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Original Studies |
Department of Medical Genetics (A.A.M., K.F.K., A.M., S.L., L.J.G., N.E.H.), Foresterhill, Aberdeen AB25 2ZD, Scotland, United Kingdom; and Medical Research Council Blood Pressure Group, Department of Medicine and Therapeutics (G.C.I., A.J., J.M.C.C.), Western Infirmary, Glasgow G11 6NT, Scotland, United Kingdom
Address all correspondence and requests for reprints to: A. MacConnachie, Department of Medical Genetics, University Medical Buildings, Foresterhill, Aberdeen AB25 2ZD, Scotland, United Kingdom.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The late stages of aldosterone synthesis are catalyzed by the P450 enzyme aldosterone synthase, which is capable of the 11- and 18-hydroxylation reactions necessary in the synthesis of the steroid. The enzyme is encoded by the gene CYP11B2, whose expression is limited to the zona glomerulosa of the adrenal cortex and is regulated by angiotensin II and K+. In contrast, the final 11ß hydroxylation in the synthesis of cortisol in the zona fasciculata is regulated by 11ß-hydroxylase, which is encoded by the gene CYP11B1. The expression of this gene is principally regulated by ACTH. CYP11B1 and CYP11B2 share a high degree of nucleotide homology (>93% for exons), and this results in the synthesis of proteins with great similarity at the amino acid sequence level. In GRA, the clinical and biochemical abnormalities reflect the regulation of aldosterone synthase activity by ACTH rather than angiotensin II.
In 1992 Lifton et al. (2, 3) described the presence of a chimeric CYP11B1/CYP11B2 gene in patients with GRA. The chimeric gene contains the 5' regulatory sequence of CYP11B1 fused to the 3' structural sequence of CYP11B2, so that the chimeric gene produces a protein with aldosterone synthase activity in response to ACTH. The consequent abnormal expression of aldosterone synthase results in primary aldosteronism, which can be inhibited by glucocorticoids.
In GRA the cross-over region between the CYP11B1 and CYP11B2 genes can occur anywhere from the start of intron 2 to the end of exon 4 of the corresponding genes. Studies with complementary DNA constructs of CYP11B2 show that sequences encoded by exons 3' of exon 5 are necessary for the preservation of aldosterone synthase activity (4, 5). The diagnosis of GRA has depended on identification by Southern blotting of an abnormal fragment in genomic DNA from affected individuals (2). However, this is a time-consuming method that is unsuitable for rapid screening of large numbers of samples.
Recently, a two-tube (externally controlled) long PCR method was described that allowed the diagnosis of GRA in the members of one GRA kindred in Australia (6). We used a similar method to confirm the diagnosis of GRA in the members of five families from northeast Scotland previously identified by Southern blotting. This method was also used to positively identify affected members of five additional GRA families previously described in Glasgow (7).
We also describe here a multiplex PCR protocol that allows the control aldosterone synthase amplification to be carried out in the same tube as the test chimeric gene amplification. This test has proven as reliable as the original two-tube long PCR protocol but has the advantage of using an internal control.
To identify the region of cross-over in each of the 10 kindreds, the chimeric long PCR product was cloned and sequenced. Five cross-over sites were identified ranging from intron 2 to exon 4, indicating the reliability of the method in identifying chimeric genes resulting from different sites of cross-over.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Genomic DNA was extracted from peripheral blood leukocytes using
a commercial kit following the protocol of Jonsson et al.
(6) (Genomix, Hoefer U.K., Newcastle-Under-Lyme, U.K.).
Genomic DNA from family members previously diagnosed by Southern
blotting as having or not having GRA was subjected to amplification.
Primers (Table 1
) for the 5'
untranslated region of CYP11B1 (XL-1a) and a reverse primer hybridizing
to exon 5 of CYP11B2 (XL-2a) were used to amplify the chimeric gene. A
third primer (XL-3) was used in a separate reaction with primer XL-2a
to amplify CYP11B2 and act as a control for the integrity of the long
PCR. For PCR, the Expand High Fidelity DNA polymerase kit (Boehringer
Mannheim, Mannheim, Germany) and 2.6 U of Expand High Fidelity
PCR system enzyme mix was used. Reactions included a hot start at 95 C
for 1 min, continued with 12 cycles of 95 C for 1 min and 68 C for 5
min, followed by 18 cycles of 95 C for 1 min and 68 C for 5 mins with a
30-sec extension per cycle.
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To avoid using a separate tube containing a control reaction, we
designed a further 5' forward primer (XL-4 Table 1
), which was
incorporated into a multiplex primer set including XL-1b and 2b. In our
multiplex long PCR one set of primers (XL-4 and XL-2b) yields a
4.0-kilobase (kb) fragment from the normal CYP11B2 gene, whereas the
other set (XL-1b and XL-2b) yields a 3.9-kb fragment from the chimeric
gene. A single (4.0 kb) product indicates the presence of a normal
aldosterone synthase gene, and the presence of an additional band at
3.9 kb indicates the presence of a chimeric crossover product (Fig. 1). The PCR conditions were identical to
the two-tube assay described above.
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DNA from a GRA-positive representative from each family was
subjected to PCR amplification as described above. The 3.9-kb chimeric
PCR product was separated by excision of the DNA-containing band from
ethidium bromide-stained agarose gel. The isolated DNA was restricted
with 10 U EcoRI (Promega Corp., Madison, WI) at
37 C for 1 h. Restricted DNA was removed from the reaction using
the Qiaex II extraction matrix (Qiagen, Chatsworth, CA)
and cloned into EcoRI linearized pGEM-3Z plasmid. Ligation
was carried out at 4 C for 16 h and half of the ligation reaction
used to transform competent DH5-
cells (Life Technologies, Gaithersburg, MD). Recombinant colonies were
identified using blue/white screening and plasmid extracted using the
Qiagen Plasmid Extraction Kit (Qiagen) following the manufacturer’s
protocols.
Sequence analysis of cloned chimeric PCR product
Sequence analysis was performed using the dideoxy chain termination method (8) and an ABI automated sequencer (ABI 377, Applied Biosystems Inc., Perkin-Elmer, Norwalk, CT). Primers for the chimeric gene in forward and reverse orientations, as well as universal and reverse for the pGEM-3Z vector were used. Cross-over regions were determined by comparison of sequence obtained with the CYP11B1 and CYP11B2 sequences (9).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two-tube long PCR. DNA from members of all five kindreds in northeast Scotland as well as DNA from members of the five kindreds previously described in Glasgow were amplified using the two-tube long PCR protocol. In total, 33 individuals with GRA were found to exhibit a band at 3.9 kb on amplification using the primers specific for the chimeric gene. Twenty members of our five kindreds known not to have GRA (as determined by Southern blotting) did not produce a band when their DNA was amplified with the chimeric gene primers. No false positives were found. Furthermore, all GRA-negative patients produced a normal product from the aldosterone synthase (control) primers, indicating that the negative results were because of the absence of a chimeric gene and not PCR failure.
Multiplex long PCR. We also tested the same positive and negative family members as above using the multiplex, single-tube long PCR method. This method produced a 4.0-kb control CYP11B2 product in the same reaction as the 3.9-kb chimeric band in all known GRA-positive individuals. All confirmed GRA-negative samples produced only the 4.0-kb control product.
Identification of cross-over points in GRA kindreds
Sequencing of cloned chimeric gene amplification product from the
10 available kindreds identified five different areas of cross-over
(Fig. 2). It is impossible to describe
the exact point of cross-over in each case because of the high level of
homology between the CYP11B1 and CYP11B2 genes, so cross-over sites are
allocated to regions of each gene. Kindreds 1, 4, and 5 all have
indistinguishable cross-over regions. These kindreds have a chimeric
gene containing CYP11B1 sequence as far as the end of intron 3
and CYP11B2 sequence from then onwards. Similarly, kindreds 3,
6, 7, and 8 possess indistinguishable chimeric genes comprising exons
1–3 of CYP11B1 and exons 4–9 of CYP11B2. Kindred 2 possesses a
chimeric gene with a cross-over in intron 2. Kindreds 9 and 10 possess
mutant genes in which the cross-over occurs in the middle and end of
exon 4, respectively. [Kindreds 6–10 cross-over data was originally
published by Jamieson et al. (7)].
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Discussion |
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The novel multiplex adaptation was tested in the same way to ensure its reliability. The same known GRA-positive and -negative individuals were tested, and in each case the technique proved both specific and sensitive. This technique has the advantage over the two-tube assay in that the control reaction is incorporated into the same tube as the amplification of the chimeric gene and is a more valid measure of reaction integrity. This assay has proved reliable and is now our method of choice for routine diagnosis of GRA.
Of the five GRA-positive families identified in the northeast of Scotland, three have completely indistinguishable cross-over regions. This suggests that the families share a common ancestor. However a more detailed haplotype analysis would be required to prove this conclusively. The cross-over region identified in kindred 3 is the same as that previously described in three of the GRA families in Glasgow (7). A large proportion of the GRA families in Lifton et al.’s (2, 3) report in the United States were of Irish ancestry; the Irish and Scots have common ancestors in the Celtic races of the 6th century, and it is possible that because of this the chimeric gene linked to GRA occurs from a series of ancient mutations that have a higher incidence in people of Celtic ancestry. The fact that we identified similar cross-over regions in our families would seem to support this theory. It is of interest that kindred 2 had a cross-over region upstream of any previously described in any Scottish family; kindred 2 originated in the Shetland Isles and may be of Nordic rather than Celtic descent. This would again support the theory of a series of founder effects for GRA, because people inheriting the chimeric gene from different populations would have different sites of cross-over.
Received May 6, 1998.
Revised August 24, 1998.
Accepted August 25, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This article has been cited by other articles:
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  M. Hampf, N. T. N. Dao, N. T. Hoan, and R. Bernhardt Unequal Crossing-Over between Aldosterone Synthase and 11{beta}-Hydroxylase Genes Causes Congenital Adrenal Hyperplasia J. Clin. Endocrinol. Metab., September 1, 2001; 86(9): 4445 - 4452. [Abstract] [Full Text] [PDF]
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