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BRIEF REPORT |
Endocrinology Clinic (A.G.S.), First Pediatric Clinic (P.G.S.), Iuliu Hatieganu University of Medicine and Pharmacy, 400476 Cluj, Romania; First Clinic of Internal Medicine (A.G.S., M.M.W.), Department of Endocrinology, Johannes Gutenberg University, 55131 Mainz, Germany; Laboratory of Molecular Genetics (S.C., E.S.), 69121 Heidelberg, Germany; and University Children’s Hospital (U.H.), Ruprecht-Karls University, 69120 Heidelberg, Germany
Address all correspondence and requests for reprints to: Anca Grigorescu Sido, M.D., First Clinic of Internal Medicine, Department of Endocrinology, Johannes Gutenberg University, Langenbeckstr. 1, 55131 Mainz, Germany. E-mail: agsido{at}yahoo.co.uk.
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Abstract |
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Objective: The study aimed to perform molecular characterization in 43 Romanian patients with classical CAH forms diagnosed at the Center for Genetic Diseases of the Pediatric Clinic/University Cluj (38 with 21-hydroxylase deficiency, five with 11ß-hydroxylase deficiency), to determine the frequency of mutations in the CYP21A2 gene and attempt a genotype-phenotype correlation in patients with 21-hydroxylase deficiency.
Design: Molecular analysis was performed by direct sequencing of PCR amplified products of the CYP21A2 and CYP11B1 genes.
Results: The most frequent mutation in Romanian patients with 21-hydroxylase deficiency was I2G (43.9%), followed by deletions and large conversions (16.7%), I172N and the triple mutation (P30L+I2G+del8bp), accounting for 12.1% each, P30L (7.6%) and R356W (1.5%). Genotypes were categorized in three mutation groups (0, A, and B), according to their predicted functional consequences, and compared with clinical phenotype. Positive predictive values were 100, 75, and 100% for groups 0, A, and B, respectively. Overall genotype-phenotype correlation was 87.88%. In the five patients with 11ß-hydroxylase deficiency, the following homozygous mutations were identified: T318R in two related patients; R448H in two unrelated patients; and P94L, a new, yet-undescribed mutation.
Conclusion: The present study is the first countrywide report of mutational analysis in a Romanian patient population with 21-hydroxylase deficiency. Molecular diagnosis was performed in a small number of CAH patients proved not to suffer from 21-hydroxylase deficiency but from 11ß-hydroxylase deficiency, and a new mutation was identified.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The diversity of the clinical picture in patients with 21-hydroxylase deficiency reflects varying degrees of enzyme inactivation caused by combinations of a panel of different mutations of the structural CYP21 gene (CYP21A2) (3).
Data about the mutational spectrum of 21-hydroxylase deficiency in Eastern Europe are scarce (4, 5, 6). Romania is the only Latin country in Central-Eastern Europe, and the mutational frequency and spectrum might differ from those encountered in other neighboring countries, due to specific ethnic features.
Therefore, we aimed to analyze the types and frequencies of mutations in Romanian patients with 21-hydroxylase deficiency and to study the correlation between genotype and phenotype. In five patients referred with the clinical diagnosis of 21-hydroxylase deficiency, a suspected 11ß-hydroxylase deficiency was confirmed by molecular analysis, and a new mutation was identified. This is the first countrywide study presenting molecular data from patients with 21-hydroxylase deficiency and the first report of molecular diagnosis in Romanian patients with 11ß-hydroxylase deficiency.
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Patients and Methods |
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We studied 43 patients with classic CAH presumably due to 21-hydroxylase deficiency, referred from all over the country to the Center for Genetic Diseases, First Pediatric Clinic, University Hospital of Cluj/Romania. Thirty-eight patients were confirmed with 21-hydroxylase deficiency (13 males, 25 females; present age range, 0.75–37.66 yr; mean age, 10.54 ± 8.47 yr). The patients came from 33 unrelated families. In five families, there were two patients with 21-hydroxylase deficiency. Consanguinity was documented in only one family. Eighteen patients presented with the salt-wasting (SW) form, whereas 20 manifested the simple-virilizing (SV) form. In the remaining five patients (four males, one female; present age, 5–23.5 yr; mean age, 15 ± 7.13 yr) from the initial group of 43 referred to our clinic with the suspicion of 21-hydroxylase deficiency, 11ß-hydroxylase deficiency was diagnosed. These patients came from four unrelated families.
Diagnosis of CAH was based on clinical, hormonal, radiological, cytogenetical, and sonographical criteria.
Clinical suspicion of 11ß-hydroxylase deficiency arose in patients with signs of hyperandrogenism associated with elevated blood pressure levels. The diagnostic procedure was similar, except detection of markedly elevated 11-deoxycortisol (11DC) and deoxycorticosterone (DOC) levels.
In both enzymatic deficiencies, the diagnosis was confirmed by molecular analysis.
Mutation analysis
The genetic study was formally approved by the Ethics Committee of the College of Physicians Cluj/Romania. Informed consent for mutation analysis was obtained from all patients/parents and family members.
Genomic DNA was prepared from peripheral blood leukocytes using the QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions.
We studied 33 unrelated patients with 21-hydroxylase deficiency (66 unrelated alleles) and four unrelated patients with 11ß-hydroxylase deficiency (eight alleles). For 28 patients with 21-hydroxylase deficiency, analysis of respective family members was possible and included 50 parents (28 mothers and 22 fathers; 100 alleles) as well as 20 other family members (15 siblings, two children of one patient, and three aunts; 40 alleles).
For the molecular diagnosis in patients with 21-hydroxylase deficiency, four fragments of the CYP21A2 gene were specifically amplified by PCR using the Expand High Fidelity PCR System (Roche, Mannheim, Germany). Selective PCR primers differentiating CYP21A2 from CYP21A1P by the 8-bp deletion in exon 3 or by a sequence in exon 6 as well as primers for sequencing reactions were as described previously (7, 8, 9). The PCR fragments were used for direct sequencing of all exons, the exon/intron junctions, intron 2, intron 7, and 380 bp of the promotor region of the CYP21A2 gene, using the Thermo Sequenase cycle sequencing kit (Amersham, Freiburg, Germany) with IRD700- and IRD800-labeled oligonucleotides (MWG Biotech, Ebersberg, Germany) on a Licor 4200L-2 automatic DNA sequencer (LI-COR Biosciences, Inc., Biotechnology, Lincoln, NE). Deletions of the CYP21A2 gene were screened and quantified by polyacrylamide electrophoresis of IRD41-labeled PCR products of exon 3, according to Day et al. (9). The reference sequence reported by White et al. (10) was used for numbering of nucleotides and amino acids.
To avoid misinterpretations, parents were genotyped whenever available to verify the segregation of mutations in compound heterozygotes.
The patients were divided into three genotype groups (0, A, and B), according to the predicted functional consequences of the underlying enzymatic deficiency (11). Group 0 had no 21-hydroxylase activity; group A should exhibit minimal residual activity. Patients assigned to these genotype groups are expected to present the SW form. Patients in group B should preserve sufficient residual enzymatic activity to preclude SW and lead to a SV phenotype.
For the molecular diagnosis in patients with 11ß-hydroxylase deficiency, three fragments of the CYP11B1 gene (EMBL Nucleotide Sequence Database accession no. J05140) were amplified by PCR (12). After purification, the amplified fragments were sequenced on an ABI PRISM 3100 3.7 genetic analyzer.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Although small, our patient group with 21-hydroxylase deficiency appears to be representative for Romania, because the patients came from 19 of the 40 Romanian counties and were born in 21 different years of a 37-yr period (1967-2004). When related to all live births registered in these years and these counties (471,823), the resulting incidence of classic CAH patients was one of 14,300, in accordance with worldwide literature data.
We found nine mutational abnormalities in the 66 unrelated alleles from patients with 21-hydroxylase deficiency. The most frequent mutation was I2G (29 of 66 alleles; 43.9%), followed by deletions (11 of 66 alleles; 16.7%), I172N and a triple mutation (P30L+I2G+del8bp) (each 8 of 66 alleles; 12.1%), P30L (5 of 66 alleles; 7.6%), R356W (1 of 66 alleles; 1.5%), and three double mutations. A total of 33.3% of patients (11 of 33) had a homozygous genotype, whereas 66.6% (22 of 33) were compound heterozygotes. Eight of 17 of the patients with the SW form, but only three of 16 patients with the SV form, had a homozygous genotype.
To correlate genotype with phenotype, we included in group 0 the deletions, R356W and the double and triple mutations. To group A we assigned the I2G mutation, to group B the I172N and the P30L mutation, initially reported as a mutation leading to late-onset forms. However, conclusive literature data indicate that the P30L mutation often results in a SV phenotype (11). Accordingly, group 0 included six patients, all manifesting the SW form. Sixteen patients (12 SW, 4 SV), either homozygous for I2G or compound heterozygous with mutations of group 0, were assigned to group A. Group B included 16 patients with the SV form, all compound heterozygotes for I172N or P30L with a more severe mutation. The calculated positive predictive values for each genotype group were: 100, 75, and 100% for groups 0, A, and B, respectively. The overall concordance between genotype and phenotype was 87.88%. Phenotype, genotype, clinical features, and hormonal values at diagnosis are shown in Table 1
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In five patients erroneously diagnosed with 21-hydroxylase deficiency, 11ß-hydroxylase deficiency was suspected clinically and was confirmed by elevated 11DC and DOC serum concentrations and by molecular analysis of the CYP11B1 gene. The clinical, hormonal, and molecular data of these patients are presented in Table 2. In patient V, we identified a new mutation, consisting of a C to T transition at nucleotide 1296 in a homozygous form, which results in the substitution of proline to leucine at codon 94 in exon 2. The boy came from a nonconsanguineous family, in which both parents were heterozygous for P94L.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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In Table 1
, genotypes are classified according to their predicted functional consequences and compared with the clinical phenotype. In group A, predicted as SW, a mixed distribution of phenotypes was found (12 with SW and four with SV CAH). The I2G/I2G genotype resulted in a SW phenotype in five unrelated patients and in a SV phenotype in another three. A divergence between this particular genotype and phenotype has already been reported (17, 18, 19). Furthermore, patient 37 in the SV group had a I2G/del genotype, usually associated with the SW phenotype (Table 1). This discordance may be the result of postulated extraadrenal 21-hydroxylase activity.
As a peculiarity, we observed the presence of the triple mutation (P30L+I2G+del8bp) in nine patients from eight unrelated families. The affected allele should result in null residual activity. Depending on the second allele we found, as expected, four SW (one with a large deletion, three with I2G) and five SV patients (one with the I172N and four with the P30L mutation on the second allele).
In our opinion, phenotype prediction should be made with caution when prenatal diagnosis is concerned.
In one patient with 11ß-hydroxylase deficiency, a new point mutation (P94L in exon 2) was detected. The proline residue is found in other known eukaryotic mitochondrial P450 enzymes of the family, such as CYP11A gene encoding cholesterol desmolase, in a highly conserved amino acid sequence. Therefore, it seems likely that a mutation of this residue would adversely affect enzymatic activity (20), as it is clearly mirrored by the clinical and hormonal features of patient V (Table 2).
The present study is the first countrywide report of mutational analysis in Romanian patients with 21-hydroxylase deficiency. Molecular diagnosis was established in a small number of patients with 11ß-hydroxylase deficiency, and a new mutation was identified.
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Footnotes |
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First Published Online July 26, 2005
Abbreviations: CAH, Congenital adrenal hyperplasia; 11DC, 11-deoxycortisol; DOC, deoxycorticosterone; SV, simple-virilizing; SW, salt-wasting.
Received February 22, 2005.
Accepted July 14, 2005.
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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