation of the 21-hydroxylase enzyme. The tandemly duplicated C4 and CYP21 genes allow for possible misalignment during meiotic metaphase and unequal crossing over between sister chromatids, sometimes resulting in a complete deletion of C4B and a net deletion of CYP21 (6, 7).

View larger version (38K): [in this window] [in a new window]   FIG. 1. The CYP21 gene structure. Southern probe hybridization sites and primer annealing sites for semiquantitative PCR on both genes are shown. a, CYP21-Q-5-F; b, CYP21-Q-5-R; c, CYP21-Q-3-F; d, CYP21-Q-3-R. For sequence of the primers, see the text. The lower portion of the diagram shows the TaqI restriction sites of a 30-kb deletion that results in a CYP21P/CYP21 hybrid gene.

In this study we screened 31 Turkish families with 21-hydroxylase deficiency for the 8 most common point mutations in the CYP21 gene using allele-specific PCR. Southern blot analysis was used to detect large scale deletions/conversions of the gene. Additionally, a novel semiquantitative PCR/enzyme digestion-based approach for detection of large scale deletions/conversions of the gene was developed for routine diagnostic purposes, and its accuracy was shown by comparison with the results of Southern blot analysis.

	Subjects and Methods

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Subjects

Informed consent was obtained from each patient, and blood samples were collected for DNA isolation. Blood samples were obtained from 29 Turkish patients and their families who were diagnosed with 21- hydroxylase deficiency. Additionally, the parents of 2 families in which index cases died because of adrenal crisis during the neonatal period were included in the study. CAH diagnosis and subtype determination were based on clinical manifestations and detection of the relevant steroid metabolites in plasma and urine. DNA was extracted from peripheral blood using a commercially available kit (DNA Isolation Kit for Mammalian Blood, Roche, Istanbul, Turkey).

Allele-specific PCR

The eight most common mutations in the CYP21 gene were detected by allele-specific PCR assay as described previously (11), with the following exceptions. A third primer of a normal allele was included in the PCR as an internal positive control. For P30L and I172N, the exon 3 primer was used; for intron 2, the P30 primer was used; for exon 3 (8-bp deletion), the I172 primer was used; for the exon 6 cluster mutation, the CYP5’s 5'-GAGCTATAAGTGGCACCTCAGGGC-3' primer was used; for V281L, the R356 primer was used; for Q318X and R356W, the V281 primer was used. In addition, the following primers were used to improve specificity: Ex4s WT, 5'-TTCTCTCTCCTCACCTGCAGCATCAT-3'; Ex4s Mut, 5'-TTCTCTCTCCTCACCT GCAGCATCAA-3'; and Ex6s WT, 5'-AGCTGCATCTCCACGATGTGA-3'.

Southern blot

Southern blot analysis was performed according to standard procedures. Briefly, 10 µg genomic DNA were digested overnight at 65 C using TaqI. After electrophoresis on an 0.8% agarose gel, blotting and hybridization were performed according to standard procedures. The probe for the blot was generated by PCR using plasmid pH21B as a template in the presence of digoxygenin-labeled deoxy (d)-UTP (Roche, Indianapolis, IN). Plasmid DNA was denatured at 98 C for 10 min, and then 200 µM dATP, dCTP, and dGTP each; 133 µM dTTP; 67 µM digoxygenin-labeled dUTP; 0.3 µM primers; 2.5 U Taq polymerase (Invitrogen, Carlsbad, CA); 1x PCR buffer; and 2.5 mM MgCl₂ were added in a final volume of 50 µl. The PCR program was 94 C for 5 min, followed by 30 cycles of 30 sec at 95 C, 30 sec at 60 C, and 30 sec at 72 C, with a 72 C final extension for 7 min in the last cycle. The sequences of the primers were as follows: CYP21 probe forward, 5'-ACCCGATCATTCCCCAGA-3'; and CYP21 probe reverse, 5'-CCTGCACAGCCTGACACA-3'.

Semiquantitative PCR/enzyme digestion

One hundred nanograms of genomic DNA were denatured at 98 C for 10 min, and then 400 µM dNTPs, 0.3 µM primers, 0.75 U Taq polymerase (Expand Long Template PCR System, Roche, Istanbul, Turkey), and 1x buffer (Expand Long Template PCR system Buffer 3, Roche) were added in a final volume of 25 µl. The PCR program was 94 C for 1 min, followed by 10 cycles of 1 min at 95 C, 30 sec at 58 C, and 3 min at 68 C, and 25 cycles of 1 min at 95 C, 30 sec at 56 C, and 3 min at 68 C, with a 68 C final extension for 10 min in the last cycle. Five microliters of the PCR product were run on 1% agarose gel to verify the amplification. Three units (0.7 µl) of TaqI (MBI Fermentas, Elips, Turkey) and 2.3 µl 10x buffer were added to the tube, and samples were incubated for 3 h (or overnight) at 65 C. After digestion the samples were run on 0.8% agarose gel, stained with ethidium bromide, and visualized under UV light. For the 3' end, the PCR conditions were as described above. PCR products were run without any digestion on 2% agarose gel and visualized as described. The sequences of the primers were as follows: CYP21-Q-5-F, 5'-GAGAGGACCAGAGCGAGGAA-3'; CYP21Q-5-R, 5'-CCTCAATGGTCCTCTTGGAG-3'; CYP21-Q-3-F, 5'-GGAGCCTCAGAGT GTGCAG-3'; and CYP21-Q-3-R, 5'-CTGAACAAGTCCCCTCCAGA-3'. The annealing sites of the primers are shown in Fig. 1.

Sequencing

The complete gene was amplified as two partially overlapping fragments using CYP5’s (see above)/Ex 6 a WT and Ex 3 s WT/CYP21B51 primer pairs, and the fragments were separated on 1% agarose gel. The fragments were purified with a commercially available purification kit (PCR Purification Kit, Roche) and sequenced using an automated sequencer (ABI PRISM 310 DNA sequencer, PerkinElmer-Cetus, Norwalk, CT). The sequencing primers were as follows: CYP21Ex1 a Seq, 5'-ACAGCCAAAGCAGCGTCA-3'; CYP21Ex2 a Seq, 5'-CAACATAGCAA GAACCCA-3'; CYP21Ex3 s Seq, 5'-AAGAAGGTCAGGCCCTCAG-3'; CYP21Ex4 a Seq, 5'-AGGACAAGGAGAGGCTCA-3'; CYP21Ex5 s Seq, 5'-TGCAGCATCAT CTGTTACCT-3'; CYP21Ex6 a Seq, 5'-AGAACCCGCCTCATAGCA-3'; CYP21Ex7 s Seq, 5'-ACTCTGTACTCCTCTCCCCA-3'; CYP21Ex8 s Seq, 5'-CTCA CTGGGTTGCTGAGGGA-3'; CYP21Ex9 s Seq, 5'-ATGAGTGAGGAAAGC CCGA-3'; CYP21Ex10 a Seq, 5'-GGAGCAATAAAGGAGAAAC-3'; and CYP21B51 a Seq, 5'-GATCTCGCAGCACTGTGTTTACAG-3'.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The clinical subtypes of the patients were determined based on the clinical manifestations and the levels of the relevant steroid metabolites and electrolytes in plasma and urine (13). Accordingly, 17 (54.8%) families were classified as SW, 12 (38.8%) as SV, and 2 (6.4%) as NC.

Allele-specific PCR was used to screen the patients and their families for the eight most common point mutations in the CYP21 gene. For large gene deletions and 5' end conversions, which are usually detected by Southern blot analysis, we developed a novel two-step semiquantitative PCR/enzyme digestion method that allows the identification of deletions and conversions in a fast and accurate manner. A primer pair was designed to amplify a fragment of the 5' ends of both active and pseudogenes simultaneously. The amplified fragments contained a common TaqI restriction site and an additional TaqI site only located on the pseudogene (Fig. 1). The common TaqI restriction site served as an internal control to exclude an incomplete digestion. The primer pair for amplification was specially designed to generate two fragments of the same size after digestion of the pseudogene, which migrated as a single band on agarose gel. The bands corresponding to the CYP21 and CYP21P genes could easily be distinguished by electrophoresis (Fig. 2), and their intensities could be compared in a semiquantitative manner. Different PCR conditions and varying numbers of PCR cycles were evaluated to determine the ideal and stable conditions for the quantitative amplification of the fragments.

View larger version (123K): [in this window] [in a new window]   FIG. 2. The comparison of the results revealed by semiquantitative PCR/enzyme digestion and Southern blot analysis. The upper panel shows the Southern blot results, the middle panel shows the 5' end PCR/enzyme digestion results, and the lower panel shows the 3' end PCR. A, Normal. B, Homozygous CYP21 deletion. C, Homozygous large gene conversion. D, Heterozygous CYP21 deletion. E, Heterozygous large gene conversion. F, Heterozygous CYP21 duplication. G, Heterozygous CYP21P duplication. H, Compound heterozygous CYP21 deletion/large gene conversion. For the 3' end of the gene, the 490- and 450-bp bands are compared with each other. The 370-bp band serves as a control for the 450-bp band (see Discussion). The samples were specially chosen to demonstrate all possible combinations of gene conversion and gene deletion/duplication events, and some of them do not represent affected patients (lanes F and G).

With the help of this novel method, we were able to determine various combinations of gene deletions and conversions. The accuracy of the method was shown by analysis of sample patients with known mutation patterns, which were determined by Southern blot analysis. Interpretations of the PCR-based method were performed blind to the Southern blot analysis. Comparison of the results subsequently proved the reliability and accuracy of the PCR-based approach.

Using allele-specific PCR for known point mutations and the novel semiquantitative PCR/enzyme digestion method, we were able to identify the disease-causing mutations on both alleles in 25 of 31 families. Among them, in 2 families with deceased index cases, the mutations were identified in both parents. In 6 index cases a mutation on only 1 allele could be identified. The allele frequencies of the point mutations were as follows: P30L, 0 (0%); IVS2 (AS,A/C-G,-13), 14 (22.5%); G1108nt, 2 (3.2%); I172N, 7 (11.4%); exon 6 cluster (I236N, V237E, M239K), 2 (3.2%); V281L, 0 (0%); Q318X, 5 (8%); and R356W, 6 (9.6%). Deletions and large gene conversions were found on 6 (9.6%) and 14 (22.5%) alleles, respectively. Sequence analysis of the complete gene, performed on 6 index cases with only 1 common mutation, revealed 2 missense mutations (R339H and P435S) described previously (14). In 6.4% of disease-causing alleles, no mutation was found. The clinical classification and the mutations of the patients are shown in Table 1.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

This is the first report on mutation screening in Turkish families with 21-hydroxylase deficiency. Using the described screening method for most common mutations, we have identified the disease-causing mutation on 58 of 62 alleles (93.5%). These data are mainly in concordance with other studies (reviewed in Ref.12). The frequencies of some of the mutations differ in various studies. We believe that this is mainly caused by the fact that the patient groups studied are not homogeneous and are not comparable regarding the clinical type of the disease. The reported frequencies (0–17%) of the V281L mutation, which is responsible for a NC phenotype, may reflect the heterogeneity of the patient groups included in various studies. In our patient group, mainly consisting of the classical form of the disease diagnosed in childhood, we could not find the V281L and P30L mutations; the latter one is also known as the cause of the NC phenotype. The frequencies of the IVS2 (AS,A/C-G,-13) mutation reported in southern Italy (15) and Mexico (16) are higher than expected (56% and 48%, respectively). These studies also reported a low frequency for gene deletion/large gene conversion events (8% and 1%, respectively). It is interesting to note that the frequencies reported from southern Italy differ from those reported by another group in Italy (17). This probably reflects the differences in clinical types in different patient populations. Furthermore, different methods were used for the mutation screening and might therefore not detect some of the mutations. Most of the allele-specific PCR-based detection methods, which use primers covering the 8-bp deletion region to specifically eliminate the amplification of the pseudogene, cannot differentiate between large scale gene deletion/5' end conversions and 8-bp deletion in exon 3 in homozygous state (in both cases there would be no PCR product that shows the presence of those alleles on both chromosomes indirectly) and cannot detect them at all in the heterozygous state without the help of Southern blot analysis, as only the allele without the 8-bp deletion or large scale gene deletion/5' end conversion would get amplified.

Semiquantitative PCR/enzyme digestion

To date, Southern blot analysis is the main method described to detect gene deletions and large gene conversions in patients with 21-hydroxylase deficiency (7). This method, however, is very time consuming and labor intensive and might not be able to be performed in all laboratories. Furthermore, it might be too expensive in some cases, especially in developing countries. Therefore, we have established a novel method that allows a fast and accurate deletion/conversion analysis. The method is based on a two-step semiquantitative PCR/TaqI enzyme digestion strategy. Classical Southern blot analysis detects and compares the intensities of the 3.7-kb/3.2-kb and 2.5-kb/2.4-kb TaqI restriction fragments located in the 5' and 3' regions of the CYP21 and CYP21P genes. As both regions are differently affected by large deletions or conversions, semiquantitative comparison of their band intensities allows an accurate determination of the underlying genotype. Basically, our novel approach was based on the same strategy, but instead of digestion of genomic DNA with TaqI, we amplified the two regions of interest located at the 5' and 3' ends of the CYP21 and CYP21P genes by PCR. The 5' and 3' ends were amplified separately but simultaneously for both genes in the same PCR reaction using the same primer pair. This allowed quantitative amplification of both genes. The analysis of the 5' fragments used a TaqI restriction digestion, whereas the 3' analysis took advantage of a 120-bp deletion in the CYP21P gene for the distinction of CYP21 and CYP21P fragments. Comparison of the fragment intensities between the CYP21 and CYP21P genes could detect all different combinations of deletions and conversions. The problem of comparing the intensities of digestion products with different lengths was circumvented by designing primers that allowed same-sized fragments after digestion, so that they migrated as a single band on agarose gel, and the amount of the DNA representing the particular gene (CYP21P) could be kept the same as before digestion. For the 3' end of the gene, the same problem was solved by comparing one fragment (490 bp) with the heteroduplex of the two fragments (migrating as a 450-bp band), instead of comparing the two fragments of interest (490 and 370 bp) with each other. In rare instances where it might be difficult to interpret the intensities of 490- and 450-bp bands, the 370-bp band serves as a control. Although the 370-bp band is more intense than the 490- and 450-bp bands, when these two bands are of equal intensity (Fig. 2, lanes A, C, and E), this difference disappears as the 450-bp band gets fainter (Fig. 2, lanes D and H). On the contrary, this difference becomes more striking when the 490-bp band loses its intensity (Fig. 2, lanes F and G). For a gel with multiple lanes, we strongly suggest that two or three pictures with different exposures be taken. The different product yields of different PCRs, if run on the same gel, may lead to difficulties in finding a single exposure that is optimum for each lane.

Recently, Olney et al. (18) published a real-time quantitative PCR method for the detection of deletions/conversions of the CYP21 gene. The described method uses the advantage of real quantification of amplified PCR products, compared with the semiquantitative analysis used in the classical Southern blot or our novel method presented here. Although the real-time quantitative PCR method for the detection of deletions/conversions was shown to be sensitive and reliable, different combinations of deletion/conversion events could not be detected with this method, which represents a disadvantage for the molecular screening test. Furthermore, it requires the use of real-time PCR instrumentation and therefore is not affordable in all laboratories.

In summary, we believe that the semiquantitative PCR/enzyme digestion method we developed is an easy, rapid, and reliable substitution for Southern blot analysis in routine diagnosis. The comparison of the results revealed by semiquantitative PCR/enzyme digestion and Southern blot analysis demonstrated the accuracy of the newly established method.

	Acknowledgments

 
We thank Brian Betensky for reviewing the manuscript.

	Footnotes

 
This work was supported by the Research Fund of Istanbul University (Project 1246/181298).

Abbreviations: CAH, Congenital adrenal hyperplasia; d, deoxy; NC, nonclassical; SV, simple virilizing; SW, salt-wasting.

Received May 12, 2003.

Accepted September 10, 2003.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

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