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BRIEF REPORT |
Department of Internal Medicine III, Division of Clinical Endocrinology and Metabolism (S.M.B.-P., H.V.) and the Department of Blood Group Serology (G.F.), Medical University of Vienna, A-1090 Vienna, Austria
Address all correspondence and requests for reprints to: S. M. Baumgartner-Parzer, Department of Internal Medicine III, Division of Endocrinology & Metabolism, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail: sabina.baumgartner-parzer{at}meduniwien.ac.at.
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Abstract |
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Subjects and Methods: As part of routine diagnostic procedures, CYP21 genotyping has been performed in two unrelated female CAH index patients and in their clinically asymptomatic parents and siblings.
Results: Both patients have inherited the paternal Intron2splice mutation and have harbored a de novo gene aberration (large deletion and I271N/exon 4) on their maternal haplotype. Surprisingly, both mothers were carriers of rare duplicated CYP21A2 haplotypes carrying CAH alleles, which were not detected in the daughters. Among 133 CAH alleles that were detected in patients and that could be traced to the respective family members by genotyping, these two de novo aberrations (representing 1.5% of 133 traced CAH alleles) were the only ones identified.
Conclusion: Because both de novo CYP21A2 gene aberrations so far identified in our laboratory occurred in the gametes of mothers carrying rare duplicated CYP21A2 haplotypes, we hypothesize that duplicated CYP21A2 genes could predispose for de novo mutations in the offspring, which is of relevance for prenatal CYP21 genotyping and genetic counseling.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Steroid 21-hydroxylase deficiency is unusual among genetic diseases in that approximately 95% of mutant alleles have apparently been generated through intergenic recombination (1). This phenomenon relates to the presence of two 21-hydroxylase genes, the functional CYP21A2 and the highly homologous pseudogene CYP21A1P, which is inactive as a result of small deletions, insertions, and point mutations (1, 2). Both 21-hydroxylase genes are lying within the histocompatibility leukocyte antigen (HLA) major histocompatibility complex locus on chromosome 6p21.3. With their neighboring genes tenascin TNXA/B, complement C4A/B, and the serine/threonine nuclear protein kinase RP plus small truncated sections of TNX and RP, they form a genetic unit designated as the RCCX module (1, 4, 5).
Each chromosome 6 has at least one RCCX module, two modules representing the standard and three or even four modules being rare cases (Fig. 1
) (4, 5). Monomodular haplotypes, which lack one of the C4 genes as well as the CYP21A1P pseudogene, have also been described (1, 4, 5). The deletion of such CYP21A1 pseudogene (monomodular RCCX unit) has been proposed to represent a premutation for a de novo deletion of CYP21A2 resulting from unequal crossover between a standard bimodular chromosome and a monomodular chromosome (6); this notion of the common monomodular CYP21A1P deletion chromosome serving as a premutation was strengthened by reports (7, 8) describing reciprocal TNXB/TNXA and TNXA/TNXB hybrids in unrelated persons and a high rate of de novo unequal crossing over in sperm DNA of an individual carrying a CYP21A1P deletion (9). In contrast, Koppens et al. (10) described one de novo CYP21A2 deletion resulting from unequal crossover occurring between chromosomes with equal numbers of RCCX modules, indicating that an unequal number of RCCX modules is not an absolute prerequisite for de novo mutations in the offspring.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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CYP21 genotyping was performed in two unrelated female CAH index patients presenting with simply virilizing CAH. Patient 1 (Prader stage 3 without salt loss) was described previously in more detail (11); patient 2 was referred to our department at the age of 23 yr. Definite information about the original genital status was not available. However, the patient had undergone two corrective operations. Although no initial salt-wasting crises were reported, the patient, when first seen in our outpatient service, was under substitution therapy with both glucocorticoids (hydrocortisone, 10 mg in the morning; dexamethasone, 0.125 mg in the evening) and mineralocorticoids (fludrocortisone, 0.05 in the morning and 0.025 mg in the evening). While on this therapy, the patient’s plasma concentrations of 17-hydroxyprogesterone and testosterone were 33.0 and 0.69 nmol/liter, respectively. The parents as well as the patients’ siblings (patient 1: sister; patient 2: brother) were clinically asymptomatic, and their basal 17-hydroxyprogesterone and testosterone plasma concentrations were within the normal range. Written informed consent for CYP21 genotyping was obtained from all individuals studied.
HLA typing
Low-resolution typing of HLA-A, B, C alleles was performed by DNA typing using a commercial kit (Dynal AutoReli; Dynal, Bromborough, UK) and by serology.
CYP21A2 genotyping
Genomic DNA was extracted from peripheral blood leukocytes and genotyping for large gene deletions/conversions was by Southern blot analysis as described previously (11, 12, 13). In brief, TaqI- and BglII-digested DNAs, immobilized on nylon membranes, were hybridized with a 32P-dCTP-labeled CYP21 probe (American Type Culture Collection, Manassas, VA). Heterozygous and homozygous deletions/conversions as well as duplications were distinguished on the basis of the intensities and ratios of TaqI and BglII fragments.
The common mutations P30L, Intron2Splice (IVS2), I172N, Cluster E6, V281L, F307insT, G291S, Q318X, R356W, G424S, and P453S were detected by direct sequencing of three fragments (I–III) specifically amplified using selective PCR primers differentiating the functional CYP21A2 gene from the CYP21A1P pseudogene by the 8-bp deletion located in exon 3 of CYP21A1P, as described previously (11, 12, 13, 14). The OMIM number is 201910.
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Results |
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Paternity and maternity were ascertained by HLA typing performed in the index patients as well as in the respective family members (Fig. 2).
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Patient 1 has previously been described (11) to be compound heterozygous for an IVS2 and an I172N (exon 4) mutation, whereas patient 2 carries an IVS2 mutation on one haplotype and a large deletion (as identified by Southern blot analysis) on the other haplotype (Fig. 2
). CYP21A2 and HLA family analyses showed that both index patients have inherited the IVS2 mutation from their fathers. Because the I172N mutation in patient 1 and the large deletion in patient 2 were not found in the respective mother, both CYP21A2 aberrations were classified as de novo mutations.
Of note, a duplicated CYP21A2 gene (compare Figs. 1 and 2) carrying the Q318X mutation has been seen in the mother of patient 1, whereas the mother of patient 2 was heterozygous for the P30L, I172N, V281L, Cluster, and Q318X mutations, showing a 2:1 ratio of the intensity of the normal vs. the mutated sequence. This ratio and the presence of the nucleotides A, C, and G (mutated nucleotide) at the same position at the IVS2 locus indicate the presence of three CYP21A2 genes in this individual (Fig. 2). The presence of all these mutations on one allele indicates a large gene conversion event, because all these substitutions are assumed to originate from the functionally inactive pseudogene (1).
In contrast to her brother, patient 1 has not inherited the duplicated but has inherited the unaffected maternal allele on which she harbored a de novo exon 4 (I172N) mutation. The sister of index patient 2 inherited the maternal standard bimodular allele (one CYP21A2 and one CYP21A1 gene) associated with HLA A2 B15 Cw3 and the nucleotide C at the IVS2 locus. In index patient 2, the maternal duplicated CYP21A2 gene, associated with HLA A1 B8 Cw7 DRB1* 15 DQB1*06 and A/G at the IVS2 locus, was not detectable by sequence analysis as a result of a de novo deletion. The latter was identified by Southern blot analysis in patient 2, but not in her sister or in her parents. Moreover, in none of the members of family II did Southern blot analysis give any hint on a heterozygous deletion of the CYP21A1P pseudogene known to occur in association with HLA A1 B8 DRB1*03 DQB1* 02 (15), which is similar to the HLA haplotype associated with the duplicated maternal CYP21A2 allele.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Among 133 CAH alleles, which were detected in patients and could be traced to the respective family members by CYP21 genotyping, we identified these two de novo CYP21A2 aberrations representing 1.5% of the 133 traced CAH alleles. This percentage is in line with previous reports (1, 16). Of note, only for 133 of more than 1700 CAH alleles, detected in our laboratory, parents and/or siblings were available for genotyping allowing to trace the origin (inherited vs. de novo) of these alleles.
Because in both index patients the de novo mutation occurred on the maternal allele, it is of interest that both mothers are carriers of rare duplicated CYP21A2 haplotypes, the latter representing trimodular RCCX units. In that context, it is of note that deletion of the CYP21A1 pseudogene (monomodular RCCX unit) has been assumed to predispose to de novo gene deletions as a result of unequal crossover between chromosomes with a monomodular and a standard bimodular form. It has been suggested (6) that such a bimodular chromosome has no equally sized homolog to align to during meiosis resulting in misalignment of a CYP21A1P pseudogene to the functional CYP21A2 gene and causing a deletion of a 30-kb region, including the CYP21A2 gene and the 3' portion of the TNXB gene. These findings suggest that not only a heterozygous combination of monomodular and bimodular, but also the combination of a bimodular with a trimodular RCCX unit (as observed in the mother of index patient 2) could predispose to unequal crossover and result in CYP21A2 deletion.
It is unknown whether such variations of the RCCX modules also play a role for the occurrence of de novo CYP21A2 large gene conversions or single nucleotide substitutions, the majority of the latter representing microconversions (including P30L, IVS2, I172N, Cluster, V281L, Q318X) between the functional CYP21A2 gene and the functionally inactive CYP21A1 pseudogene. Only recently, Porzio et al. (17) observed a large gene conversion occurring de novo on the patient’s paternal allele, which carried a V281L mutation and which was associated with a HLA A2 B14 DRB1*01 haplotype. Although this HLA haplotype is known to be linked to the V281L mutation and a duplicated C4A and CYP21A1P pseudogene (1), the presence/absence of a CYP21A1P duplication was, however, not specifically addressed in that report (17). So far, only one I172N de novo mutation was shown to represent a microconversion resulting from transfer of a maternal CYP21A1P sequence in the son’s CYP21A2 allele during maternal meiosis (18). Tusie-Luna and White (9), however, suggest that although CYP21A2 deletions occur during meiosis, conversions may happen primarily during mitosis. This assumption is based on data obtained in sperm vs. leukocyte DNA and hence the mechanisms accounting for de novo microconversions within the female gamete (i.e. on maternal CYP21A2 genes) remain speculative.
In contrast to heterozygous CYP21A1 pseudogene deletions (monomodular RCCX unit) occurring with a frequency of 5 to 20% (1, 13), duplicated CYP21A2 haplotypes have only rarely been described (11, 20) and have not been reported in relation with de novo aberrations in the offspring by others. The presence of one duplicated haplotype (three functional CYP21A2 genes) is easily detected by the presence of three different nucleotides (A/C/G) at the polymorphic IVS2 locus (A/C, normal; G, mutated sequence, like in the mother of patient 2 and the brother of patient 1). For all other nonpolymorphic sites or other constellations in IVS2—even by sequence analysis—the identification of duplicated haplotypes is difficult [e.g. A/A/C (like in the mother of patient 1), C/C/A, etc.] or almost impossible (A/A/A, C/C/C). Thus, the true frequency of such duplicated CYP21A2 haplotypes could be higher than so far assumed, and their potential role for de novo aberrations in the offspring remains to be determined.
As a result of our observations of duplicated CYP21A2 haplotypes (trimodular RCCX unit) in the mothers of both patients with CAH exhibiting de novo aberrations on their maternal allele, it is tempting to speculate that haplotypes carrying such duplicated CYP21A2 genes could have a predisposing role for de novo aberrations as previously assumed for heterozygous CYP21A1 deletions (monomodular RCCX unit) in combination with standard bimodular RCCX units.
On the basis of our findings, this brief report encourages the search for haplotypes and constellations, which could predispose for CYP21A2 de novo aberrations, because the observations described here could influence the strategy for prenatal CYP21A2 genotyping and genetic counseling.
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Acknowledgments |
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Footnotes |
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First Published Online December 12, 2006
Abbreviations: CAH, Congenital adrenal hyperplasia; HLA, histocompatibility leukocyte antigen; IVS2, Intron2Splice.
Received October 6, 2006.
Accepted December 1, 2006.
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