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BRIEF REPORT |
Genetics Institute (T.P., R.G.-B., K.B., L.K.) and Division of Endocrinology, Meyer Children’s Hospital, Rambam Medical Center (Z.H.), Technion–Israel Institute of Technology (T.P., R.G.-B., Z.H.), Haifa 31096, Israel
Address all correspondence and requests for reprints to: Dr. Ze’ev Hochberg, Meyer Children’s Hospital, P.O.B. 9602, Haifa 31096, Israel. E-mail: z_hochberg{at}rambam.health.gov.il.
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Abstract |
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Study Subjects: We screened 236 healthy MJ for R448H.
Results: Only two of the subjects screened were found to be carriers, suggesting that the R448H allele frequency is lower than was assumed previously. An R448H/R448C compound heterozygote patient, diagnosed with 11-OHD, was identified. However, a subsequent screen of MJ subjects for R448C failed to detect any carriers, suggesting that this was a private mutation of this family.
Conclusion: The high incidence of 11-OHD in MJ, therefore, is only partially explained by the presence of R448H as a founder mutation.
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Introduction |
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More than 30 different mutations and polymorphisms have been described in CYP11B1 (8, 9, 10, 11, 12, 13, 14); most are missense mutations, which are distributed across the entire coding sequence, and are family-specific mutations. The presence of mutation hot spots or a founder effect was suggested for a few of these mutations (9). The incidence of 11-OHD was reported to be one in 100,000 among Caucasians (15).
The incidence in Israeli MJ is higher and was estimated at one in 5000 to one in 7000 (16), a figure close to population frequency for classical 21-OHD. A molecular analysis of CYP11B1 in MJ patients with 11-OHD identified the R448H mutation in exon 8 in 11 of 12 of the disease alleles (17), suggesting a founder effect in MJ (16). The equivalent of Arg448 is found in every known eukaryotic P450, and the R448H mutation was confirmed to interfere with enzymatic activity (8). Thus, these studies defined an ethnic group at risk, with a proposed carrier rate of approximately one in 40, for whom a single mutation accounts for most of the disease alleles. This estimate for carrier rate of 11-OHD and the fact that a single mutation has been described for the well-defined subgroup of MJ may merit the inclusion of this mutation within a genetic screening program in MJ.
To estimate the carrier rate for the R448H mutation in the healthy MJ population in Israel, we screened 236 healthy MJ and studied 11 MJ patients with 11-OHD.
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Subjects and Methods |
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Study subjects (n = 242) were recruited from MJ individuals, referred for various genetic screening tests. Subjects were either self-referred or referred by their obstetrician as part of a routine prenatal or preconception care, or were referred for molecular diagnosis of familial Mediterranean fever, which is common in MJ. None of the subjects reportedly had a family history of CAH. To the best of our knowledge, subjects were not related to one another. MJ CAH patients carried a clinical and biochemical diagnosis of 21-OHD or 11-OHD (n = 13). Subjects were defined as MJ, according to self-testimony, only when both parents were of MJ descent, except for one 11-OHD CAH patient whose parents were of Moroccan/Iraqi descent. Informed consent was obtained from all subjects studied or their guardians.
Mutation analysis
Genomic DNA was prepared from whole blood collected in EDTA, according to a standard salting-out procedure (18), or using the Puregene kit (Gentra, Minneapolis, MN). Exon 8 was amplified by PCR with primers 11-OH forward 1 (5'-gcc ctt tgg ctt tgg cgt g, mismatched nucleotide is underlined) and 11-OH reverse 1 (5'-gag gcc agt ccc aca ttg ct). The position of the mismatched nucleotide corresponds to codon 447, and primer 11-OH forward 1 does not extend into codon 448. PCR was performed in 1.5 mM Mg, using 1 U Taq polymerase (Sigma-Aldrich Corp., Rehovot, Israel) and buffer supplied by the manufacturer. Reactions were performed in a thermocycler (Biometra, Niedersachsen, Germany) under the following conditions: initial denaturation at 94 C for 4 min, then 35 cycles of 94 C denaturing for 30 sec, 62 C annealing for 30 sec, 72 C extension for 30 sec, and a final step of 10 min at 72 C. For R448H mutation analysis, the PCR product was digested with ApaLI (New England Biolabs, Beverly, MA) at 37 C and analyzed on a 3% agarose gel. For detection of other mutations in the R448 codon, the same amplified fragment was subjected to digestion with the enzyme HhaI (New England Biolabs, Minneapolis, MN) at 37 C and analyzed on a 10% polyacrylamide gel.
For sequence analysis, PCR-amplified exon 8 fragments were enzymatically purified by a sequential incubation of 15 min at 37 C and heat inactivation at 80 C with exonuclease 1, followed by incubation with shrimp alkaline phosphatase (U.S. Biochemical Corp., Cleveland, OH). Purified fragments were sequenced with the BigDye terminator cycle-sequencing kit, on an ABI PRISM 310 sequencer according to standard procedures as recommended by the manufacturer (Applied Biosystems, Foster City, CA).
Mutation analysis of the CYP21 gene in patients with 21-OHD included P30L, I2 splice, V281L, E6 cluster, Q318, exon 3 del 8bp, I172A, as previously described (19), and the F306 + 1nt frameshift mutation, analyzed by the laboratory of Dr. I. Morel (Hôpital Debrousse, Lyon, France).
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Results |
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A diagnostic test based on PCR and restriction enzyme analysis was developed for specific detection of R448H. The test relies on a mismatched primer designed to introduce a novel ApaI restriction enzyme site when the R448H mutation is present and a primer that distinguishes CYP11B1 from CYP11B2 (Fig. 1
). This test is specific for the R448H mutation and will not detect other changes in the R448 codon. Sequencing-confirmed homozygote or heterozygote R448H samples were used as controls in each set of reactions. A population screening of 236 healthy MJ subjects for R448H yielded two carriers (0.85%), compared with one of 40 (2.5%) in the previous report (20).
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The patient was diagnosed, at the age of 1 yr, with CAH attributable to 11ß-OHD. She was the full-term product of a normal pregnancy. The parents were unrelated; the father was of MJ origin, and the mother was from an Iraqi-Jewish family. Referred for precocious thelarche, she was found to have hyperpigmentation, clitoris enlargement, and posterior fusion of the labia minor. She had Tanner stage 2 pubic hair and breasts of 2-cm diameter. The bone age was advanced to 21 months. Basal and ACTH-stimulated serum cortisol levels were normal at 378 and 591 nmol/liter, respectively. Basal and ACTH-stimulated serum 11-deoxycortisol levels were 434 and 462 nmol/liter, respectively (normal, <30 nmol/liter). Basal and ACTH-stimulated serum 17-hydroxyprogesterone levels were 19 and 42 nmol/liter, respectively (normal, <10 and 30 nmol/liter, respectively).
One allele with the common MJ R448H mutation was detected in this patient, but not in her mother’s DNA sample (Fig. 1
; family B). Because codon 448 was reported previously to harbor another mutation and was suggested to be a mutation hot spot, we analyzed this codon by HhaI digestion for additional mutations (Fig. 2A). Any mutation in codon 448 will destroy the endogenous HhaI site present in the wild-type allele. The presence of the 34-bp product in the mother, who did not harbor the R448H mutation, indicated another mutation in codon 448. This mutation was confirmed by direct sequencing to be C>T, leading to R448C (Fig. 2B, antisense strand sequence is depicted) and was confirmed in the patient. R448C is a loss of function mutation (9), and its combination with R448H in this patient fully accounts for the deficiency phenotype. We did not detect any other R448C carriers among 242 Israeli MJ.
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We also studied 13 unrelated MJ patients referred for CAH with a clinical and biochemical diagnosis of 21-OHD. We reasoned that in MJ patients, some might be incorrectly diagnosed, or they may carry a heterozygote mutation. Two R448H heterozygotes were identified in this group; one was homozygous for the I2 splice mutation in CYP21, and the other was homozygous for an F306 + 1nt frameshift mutation.
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Discussion |
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The present report describes a second mutation in CYP11B1, R448C, in an MJ/Iraqi patient with 11-OHD. This mutation has been reported previously in a non-Jewish, Iranian patient (9) and was shown to abolish enzyme activity. However, this mutation was not observed in any MJ chromosome in our sample and therefore should be regarded as a private mutation in this family. This finding supports the idea of codon R448 as a mutation hot spot.
Although the carrier frequency reported here for R448H, does not justify a broad recommendation for screening MJ families, meticulous genetic counseling of such couples, aimed at identifying a family history of CAH, is advisable. Couples at risk and suspected CAH patients of MJ descent can be screened for the common R448H mutation. When this mutation is not present, the presence of other mutations in clinically diagnosed patients should be explored.
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Footnotes |
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First Published Online July 19, 2005
Abbreviations: CAH, Congenital adrenal hyperplasia; MJ, Moroccan Jew; 11-OHD, 11ß-hydroxylase deficiency.
Received May 20, 2005.
Accepted June 20, 2005.
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