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duplications in Congenital Adrenal Hyperplasia: First technical report
Clinica Chimica Acta 402 (2009) 164–170
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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c l i n c h i m
Multiplex ligation-dependent probe amplification (MLPA) assay for the detection of CYP21A2 gene deletions/duplications in Congenital Adrenal Hyperplasia: First technical report Paola Concolino a,⁎, Enrica Mello a, Vincenzo Toscano b, Franco Ameglio a, Cecilia Zuppi a, Ettore Capoluongo a a b
Laboratory of Molecular Biology, Institute of Biochemistry and Clinical Biochemistry, Catholic University, Largo A. Gemelli 8, 00168 Rome, Italy Department of Endocrinology, II Faculty of Medicine, University ‘La Sapienza’, S. Andrea Hospital, Rome, Italy
a r t i c l e
i n f o
Article history: Received 2 December 2008 Received in revised form 7 January 2009 Accepted 12 January 2009 Available online 20 January 2009 Keywords: Multiplex ligation-dependent probe amplification (MLPA) CYP21A2 CAH diagnosis Duplication Deletion
a b s t r a c t Background: More than 90% of the cases of Congenital Adrenal Hyperplasia (CAH) are associated with mutations in 21-hydroxylase gene (CYP21A2). Up to now, large CYP21A2 rearrangements have been mainly detected by Southern blot analysis, although more rapid methods have been alternatively proposed. In this paper, we report the use of a multiplex ligation-dependent probe amplification (MLPA) method for easy and rapid detection of deletions/ duplications in the CYP21A2 gene. Methods: We collected 18 CAH Italian patients previously analyzed by gene sequencing and Southern blot technique. In addition, a prenatal diagnosis study was performed. Results: Of the 7 known subjects with CYP21A2 deletions and 2 with gene duplications previously characterized in our laboratory, all were successfully identified by the MLPA analysis. In the prenatal diagnosis study, the MLPA assay was able to identify the presence of a CYP21A2 gene duplication in the fetus, as well in other two family members. Conclusion: MLPA analysis represents a simple, rapid and sensitive tool for the detection of CYP21A2/ CYP21A1P deletions/duplications in CAH molecular diagnosis. Compared to Southern blot, MLPA may be considered a high throughput analysis, allowing the simultaneous study of several samples in the same experiment and the investigation of both gene (CYP21A2) and pseudogene (CYP21A1P) in each patient. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Congenital Adrenal Hyperplasia (CAH) is one of the most frequent inborn metabolic errors, inherited in an autosomal recessive manner. More than 90% of cases of CAH are due to 21-hydroxylase deficiency [1]. In the adrenal cortex, steroid 21-hydroxylase normally converts 17-hydroxyprogesterone (17-OHP) into 11-deoxycortisol and progesterone into 11-deoxycorticosterone. These steroids are subsequently converted into cortisol and aldosterone, respectively [1,2]. CAH includes a wide spectrum of clinical manifestation [3,4]. The gene encoding for the steroid 21-hydroxylase enzyme, CYP21A2, is located within the HLA class III region of the major histocompatibility complex locus on chromosome 6 [5]. In this region, four tandemly arranged genes – serine/threonine kinase RP, complement C4, steroid 21-hydroxylase CYP21, and tenascin TNX – are organized as a genetic unit designated as a RCCX module. In a RCCX bimodular haplotype, duplication of the RCCX module occurs and the orientation of genes,
⁎ Corresponding author. Catholic University, Largo A. Gemelli 8, 00168 Rome, Italy. Tel.: +39 0630154250; fax: +39 0630156706. E-mail address: [email protected] (P. Concolino). 0009-8981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2009.01.008
from telomere to centromere, is: RP1–C4A–CYP21A1P–TNXA–RP2–C4B– CYP21A2–TNXB. The three pseudogenes, CYP21A1P–TNXA and RP2, located between the two C4 loci, do not encode functional proteins [6,7]. In Caucasian populations, bimodular and monomodular RCCX organizations are present in about 69% and 17% of chromosome 6, respectively; while trimodular RCCX haplotypes have a frequency of about 14% [8]. Both CYP21A2 gene and CYP21A1P pseudogene each contain 10 exons spaced over 3.1 kb, their nucleotide sequence are 98% identical in exons and approximately 96% identical in introns [9]. Most chromosomes bear 1 CYP21A1P pseudogene and 1 CYP21A2 gene, although deletion and duplications have been described [10]. Intergenic recombinations are responsible for 95% of the mutations associated with 21hydroxylase deficiency; the remaining 5% of mutations do not appear to be the result of gene conversion events [11]. Among the intergenic recombinations, approximately the 75% is represented by mutations normally present in the pseudogene possibly transferred to the functional gene by microconversion events [12]. The remaining 20%– 25% of mutations are represented by CYP21A2 gene deletions or CYP21A1P/CYP21A2 chimeric genes. In fact, an unequal crossing over event during meiosis can determine a complete deletion of C4B and a net
P. Concolino et al. / Clinica Chimica Acta 402 (2009) 164–170 Table 1 Clinical and genetic data of 18 unrelated Italian CAH patients Case Sex Clinical Genotype diagnosis (paternal allele/maternal allele) 1 2 3 4 5 6 7
F F F F F M M
NC NC NC NC NC NC NC
8 9 10 11 12 13 14 15 16 17 18
F F M F F M M F F F F
NC NC SW SW NC NC SV SV NC SW SV
−/p.P453S p.V281L/− p.V281L/p.V281L p.V281L/− p.V281L/p.V281L p.P453S/p.V281L p.V281L +p.P453S +p.R483P/p. V281L p.P482S/− p.V281L/p.R356W IVS2-13A/CNG/− −/p.W22X p.P453S/IVS2-13A/CNG dup p.Q318X/p.R356W p.Q318X/p.I172N p.I172N/p.I172N p.V281L/IVS2-13A/CNG dup IVS2-13A/CNG/IVS2-13A/CNG p.I172N/−
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Table 2 Prenatal diagnosis study
CYP21A1Pa CYP21A2a copy number copy number
Case
CYP21A1Pa Clinical Genotype CYP21A2a phenotype (paternal allele/maternal allele) copy number copy number
1 1 2 1 2 2 2
2 3 3 2 3 2 1
Father Mother Daughter Fetus
SV AS AS ND
1 2 1 1 3 2 2 2 3 2 1
2 2 3 2 2 2 1 2 2 1 2
F: female, M: male, NC: nonclassic, SV: simple virilising, SW: salt wasting, wt: wild type, dup: gene duplication. The indent (−) indicates the loss of pathernal/mathernal wild type CYP21A2 allele. a These results were formerly obtained by Southern blot analysis and successively confirmed by MLPA method.
deletion of CYP21A2 gene. In addition, a 30 kb deletion, involving the 3′ end of CYP21A1P, all the C4B, and the 5′ end of CYP21A2, produces a single nonfunctional chimeric gene with 5′ and 3′ ends corresponding to CYP21A1P and CYP21A2, respectively [1,2,12]. Other unequal meiotic crossover arrangements can produce duplicated CYP21A2 genes, which have been found in Dutch [13], Swedish [14,15], Italian [16], and other populations, mainly associated with the presence of the severe p.Q318X or IVS2 (c.293-13 A/CNG) mutations in one of the CYP21A2 genes. Consequently, to provide a correct result, the assessment of CYP21A2 gene copy number is very important in CAH molecular diagnosis, especially when the p.Q318X or IVS2 mutations are involved. Until now large CYP21A2 rearrangements have been mainly detected by Southern blot analysis [17,18] although PCR-based amplification analyses have been developed and described [19–21]. Recently, a real-time quantitative PCR (q-PCR)-based method for the CYP21A2 copy number detection has also been described [22]. In this paper we report the results of an optimized protocol for a multiplex ligation-dependent probe amplification assay (MLPA), capable of obtaining easy and rapid detection of deletions/duplications in the CYP21A2 gene. We therefore tested 18 CAH Italian patients previously analyzed in our laboratory by Southern blot and direct CYP21A2 gene sequencing. In addition, an example of prenatal CAH diagnosis is provided to document the value of this technique in diagnosis and genetic counselling. 2. Materials and methods 2.1. Genotyping of the samples Eighteen DNA samples from Italian subjects were used in the study. The patients were enrolled after endocrine evaluation and clinical CAH diagnosis. Genomic DNA was isolated from peripheral blood samples using High Pure PCR Template Preparation Kits (Roche Diagnostic, USA). DNA was eluted in 80 μl of water, quantified by spectrophotometer at 260 nm and stored at −20 °C until use. A prenatal DNA sample (obtained from the chorionic villi) and the blood samples of fetus' parents and sister were sent to our laboratory for molecular diagnosis of 21hydroxylase deficiency by specialists of outside hospital. The CYP21A2 gene sequencing and Southern blot analysis were carried out as previously described [23,24]. 2.2. SALSA MLPA P050B CAH probemix The SALSA MLPA KIT P050B CAH (see Supplemental Data Table 1) is commercially available from MRC Holland, Amsterdam, The Netherlands. The probemix included in
p.R341P/IVS2-13A/CNG p.Q318X dup/wt IVS2-13A/CNG/p.Q318X dup p.R341P/p.Q318X dup
2 3 3 3
2 2 2 2
SV: simple virilising, AS= asymptomatic, ND= not determined. a These results were formerly obtained by Southern blot analysis and successively confirmed by MLPA method.
this MLPA kit contains 33 different probes with amplification products between 130 and 391 bp. Five probes are specific for the CYP21A2 gene and recognize the 5′ region and the exons 3, 4, 6 and 8. The specific probes for exons 3, 4, 6 and 8 contain the wild type sequences for the Del8bp, p.I172N, Cluster E6 and p.Q318X mutations respectively (see Supplemental Data Figure 1). Furthermore, there are 3 CYP21A1P specific probes, 3 different TNXB probes, 1 probe for each complement factor and finally 1 probe for the CREBL1 gene. These 8 probes are specific to the 6p21 region and none must be used as control probe since large genomic rearrangements are known to occur within this region. In fact, 19 probes specific for human genes are included as controls for copy number quantification, two of these are located on the chromosome 6 (outside the chromosome 6p21.3 region) and one is specific for the chromosome Y. In addition, 4 DQ (DNA Quantity) control fragments (64, 70, 76 and 82 bp) are included. Their peak sizes are inversely correlated with the amount of DNA present in the sample: in fact, when more than 100 ng of DNA are used, the four DQ amplification products are hardly visible. The SALSA MLPA probemix also contains three control probes of 92, 88 and 96 bp. The peak area of these fragments should be of similar size as most of the other MLPA amplification products. A low signal of 88 and 96 bp probes indicates an incomplete DNA denaturation while a low signal of 92 bp probe records a failure of the ligation reaction. Finally, the 100 and 105 bp probes are specific for the X and Y chromosomes, respectively. 2.3. Setup of MLPA reaction DNA quality represents the most important variable when the MLPA reaction is employed. In fact, DNA extraction method and its subsequent treatment (such as storage) influence the width of MLPA peak. Regarding our experience, the use of commercial columns may represent the highest-quality method for DNA blood extraction in order to obtain a good amount of pure and intact nucleic acid. In addition, differently from that reported by the kit protocol provided by the manufacturer, we suggest to use at least 200 ng of DNA template. We highlight that all the other indications included in the manufacturer protocol should be carefully followed. Briefly, the DNA was denatured (5 min at 98 °C) and hybridized overnight at 60 °C with the SALSA probemix. Samples were then treated with Ligase-65 enzyme for 15 min at 54 °C. The reactions were stopped by incubation at 98 °C for 5 min. Finally, PCR amplification was carried out with the specific SALSA PCR primers for 35 cycles (95 °C for 30 s; 60 °C for 30 s; 72 °C for 1 min). Amplification products were run on a SEQ8000 Genetic Analyzer (Beckman Coulter Fullerton, CA) loading in each well: 0.8 µl of the PCR reaction + 0.5 µl of the FRAG-600 size standard (Beckman Coulter Fullerton, CA) + 32 µl of Beckman Sample Loading Solution (Beckman Coulter Fullerton, CA). The setting of the capillary electrophoresis was: capillary temperature: 50 °C; denaturation: 90 °C for 120 s; injection time: 2.0 kV for 30 s and runtime: 60 min at 4.8 kV. The peaks obtained after capillary electrophoresis could easily be identified and assigned to specific probes on the basis of their different lengths. All peaks matched with the expected sizes, with a deviation up to ± 3 bases. Each result was repeated and confirmed by three independent experiments. 2.4. MLPA data analysis To analyze MLPA data we used Coffalyser 8.0 Software (MRC Holland, Amsterdam, The Netherlands), an Excel-based program. Three healthy males and three healthy females were included in the analysis as controls. The relative peak area (RPA) of each sample's probe was obtained by dividing each measured peak area (As) by the sum of the area of all peaks of that sample (∑As). To obtain the relative peak ratio (RPR), the RPA (As/∑As) was then divided by the mean RPA of the corresponding probe obtained from three control wild type DNA samples from subjects of the same sex. The Coffalyser program identifies a peak as follows: a) normal when the RPR results within a range from 0.7 to 1.3, b) deleted when RPR is b 0.7, and c) duplicated when RPR is N1.3.
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Fig. 2. MLPA data from case 1 ( Table 1). The patient carried only one copy of CYP21A2 gene: (a) capillary electrophoresis pattern. The arrows show the peaks of CYP21A2 specific probes. (b) relative Peak ratio (RPR) of all CYP21A2 probes was b0.7.
3. Results 3.1. Mutation analysis Clinical diagnosis and molecular data (genotype and CYP21A2, CYP21A1P copy number) of the 18 CAH Italian patients are summarized in Table 1. 3.1.1. Prenatal diagnosis Table 2 shows the data regarding the CYP21A2 genetic study performed on an Italian family in order to carry out a prenatal CAH diagnosis. Based on the sequencing results, the father, suffering from classical CAH form, was classified as a compound heterozygote for the p.R341P (exon 8) and intron 2 splicing mutations. The mother and the first daughter, clinically asymptomatic, were classified as heterozygote for the p.Q318X mutation and compound heterozygote for the p. Q318X and the intron 2 splicing mutations, respectively. Finally, the fetus resulted compound heterozygote for the p.Q318X and p.R341P mutations. The presence of three bases A/C/G at 655 intron 2 position in the first daughter and the absence of clinical symptoms suggested the presence of a CYP21A2 duplication in this subject. This hypothesis was also supported by the results of Southern blot analysis, showing increased intensity of the 3.7-kb TaqI band (in the mother, daughter and fetus) representing the functional CYP21A2 gene (data not shown).
The segregation analysis revealed that in the first daughter and in the fetus the p.Q318X mutation was located in one of the two copies of CYP21A2 gene inherited from the mother. 3.2. MLPA results Fig. 1 reports the MLPA electropherograms from one male and one female control subjects, RPR for each probe was close to 1 indicating the absence of deletions/duplications in the DNA. MLPA confirmed the deletion of one CYP21A2 allele in 7 out of 18 CAH patients (cases 1, 2, 4, 8, 10, 11 and 18, in Table 1). In fact, all the specific peaks for CYP21A2 exons showed a RPR b 0.7, as compared to the normal controls. Four of these patients were affected from NC-CAH form: two carrying the p.V281L mutation in exon 7 (cases 2 and 4), one the p.P453S mutation in exon 10 (case 1) and another one the p. P482S mutation in exon 10 (case 8). Cases 10, 11 and 18 regard three patients suffering from classic CAH form and carrying the IVS2-13A/ CNG, the p.W22X and p.I172N mutations, respectively. In addition, MLPA analysis showed that two of these CAH patients (cases 2 and 10) carried three copies of CYP21A1P pseudogene (Table 1). The MLPA data regarding the case 1 are reported in Fig. 2 where the CYP21A2 specific probes show the ratio values of: 0.49, 0.53, 0.48, 0.58, and 0.51 for the 5′ gene probe and exons 3, 4, 6 and 8, respectively. Nine CAH patients (cases 3, 5, 6, 7, 9, 13, 14, 15 and 17; Table 1) carried 2 copies of CYP21A2 gene: 6 out of 9 were affected from
Fig. 1. MLPA data from male (a, b) and female (c, d) control subjects: (a), (c) capillary electrophoresis patterns. The black arrows show the peaks of CYP21A2 specific probes while the dashed arrow shows the chromosome Y specific probe. The curly bracket includes the reaction control peaks of 86, 92, 96, 100 and 105 bp. In female subjects, the deletion of Y-specific probe results in the absence of the relative peak. (b), (d) Relative peak ratio (RPR) of all probes was close to one. The dashed arrow shows the Y probe RPR.
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nonclassic (NC) form, 2 from simple virilising (SV) and the last one from salt wasting (SW). Fig. 3 shows the MLPA data of case 15. The subject was found to be homozygote for the p.I172N mutation in the exon 4 of the CYP21A2 gene; consequently the MLPA exon 4 specific probe was unable to bind its target. In fact, in the MLPA electropherogram (Fig. 3) the peak corresponding to the CYP21A2 exon 4 probe was absent (ratio = 0). For the same reason, in the electropherogram of case 14 (genotype: p. Q318X/p.I172N) the exon 4 and exon 8 specific probes showed a ratio b0.7 while in case 13 (genotype: p.Q318X/p.R356W) only the exon 8 specific probe presented a ratiob0.7. The MLPA CYP21A1P specific probes showed a ratio b0.7 in cases 7, 14 and 17, indicating that only one copy of the pseudogene was present in these patients. Three CYP21A1P copies were present in cases 3 and 5 (ratio N 1.3) while the other patients (cases 6, 7, 9, and 13) carried two copies. Finally, two nonconsanguineous NC-CAH patients (cases 12 and 16) resulted to be heterozygote for the CYP21A2 gene duplication. In fact, they carried the IVS2-13A/CNG mutation on a duplicate CYP21A2 gene inherited, in both cases, by the respective mothers. 3.2.1. Prenatal diagnosis Fig. 4 reports the MLPA results regarding the analysis performed on the fetus. All CYP21A2 specific probes show a ratio N1.3, indicating a heterozygote gene duplication, except for the exon 8 probe. In fact, since this probe contains the wild type sequence for the p.Q318X nonsense mutation, it is unable to bind the mutated allele. For this reason, the specific ratio (1.1) was in the range of normality (0.7–1.3). Similar MLPA results were obtained on the mother and daughter
which presented the following genotypes: p.Q318X dup/wt and IVS213A/CNG/p.Q318X dup, respectively (Table 2). Finally, the father was found to be heterozygote at CYP21A2 locus. 4. Discussion The CYP21A2 gene, which encodes the adrenocortical enzyme steroid 21-hydroxylase, lies in the central (class III) region of the human major histocompatibility complex on the short arm of chromosome 6 (6p21.3). Together with the adjacent complement C4 gene and truncated parts of the RP1 and TNXB genes, CYP21A2 constitutes a highly variable DNA region named RCCX module [8]. Generally, the RCCX module has three possible forms: monomodular, bimodular and trimodular. Because of the high degree of sequence homology and tandem repeating order of these genes, misalignments may occur during meiosis and generate illegitimate genetic recombinations or unequal crossovers. CYP21A2 copy number variations, such as deletions or gene duplications, have been largely described in literature [25]. Southern blot analysis has been the reference method for the detection of large CYP21A2 deletions/duplications. However, this method requires large quantities of DNA and is too laborious and time-consuming to be routinely used for 21-hydroxylase deficiency molecular diagnosis [17–18]. For this reason, other strategies have been developed up to now; most of these methods are based on PCR [19–22,26–28] or real-time qPCR [22,29], although they often fail the detection of CYP21A2 gene duplications [19–21,29]. Recently, a new technique [30–31], MLPA, has been described to detect gene dosage abnormalities in a wide range of pathologies [32–
Fig. 3. MLPA data regarding case 15 (Table 1). The patient resulted homozygote for the exon 4 p.I172N mutation: (a) capillary electrophoresis pattern, the arrows show the peaks of CYP21A2 specific probes. The peak relative to the CYP21A2 exon 4 probe was absent. (b) Relative peak ratio (RPR) of all other probes is close to one.
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Fig. 4. CAH prenatal diagnosis. MLPA data from the fetus carrying a duplicated copy of CYP21A2 on maternal chromosome 6 (Table 2): (a) the arrows show the peaks of CYP21A2 specific probes in the capillary electrophoresis pattern. Deletion of Y-specific probes (105 and 238 bp) indicated the fetus was female. (b) Relative peak ratio (RPR) of CYP21A2 probes was N 1.3, except for the CYP21A2 exon 8 probe containing the wild type sequence of p.Q318X mutation.
35]. It is based on a comparative quantification of hybridized probes that are amplified by PCR with a single pair of universal primers. It thus gets round of the intrinsic obstacle of multiplex PCR and makes simultaneous interrogation of gene dosage at multiple target loci a much easier job. In the present study, we investigated the usefulness and the performances of MLPA commercially available assay for the identification of CYP21A2/CYP21A1P deletions/duplications. In this regard, we tested 18 CAH Italian patients previously analyzed by gene sequencing and Southern blot techniques. Of the 7 known CYP21A2 deletions and 2 gene duplications previously characterized in our laboratory, all of them were successfully identified by the MLPA analysis (Table 1). In the patients carrying the p.I172N or the p.Q318X mutation, the signal of specific probes was absent or reduced (ratio b 0.7), according to the homozygote or heterozygote mutation state of each individual. In addition, the method was able to discover three CYP21A1P heterozygous deletion and four pseudogene duplications (Table 1). We also report a case of CAH prenatal diagnosis, where the MLPA assay was used to confirm the presence of a duplicated CYP21A2 allele, carrying the p.Q318X severe mutation, both in the fetus and in other two family members (Table 2). Prenatal treatment to prevent virilisation caused by the classic 21hydroxylase deficiency, is offered to couples whose developing child is at-risk of suffering from this disease [1]. Molecular analysis of prenatal DNA samples obtained after the start of preventive treatment, indicates whether or not it should be discontinued. For this purpose, it is important to determine the exact copy number of the gene in the
fetus, in particular when the p.Q318X and/or the IVS2-13A/CNG mutations are present in the analyzed family. In fact, in our CAH family, the finding of a duplicated CYP21A2 gene in the fetus was considered as a marker of better prognosis and supported the endocrinologist in the choice of a correct prenatal drug dosage. Our results indicate that MLPA analysis is a very informative tool in the molecular diagnosis of CAH. However, we stress that the use of this methodology requires a deep knowledge of CYP21A2 genetics. In fact, the 21-hydroxylase gene locus has a complex structure and the CYP21A2 gene is considered as one of the most polymorphic human genes. A large number of known mutations and polymorphisms occur along the coding gene sequence and micro-deletions/insertions are very frequent in intronic regions, especially in intron 2. False positive results could arise because the mutations/polymorphisms very close to the probe binding regions and the ligation site may prevent probe hybridization and ligation. This is the major drawback of MLPA technique. Therefore, a confirmatory test by longrange PCR or direct sequencing is necessary to verify a single exon deletion detected by MLPA. However, single exon deletion/duplication results to be a very rare event in CYP21A2 genetics; in fact, to date, there are no cases reported in the literature. On the contrary, novel CYP21A2 point mutations, which may interfere with probe's binding, are continually discovered along the total coding sequence [36]. In conclusion, MLPA analysis represents a simple, rapid and more sensitive tool for detecting of CYP21A2/CYP21A1P deletions/duplications in CAH molecular diagnosis. Compared to Southern blot, MLPA results a high throughput analysis, allowing a simultaneous study of
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several samples in the same experiment and the investigation both of gene (CYP21A2) and pseudogene (CYP21A1P) in each patient. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cca.2009.01.008. References [1] White PC, Speiser PW. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr Rev 2000;21:245–91. [2] Speiser PW, White PC. Congenital adrenal hyperplasia. N Engl J Med 2003;349:776–88. [3] New MI. Extensive clinical experience: nonclassical 21-hydroxylase deficiency. J Clin Endocrinol Metab 2006;91:4205–14. [4] Frisch H, Battelino T, Schober E, Baumgartner-Parzer S, Nowotny P, Vierhapper H. Salt wasting in simple virilizing congenital adrenal hyperplasia. J Pediatr Endocrinol Metab 2001;14:1649–55. [5] White PC, Grossberger D, Onufer BJ. Two genes encoding steroid 21-hydroxylase are located near the genes encoding the fourth component of complement in man. Proc Natl Acad Sci U S A 1985;82:1089–93. [6] Yang Z, Mendoza AR, Welch TR, Zipf WB, Yu CY. Modular variations of the human major histocompatibility complex class III genes for serine/threonine kinase RP, complement component C4, steroid 21-hydroxylase CYP21, and tenascin TNX (the RCCX module). A mechanism for gene deletions and disease associations. J Biol Chem 1999;274:12147–56. [7] Haglund-Stengler B, Martin Ritzen E, Gustafsson J, Luthman H. Haplotypes of the steroid 21-hydroxylase gene region encoding mild steroid 21-hydroxylase deficiency. Proc Natl Acad Sci U S A 1991;88:8352–6. [8] Blanchong CA, Zhou B, Rupert KL. Deficiencies of human complement component C4A and C4B and heterozygosity in length variants of RP-C4-CYP21-TNX (RCCX) modules in caucasians. The load of RCCX genetic diversity on major histocompatibility complex-associated disease. J Exp Med 2000;191:2183–96. [9] White PC, New MI, Dupont B. Structure of human steroid 21-hydroxylase genes. Proc Natl Acad Sci U S A 1986;83:5111–5. [10] Werkmeister JW, New MI, Dupont B, White PC. Frequent deletion and duplication of the steroid 21-hydroxylase genes. Am J Hum Genet 1986;39:461–9. [11] Lee HH. CYP21 mutations and congenital adrenal hyperplasia. Clin Genet 2001;59:293–301. [12] Tusié-Luna MT, White PC. Gene conversions and unequal crossovers between CYP21 (steroid 21-hydroxylase gene) and CYP21P involve different mechanisms. Proc Natl Acad Sci U S A 1995;92:10796–800. [13] Koppens PF, Hoogenboezem T, Degenhart HJ. Duplication of the CYP21A2 gene complicates mutation analysis of steroid 21-hydroxylase deficiency: characteristics of three unusual haplotypes. Hum Genet 2002;111:405–10. [14] Haglund-Stengler B, Martin Ritzén E, Gustafsson J, Luthman H. Haplotypes of the steroid 21-hydroxylase gene region encoding mild steroid 21-hydroxylase deficiency. Proc Natl Acad Sci U S A 1991;88:8352–6. [15] Wedell A, Stengler B, Luthman H. Characterization of mutations on the rare duplicated C4/CYP21 haplotype in steroid 21-hydroxylase deficiency. Hum Genet 1994;94:50–4. [16] Sinnott PJ, Livieri C, Sampietro M. CYP21/C4 gene organisation in Italian 21hydroxylase deficiency families. Hum Genet 1992;88:545–51.
[17] White PC, Vitek A, Dupont B, New MI. Characterization of frequent deletions causing steroid 21-hydroxylase deficiency. Proc Natl Acad Sci U S A 1988;85:4436–40. [18] Lobato MN, Aledo R, Meseguer A. High variability of CYP21 gene rearrangements in Spanish patients with classic form of congenital adrenal hyperplasia. Hum Hered 1998;48:216–25. [19] Lee HH, Chang JG, Tsai CH, Tsai FJ, Chao HT, Chung B. Analysis of the chimeric CYP21P/ CYP21 gene in steroid 21-hydroxylase deficiency. Clin Chem 2000;46:606–11. [20] Lee HH, Lee YJ, Lin CY. PCR-based detection of the CYP21 deletion and TNXA/TNXB hybrid in the RCCX module. Genomics 2004;83:944–50. [21] Lee HH, Lee YJ, Chan P, Lin CY. Use of PCR-based amplification analysis as a substitute for the Southern blot method for CYP21 deletion detection in congenital adrenal hyperplasia. Clin Chem 2005;51:480. [22] Parajes S, Quinterio C, Domínguez F, Loidi L. A simple and robust quantitative PCR assay to determine CYP21A2 gene dose in the diagnosis of 21-hydroxylase deficiency. Clin Chem 2007;53:1577–84. [23] Concolino P, Satta MA, Santonocito C, et al. Linkage between I172N mutation, a marker of 21-hydroxylase deficiency, and a single nucleotide polymorphism in Int6 of CYP21B gene: a genetic study of Sardinian family. Clin Chim Acta 2006;364:298–302. [24] Loidi L, Quinteiro C, Parajes S, et al. High variability in CYP21A2 mutated alleles in Spanish 21-hydroxylase deficiency patients, six novel mutations and a founder effect. Clin Endocrinol (Oxf) 2006;64:330–6. [25] Parajes S, Quinteiro C, Domínguez F, Loidi L. High frequency of copy number variations and sequence variants at CYP21A2 locus: implication for the genetic diagnosis of 21-hydroxylase deficiency. PLoS ONE 2008;3:e2138. [26] Lee HH, Chang SF, Lee YJ, et al. Deletion of the C4-CYP21 repeat module leading to the formation of a chimeric CYP21P/CYP21 gene in a 9.3-kb fragment as a cause of steroid 21-hydroxylase deficiency. Clin Chem 2003;49:319–22. [27] Koppens PF, Degenhart HJ. PCR-based detection of CYP21 deletions. Clin Chem 2003;49:1555–6. [28] Tukel T, Uyguner O, Wei JQ, et al. 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. J Clin Endocrinol Metab 2003;88:5893–7. [29] Olney RC, Mougey EB, Wang J, Shulman DI, Sylvester JE. Using real-time, quantitative PCR for rapid genotyping of the steroid 21-hydroxylase gene in a north Florida population. J Clin Endocrinol Metab 2002;87:735–41. [30] Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 2002;30:e57. [31] Sellner LN, Taylor GR. MLPA and MAPH: new techniques for detection of gene deletions. Hum Mutat 2004;23:413–9. [32] Veschi S, Aceto G, Scioletti AP, et al. High prevalence of BRCA1 deletions in BRCAPRO-positive patients with high carrier probability. Ann Oncol 2007;18: vi86–92. [33] Gatta V, Antonucci I, Morizio E, et al. Identification and characterization of different SHOX gene deletions in patients with Leri–Weill dyschondrosteosys by MLPA assay. J Hum Genet 2007;52:21–7. [34] Lai KK, Lo IF, Tong TM, Cheng LY, Lam ST. Detecting exon deletions and duplications of the DMD gene using Multiplex Ligation-dependent Probe Amplification (MLPA). Clin Biochem 2006;39:367–72. [35] Pedace L, Majore S, Megiorni F, et al. Identification of a novel duplication in the APC gene using multiple ligation probe amplification in a patient with familial adenomatous polyposis. Cancer Genet Cytogenet 2008;182:130–5. [36] Database of CY21A2 by the Human Cytochrome P450 (CYP) Allele Nomenclature Committee: http://www.imm.ki.se/CYPalleles/cyp21.htm.
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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c l i n c h i m
Multiplex ligation-dependent probe amplification (MLPA) assay for the detection of CYP21A2 gene deletions/duplications in Congenital Adrenal Hyperplasia: First technical report Paola Concolino a,⁎, Enrica Mello a, Vincenzo Toscano b, Franco Ameglio a, Cecilia Zuppi a, Ettore Capoluongo a a b
Laboratory of Molecular Biology, Institute of Biochemistry and Clinical Biochemistry, Catholic University, Largo A. Gemelli 8, 00168 Rome, Italy Department of Endocrinology, II Faculty of Medicine, University ‘La Sapienza’, S. Andrea Hospital, Rome, Italy
a r t i c l e
i n f o
Article history: Received 2 December 2008 Received in revised form 7 January 2009 Accepted 12 January 2009 Available online 20 January 2009 Keywords: Multiplex ligation-dependent probe amplification (MLPA) CYP21A2 CAH diagnosis Duplication Deletion
a b s t r a c t Background: More than 90% of the cases of Congenital Adrenal Hyperplasia (CAH) are associated with mutations in 21-hydroxylase gene (CYP21A2). Up to now, large CYP21A2 rearrangements have been mainly detected by Southern blot analysis, although more rapid methods have been alternatively proposed. In this paper, we report the use of a multiplex ligation-dependent probe amplification (MLPA) method for easy and rapid detection of deletions/ duplications in the CYP21A2 gene. Methods: We collected 18 CAH Italian patients previously analyzed by gene sequencing and Southern blot technique. In addition, a prenatal diagnosis study was performed. Results: Of the 7 known subjects with CYP21A2 deletions and 2 with gene duplications previously characterized in our laboratory, all were successfully identified by the MLPA analysis. In the prenatal diagnosis study, the MLPA assay was able to identify the presence of a CYP21A2 gene duplication in the fetus, as well in other two family members. Conclusion: MLPA analysis represents a simple, rapid and sensitive tool for the detection of CYP21A2/ CYP21A1P deletions/duplications in CAH molecular diagnosis. Compared to Southern blot, MLPA may be considered a high throughput analysis, allowing the simultaneous study of several samples in the same experiment and the investigation of both gene (CYP21A2) and pseudogene (CYP21A1P) in each patient. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Congenital Adrenal Hyperplasia (CAH) is one of the most frequent inborn metabolic errors, inherited in an autosomal recessive manner. More than 90% of cases of CAH are due to 21-hydroxylase deficiency [1]. In the adrenal cortex, steroid 21-hydroxylase normally converts 17-hydroxyprogesterone (17-OHP) into 11-deoxycortisol and progesterone into 11-deoxycorticosterone. These steroids are subsequently converted into cortisol and aldosterone, respectively [1,2]. CAH includes a wide spectrum of clinical manifestation [3,4]. The gene encoding for the steroid 21-hydroxylase enzyme, CYP21A2, is located within the HLA class III region of the major histocompatibility complex locus on chromosome 6 [5]. In this region, four tandemly arranged genes – serine/threonine kinase RP, complement C4, steroid 21-hydroxylase CYP21, and tenascin TNX – are organized as a genetic unit designated as a RCCX module. In a RCCX bimodular haplotype, duplication of the RCCX module occurs and the orientation of genes,
⁎ Corresponding author. Catholic University, Largo A. Gemelli 8, 00168 Rome, Italy. Tel.: +39 0630154250; fax: +39 0630156706. E-mail address: [email protected] (P. Concolino). 0009-8981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2009.01.008
from telomere to centromere, is: RP1–C4A–CYP21A1P–TNXA–RP2–C4B– CYP21A2–TNXB. The three pseudogenes, CYP21A1P–TNXA and RP2, located between the two C4 loci, do not encode functional proteins [6,7]. In Caucasian populations, bimodular and monomodular RCCX organizations are present in about 69% and 17% of chromosome 6, respectively; while trimodular RCCX haplotypes have a frequency of about 14% [8]. Both CYP21A2 gene and CYP21A1P pseudogene each contain 10 exons spaced over 3.1 kb, their nucleotide sequence are 98% identical in exons and approximately 96% identical in introns [9]. Most chromosomes bear 1 CYP21A1P pseudogene and 1 CYP21A2 gene, although deletion and duplications have been described [10]. Intergenic recombinations are responsible for 95% of the mutations associated with 21hydroxylase deficiency; the remaining 5% of mutations do not appear to be the result of gene conversion events [11]. Among the intergenic recombinations, approximately the 75% is represented by mutations normally present in the pseudogene possibly transferred to the functional gene by microconversion events [12]. The remaining 20%– 25% of mutations are represented by CYP21A2 gene deletions or CYP21A1P/CYP21A2 chimeric genes. In fact, an unequal crossing over event during meiosis can determine a complete deletion of C4B and a net
P. Concolino et al. / Clinica Chimica Acta 402 (2009) 164–170 Table 1 Clinical and genetic data of 18 unrelated Italian CAH patients Case Sex Clinical Genotype diagnosis (paternal allele/maternal allele) 1 2 3 4 5 6 7
F F F F F M M
NC NC NC NC NC NC NC
8 9 10 11 12 13 14 15 16 17 18
F F M F F M M F F F F
NC NC SW SW NC NC SV SV NC SW SV
−/p.P453S p.V281L/− p.V281L/p.V281L p.V281L/− p.V281L/p.V281L p.P453S/p.V281L p.V281L +p.P453S +p.R483P/p. V281L p.P482S/− p.V281L/p.R356W IVS2-13A/CNG/− −/p.W22X p.P453S/IVS2-13A/CNG dup p.Q318X/p.R356W p.Q318X/p.I172N p.I172N/p.I172N p.V281L/IVS2-13A/CNG dup IVS2-13A/CNG/IVS2-13A/CNG p.I172N/−
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Table 2 Prenatal diagnosis study
CYP21A1Pa CYP21A2a copy number copy number
Case
CYP21A1Pa Clinical Genotype CYP21A2a phenotype (paternal allele/maternal allele) copy number copy number
1 1 2 1 2 2 2
2 3 3 2 3 2 1
Father Mother Daughter Fetus
SV AS AS ND
1 2 1 1 3 2 2 2 3 2 1
2 2 3 2 2 2 1 2 2 1 2
F: female, M: male, NC: nonclassic, SV: simple virilising, SW: salt wasting, wt: wild type, dup: gene duplication. The indent (−) indicates the loss of pathernal/mathernal wild type CYP21A2 allele. a These results were formerly obtained by Southern blot analysis and successively confirmed by MLPA method.
deletion of CYP21A2 gene. In addition, a 30 kb deletion, involving the 3′ end of CYP21A1P, all the C4B, and the 5′ end of CYP21A2, produces a single nonfunctional chimeric gene with 5′ and 3′ ends corresponding to CYP21A1P and CYP21A2, respectively [1,2,12]. Other unequal meiotic crossover arrangements can produce duplicated CYP21A2 genes, which have been found in Dutch [13], Swedish [14,15], Italian [16], and other populations, mainly associated with the presence of the severe p.Q318X or IVS2 (c.293-13 A/CNG) mutations in one of the CYP21A2 genes. Consequently, to provide a correct result, the assessment of CYP21A2 gene copy number is very important in CAH molecular diagnosis, especially when the p.Q318X or IVS2 mutations are involved. Until now large CYP21A2 rearrangements have been mainly detected by Southern blot analysis [17,18] although PCR-based amplification analyses have been developed and described [19–21]. Recently, a real-time quantitative PCR (q-PCR)-based method for the CYP21A2 copy number detection has also been described [22]. In this paper we report the results of an optimized protocol for a multiplex ligation-dependent probe amplification assay (MLPA), capable of obtaining easy and rapid detection of deletions/duplications in the CYP21A2 gene. We therefore tested 18 CAH Italian patients previously analyzed in our laboratory by Southern blot and direct CYP21A2 gene sequencing. In addition, an example of prenatal CAH diagnosis is provided to document the value of this technique in diagnosis and genetic counselling. 2. Materials and methods 2.1. Genotyping of the samples Eighteen DNA samples from Italian subjects were used in the study. The patients were enrolled after endocrine evaluation and clinical CAH diagnosis. Genomic DNA was isolated from peripheral blood samples using High Pure PCR Template Preparation Kits (Roche Diagnostic, USA). DNA was eluted in 80 μl of water, quantified by spectrophotometer at 260 nm and stored at −20 °C until use. A prenatal DNA sample (obtained from the chorionic villi) and the blood samples of fetus' parents and sister were sent to our laboratory for molecular diagnosis of 21hydroxylase deficiency by specialists of outside hospital. The CYP21A2 gene sequencing and Southern blot analysis were carried out as previously described [23,24]. 2.2. SALSA MLPA P050B CAH probemix The SALSA MLPA KIT P050B CAH (see Supplemental Data Table 1) is commercially available from MRC Holland, Amsterdam, The Netherlands. The probemix included in
p.R341P/IVS2-13A/CNG p.Q318X dup/wt IVS2-13A/CNG/p.Q318X dup p.R341P/p.Q318X dup
2 3 3 3
2 2 2 2
SV: simple virilising, AS= asymptomatic, ND= not determined. a These results were formerly obtained by Southern blot analysis and successively confirmed by MLPA method.
this MLPA kit contains 33 different probes with amplification products between 130 and 391 bp. Five probes are specific for the CYP21A2 gene and recognize the 5′ region and the exons 3, 4, 6 and 8. The specific probes for exons 3, 4, 6 and 8 contain the wild type sequences for the Del8bp, p.I172N, Cluster E6 and p.Q318X mutations respectively (see Supplemental Data Figure 1). Furthermore, there are 3 CYP21A1P specific probes, 3 different TNXB probes, 1 probe for each complement factor and finally 1 probe for the CREBL1 gene. These 8 probes are specific to the 6p21 region and none must be used as control probe since large genomic rearrangements are known to occur within this region. In fact, 19 probes specific for human genes are included as controls for copy number quantification, two of these are located on the chromosome 6 (outside the chromosome 6p21.3 region) and one is specific for the chromosome Y. In addition, 4 DQ (DNA Quantity) control fragments (64, 70, 76 and 82 bp) are included. Their peak sizes are inversely correlated with the amount of DNA present in the sample: in fact, when more than 100 ng of DNA are used, the four DQ amplification products are hardly visible. The SALSA MLPA probemix also contains three control probes of 92, 88 and 96 bp. The peak area of these fragments should be of similar size as most of the other MLPA amplification products. A low signal of 88 and 96 bp probes indicates an incomplete DNA denaturation while a low signal of 92 bp probe records a failure of the ligation reaction. Finally, the 100 and 105 bp probes are specific for the X and Y chromosomes, respectively. 2.3. Setup of MLPA reaction DNA quality represents the most important variable when the MLPA reaction is employed. In fact, DNA extraction method and its subsequent treatment (such as storage) influence the width of MLPA peak. Regarding our experience, the use of commercial columns may represent the highest-quality method for DNA blood extraction in order to obtain a good amount of pure and intact nucleic acid. In addition, differently from that reported by the kit protocol provided by the manufacturer, we suggest to use at least 200 ng of DNA template. We highlight that all the other indications included in the manufacturer protocol should be carefully followed. Briefly, the DNA was denatured (5 min at 98 °C) and hybridized overnight at 60 °C with the SALSA probemix. Samples were then treated with Ligase-65 enzyme for 15 min at 54 °C. The reactions were stopped by incubation at 98 °C for 5 min. Finally, PCR amplification was carried out with the specific SALSA PCR primers for 35 cycles (95 °C for 30 s; 60 °C for 30 s; 72 °C for 1 min). Amplification products were run on a SEQ8000 Genetic Analyzer (Beckman Coulter Fullerton, CA) loading in each well: 0.8 µl of the PCR reaction + 0.5 µl of the FRAG-600 size standard (Beckman Coulter Fullerton, CA) + 32 µl of Beckman Sample Loading Solution (Beckman Coulter Fullerton, CA). The setting of the capillary electrophoresis was: capillary temperature: 50 °C; denaturation: 90 °C for 120 s; injection time: 2.0 kV for 30 s and runtime: 60 min at 4.8 kV. The peaks obtained after capillary electrophoresis could easily be identified and assigned to specific probes on the basis of their different lengths. All peaks matched with the expected sizes, with a deviation up to ± 3 bases. Each result was repeated and confirmed by three independent experiments. 2.4. MLPA data analysis To analyze MLPA data we used Coffalyser 8.0 Software (MRC Holland, Amsterdam, The Netherlands), an Excel-based program. Three healthy males and three healthy females were included in the analysis as controls. The relative peak area (RPA) of each sample's probe was obtained by dividing each measured peak area (As) by the sum of the area of all peaks of that sample (∑As). To obtain the relative peak ratio (RPR), the RPA (As/∑As) was then divided by the mean RPA of the corresponding probe obtained from three control wild type DNA samples from subjects of the same sex. The Coffalyser program identifies a peak as follows: a) normal when the RPR results within a range from 0.7 to 1.3, b) deleted when RPR is b 0.7, and c) duplicated when RPR is N1.3.
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Fig. 2. MLPA data from case 1 ( Table 1). The patient carried only one copy of CYP21A2 gene: (a) capillary electrophoresis pattern. The arrows show the peaks of CYP21A2 specific probes. (b) relative Peak ratio (RPR) of all CYP21A2 probes was b0.7.
3. Results 3.1. Mutation analysis Clinical diagnosis and molecular data (genotype and CYP21A2, CYP21A1P copy number) of the 18 CAH Italian patients are summarized in Table 1. 3.1.1. Prenatal diagnosis Table 2 shows the data regarding the CYP21A2 genetic study performed on an Italian family in order to carry out a prenatal CAH diagnosis. Based on the sequencing results, the father, suffering from classical CAH form, was classified as a compound heterozygote for the p.R341P (exon 8) and intron 2 splicing mutations. The mother and the first daughter, clinically asymptomatic, were classified as heterozygote for the p.Q318X mutation and compound heterozygote for the p. Q318X and the intron 2 splicing mutations, respectively. Finally, the fetus resulted compound heterozygote for the p.Q318X and p.R341P mutations. The presence of three bases A/C/G at 655 intron 2 position in the first daughter and the absence of clinical symptoms suggested the presence of a CYP21A2 duplication in this subject. This hypothesis was also supported by the results of Southern blot analysis, showing increased intensity of the 3.7-kb TaqI band (in the mother, daughter and fetus) representing the functional CYP21A2 gene (data not shown).
The segregation analysis revealed that in the first daughter and in the fetus the p.Q318X mutation was located in one of the two copies of CYP21A2 gene inherited from the mother. 3.2. MLPA results Fig. 1 reports the MLPA electropherograms from one male and one female control subjects, RPR for each probe was close to 1 indicating the absence of deletions/duplications in the DNA. MLPA confirmed the deletion of one CYP21A2 allele in 7 out of 18 CAH patients (cases 1, 2, 4, 8, 10, 11 and 18, in Table 1). In fact, all the specific peaks for CYP21A2 exons showed a RPR b 0.7, as compared to the normal controls. Four of these patients were affected from NC-CAH form: two carrying the p.V281L mutation in exon 7 (cases 2 and 4), one the p.P453S mutation in exon 10 (case 1) and another one the p. P482S mutation in exon 10 (case 8). Cases 10, 11 and 18 regard three patients suffering from classic CAH form and carrying the IVS2-13A/ CNG, the p.W22X and p.I172N mutations, respectively. In addition, MLPA analysis showed that two of these CAH patients (cases 2 and 10) carried three copies of CYP21A1P pseudogene (Table 1). The MLPA data regarding the case 1 are reported in Fig. 2 where the CYP21A2 specific probes show the ratio values of: 0.49, 0.53, 0.48, 0.58, and 0.51 for the 5′ gene probe and exons 3, 4, 6 and 8, respectively. Nine CAH patients (cases 3, 5, 6, 7, 9, 13, 14, 15 and 17; Table 1) carried 2 copies of CYP21A2 gene: 6 out of 9 were affected from
Fig. 1. MLPA data from male (a, b) and female (c, d) control subjects: (a), (c) capillary electrophoresis patterns. The black arrows show the peaks of CYP21A2 specific probes while the dashed arrow shows the chromosome Y specific probe. The curly bracket includes the reaction control peaks of 86, 92, 96, 100 and 105 bp. In female subjects, the deletion of Y-specific probe results in the absence of the relative peak. (b), (d) Relative peak ratio (RPR) of all probes was close to one. The dashed arrow shows the Y probe RPR.
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nonclassic (NC) form, 2 from simple virilising (SV) and the last one from salt wasting (SW). Fig. 3 shows the MLPA data of case 15. The subject was found to be homozygote for the p.I172N mutation in the exon 4 of the CYP21A2 gene; consequently the MLPA exon 4 specific probe was unable to bind its target. In fact, in the MLPA electropherogram (Fig. 3) the peak corresponding to the CYP21A2 exon 4 probe was absent (ratio = 0). For the same reason, in the electropherogram of case 14 (genotype: p. Q318X/p.I172N) the exon 4 and exon 8 specific probes showed a ratio b0.7 while in case 13 (genotype: p.Q318X/p.R356W) only the exon 8 specific probe presented a ratiob0.7. The MLPA CYP21A1P specific probes showed a ratio b0.7 in cases 7, 14 and 17, indicating that only one copy of the pseudogene was present in these patients. Three CYP21A1P copies were present in cases 3 and 5 (ratio N 1.3) while the other patients (cases 6, 7, 9, and 13) carried two copies. Finally, two nonconsanguineous NC-CAH patients (cases 12 and 16) resulted to be heterozygote for the CYP21A2 gene duplication. In fact, they carried the IVS2-13A/CNG mutation on a duplicate CYP21A2 gene inherited, in both cases, by the respective mothers. 3.2.1. Prenatal diagnosis Fig. 4 reports the MLPA results regarding the analysis performed on the fetus. All CYP21A2 specific probes show a ratio N1.3, indicating a heterozygote gene duplication, except for the exon 8 probe. In fact, since this probe contains the wild type sequence for the p.Q318X nonsense mutation, it is unable to bind the mutated allele. For this reason, the specific ratio (1.1) was in the range of normality (0.7–1.3). Similar MLPA results were obtained on the mother and daughter
which presented the following genotypes: p.Q318X dup/wt and IVS213A/CNG/p.Q318X dup, respectively (Table 2). Finally, the father was found to be heterozygote at CYP21A2 locus. 4. Discussion The CYP21A2 gene, which encodes the adrenocortical enzyme steroid 21-hydroxylase, lies in the central (class III) region of the human major histocompatibility complex on the short arm of chromosome 6 (6p21.3). Together with the adjacent complement C4 gene and truncated parts of the RP1 and TNXB genes, CYP21A2 constitutes a highly variable DNA region named RCCX module [8]. Generally, the RCCX module has three possible forms: monomodular, bimodular and trimodular. Because of the high degree of sequence homology and tandem repeating order of these genes, misalignments may occur during meiosis and generate illegitimate genetic recombinations or unequal crossovers. CYP21A2 copy number variations, such as deletions or gene duplications, have been largely described in literature [25]. Southern blot analysis has been the reference method for the detection of large CYP21A2 deletions/duplications. However, this method requires large quantities of DNA and is too laborious and time-consuming to be routinely used for 21-hydroxylase deficiency molecular diagnosis [17–18]. For this reason, other strategies have been developed up to now; most of these methods are based on PCR [19–22,26–28] or real-time qPCR [22,29], although they often fail the detection of CYP21A2 gene duplications [19–21,29]. Recently, a new technique [30–31], MLPA, has been described to detect gene dosage abnormalities in a wide range of pathologies [32–
Fig. 3. MLPA data regarding case 15 (Table 1). The patient resulted homozygote for the exon 4 p.I172N mutation: (a) capillary electrophoresis pattern, the arrows show the peaks of CYP21A2 specific probes. The peak relative to the CYP21A2 exon 4 probe was absent. (b) Relative peak ratio (RPR) of all other probes is close to one.
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Fig. 4. CAH prenatal diagnosis. MLPA data from the fetus carrying a duplicated copy of CYP21A2 on maternal chromosome 6 (Table 2): (a) the arrows show the peaks of CYP21A2 specific probes in the capillary electrophoresis pattern. Deletion of Y-specific probes (105 and 238 bp) indicated the fetus was female. (b) Relative peak ratio (RPR) of CYP21A2 probes was N 1.3, except for the CYP21A2 exon 8 probe containing the wild type sequence of p.Q318X mutation.
35]. It is based on a comparative quantification of hybridized probes that are amplified by PCR with a single pair of universal primers. It thus gets round of the intrinsic obstacle of multiplex PCR and makes simultaneous interrogation of gene dosage at multiple target loci a much easier job. In the present study, we investigated the usefulness and the performances of MLPA commercially available assay for the identification of CYP21A2/CYP21A1P deletions/duplications. In this regard, we tested 18 CAH Italian patients previously analyzed by gene sequencing and Southern blot techniques. Of the 7 known CYP21A2 deletions and 2 gene duplications previously characterized in our laboratory, all of them were successfully identified by the MLPA analysis (Table 1). In the patients carrying the p.I172N or the p.Q318X mutation, the signal of specific probes was absent or reduced (ratio b 0.7), according to the homozygote or heterozygote mutation state of each individual. In addition, the method was able to discover three CYP21A1P heterozygous deletion and four pseudogene duplications (Table 1). We also report a case of CAH prenatal diagnosis, where the MLPA assay was used to confirm the presence of a duplicated CYP21A2 allele, carrying the p.Q318X severe mutation, both in the fetus and in other two family members (Table 2). Prenatal treatment to prevent virilisation caused by the classic 21hydroxylase deficiency, is offered to couples whose developing child is at-risk of suffering from this disease [1]. Molecular analysis of prenatal DNA samples obtained after the start of preventive treatment, indicates whether or not it should be discontinued. For this purpose, it is important to determine the exact copy number of the gene in the
fetus, in particular when the p.Q318X and/or the IVS2-13A/CNG mutations are present in the analyzed family. In fact, in our CAH family, the finding of a duplicated CYP21A2 gene in the fetus was considered as a marker of better prognosis and supported the endocrinologist in the choice of a correct prenatal drug dosage. Our results indicate that MLPA analysis is a very informative tool in the molecular diagnosis of CAH. However, we stress that the use of this methodology requires a deep knowledge of CYP21A2 genetics. In fact, the 21-hydroxylase gene locus has a complex structure and the CYP21A2 gene is considered as one of the most polymorphic human genes. A large number of known mutations and polymorphisms occur along the coding gene sequence and micro-deletions/insertions are very frequent in intronic regions, especially in intron 2. False positive results could arise because the mutations/polymorphisms very close to the probe binding regions and the ligation site may prevent probe hybridization and ligation. This is the major drawback of MLPA technique. Therefore, a confirmatory test by longrange PCR or direct sequencing is necessary to verify a single exon deletion detected by MLPA. However, single exon deletion/duplication results to be a very rare event in CYP21A2 genetics; in fact, to date, there are no cases reported in the literature. On the contrary, novel CYP21A2 point mutations, which may interfere with probe's binding, are continually discovered along the total coding sequence [36]. In conclusion, MLPA analysis represents a simple, rapid and more sensitive tool for detecting of CYP21A2/CYP21A1P deletions/duplications in CAH molecular diagnosis. Compared to Southern blot, MLPA results a high throughput analysis, allowing a simultaneous study of
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