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Original Studies |
Department of Biochemistry, Victoria University of Wellington, School of Biological Sciences (J.F., D.J.D.), Wellington, New Zealand; the Department of Pediatrics, North Shore University Hospital, New York University School of Medicine (N.D., R.P., P.W.S.), Manhasset, New York 11030; the National Testing Center (D.W.), Auckland, New Zealand; and the Division of Endocrinology, University Children’s Hospital (T.T.), Zurich, Switzerland
Address all correspondence and requests for reprints to: Phyllis W. Speiser, M.D., Division of Pediatric Endocrinology and Metabolism, North Shore University Hospital, 300 Community Drive, Manhasset, New York 11030. E-mail: speiser{at}nshs.edu
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Abstract |
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CYP21 genotyping indicated a heterozygote rate of 2.8% for classic mutations (excluding CYP21 deletions), and 2.0% for nonclassic mutations in New Zealanders. Ten full-term affected neonates showed a wide range of 17-OHP levels (15–1400 nmol/L). Sick or preterm infants or infants screened on the first day of life with high 17-OHP proved genetically unaffected. Genetic linkage disequilibrium was found between two CYP21 mutations and chromosome 6p markers.
Guthrie cards can be used to accurately genotype CYP21 and other relevant markers, potentially enhancing the specificity and sensitivity of congenital adrenal hyperplasia screening. CYP21 heterozygote frequency for classic mutations is higher than expected based on genotype compared with that predicted by hormonal newborn screening.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because CAH is potentially fatal, and there is a simple, cheap test available [immunoassay of 17-hydroxyprogesterone (17-OHP)], it fits the international criteria for a suitable condition for screening, and several programs include CAH in the newborn test battery (2, 11). The test sample is usually dried blood stored on filter paper (Guthrie cards) (12). The specificity of the test varies between 97% after a single test and 99.8% after a second test, depending on the assay and the protocol used for screening (11). False positives are due in large part to the variations in normal levels of 17-OHP with gestational age and to the increase in cortisol production, and hence 17-OHP, in sick or stressed infants. Repeating hormonal testing in infant screening programs involves significant financial costs to the health care system and emotional costs to the families involved. Addition of cystic fibrosis genotyping (from the original blood sample) after a positive biochemical screening test has been quite successful in reducing the false positive rate (13). A similar rapid and robust test for the CYP21 genotype would be a very useful addition to CAH-screening programs.
In this report we demonstrate that Guthrie cards can be used to accurately genotype CYP21. Genotyping could thus eliminate the need for obtaining a second blood sample for 17-OHP determination, expediting accurate diagnosis and avoiding unnecessary treatment among infants subjected to newborn CAH screening. Multiplexed analyses can also be performed for other genes, incorporating sex chromosome markers and markers linked to CYP21 on chromosome 6p.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All New Zealand samples were obtained from the National Testing Center, with the approval of the Wellington human ethics committee, and consisted of a 3-mm diameter disc (as used for hormonal testing) punched out of a dried blood spot stored on filter paper (no. 903, Schleicher & Schuell, Inc., Keene, NH). Samples were randomly selected in batches of between 50–100 during an 8-month period in 1996–1997. Blood samples had been obtained at least 1 month and as long as 6 months before genotyping and represented surplus material from the newborn screening program. The 603 samples were chosen blind to any identifiers, including hormonal data, and were thus representative of the ethnic composition of the New Zealand population (Caucasian, 78.1%; Maori, 13.5%; Pacific Islands, 4.6%; Asian, 3.8%; data taken from 1996 census, http://www.stats.govt.nz). The sexes of the infants from whom samples were derived were recorded.
The remaining 50 samples were from infants predominantly of Caucasian origin, either Swiss or North American. Twenty-one of these latter samples were sent for genotyping because of abnormally high 17-OHP levels and/or unusual clinical features. The remaining 29 samples were collected from infants in whom CAH was not suspected, but for whom hormonal measurements were available. Immunoassays for 17-OHP (14, 15) were performed using different methods at several different state-run laboratories, and thus a single set of assay parameters cannot be given. The upper normal serum equivalent levels for blood filter paper 17-OHP ranged from 30–40 nmol/L, depending on the particular screening program’s assay.
DNA extraction
Genomic DNA was extracted from a filter paper blood spot of 3-mm diameter (equivalent to 2.2 µL blood). The disc was lysed in 200 µL hemolysis buffer (20 mmol/L Tris-HCl, pH 8.0, containing 100 mmol/L NaCl, 1 mmol/L ethylenediamine tetraacetate, and 0.2% Triton X-100) for approximately 2 h at room temperature. The lysis buffer was replaced, and the disc was lysed for an additional 2 h. The supernatant was then discarded, and the disc was retained. DNA was extracted using Chelex 100 resin (Sigma Chemical Co., St. Louis, MO) essentially as previously described (16). To the lysed disc was added 75 µL 6% Chelex 100, and the DNA was extracted by incubation at 37 C for 15 min, at 56 C for 10 min, and at 99 C for 10 min. The sample was centrifuged at 6000 x g for 2 min, and the supernatant was stored at -20 C before use in PCR.
Gene-specific PCR amplification
The CYP21 gene was PCR amplified as two overlapping fragments
essentially as described by Day et al. (8), as shown in Fig. 1
. Amplifications were performed in a
total volume of 25 µL using 7.5-µL aliquots of Chelex-extracted DNA
and 1.5 U polymerase mix from the Expand Long Template PCR System
(Boehringer Mannheim, Indianapolis, IN). Amplification was achieved
using 40 cycles of 94 C for 30 s, 70 C for 30 s, and 72 C for
4 min. A final 5-min extension at 72 C completed the amplification.
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Detection of nine steroid 21-hydroxylase alleles (positions of
specific mutations shown in Fig. 1B
) was performed as previously
described (8), except that two overlapping gene-specific PCR products
were used as template. Proline 453 to serine (P453S), a relatively
uncommon mutation, was not screened in the present study. Fluorescently
labeled products were detected using an ABI 377 DNA sequencer (Applied
Biosystems, Foster City, CA) running Genescan version 2.1
software (Applied Biosystems).
Sequencing of PCR products
Confirmation of LDR typing of individuals carrying CYP21 mutations was achieved by automated fluorescent sequencing of PCR product using an ABI 377 automated fluorescent sequencer according to the manufacturer’s instructions.
Microsatellite markers
DNA markers tightly linked to CYP21 and human leukocyte antigen
(HLA) genes have been used to identify affected members of CAH families
(17, 18). It was of interest to determine whether any microsatellite
haplotypes were in genetic disequilibrium with the different forms of
21-OHD as has been found with the HLA haplotypes (19, 20) and to
determine the frequency of the microsatellite alleles in our
population. Six polymorphic microsatellite loci that flank the CYP21
gene (Fig. 1A) and the X-Y dimorphic amelogenin sex-determining gene
(21) were amplified (17) in a multiplexed PCR reaction separate from
the CYP21 amplification. PCR conditions were modified slightly to
include 1.75 mmol/L MgCl2 and 0.2 µmol/L for each of the
14 respective primers (7 primer sets). DNA was first denatured at 94 C
for 3 min. Amplification was achieved with 30 cycles of 95 C for
30 s, 60 C for 30 s, and 72 C for 30 s, with a final
extension at 72 C for 5 min. Suitably diluted product was analyzed
using an ABI 377 DNA sequencer running Genescan version 2.1
software.
The microsatellite designation (Table 1)
is given as the number of repeat elements in the PCR products for
D6S299 (22), tumor necrosis factor-
(TNF) (23), and D6S273 (22).
These were determined by sequencing homozygous individuals to calibrate
mobility on the gel. For the remaining three microsatellite loci,
D6S1663 (22), Tap1 (24), and D6S291 (22), the exact repeat number is
not known; instead, PCR product size is given.
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Comparison of expected and observed carrier frequencies for CYP21 mutations was performed using the delta test, as the expected carrier rate was derived from disease incidence using the Hardy-Weinberg equation.
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Results |
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The Guthrie card extraction method enabled genotyping of neonates
from very small samples of dried blood, including specimens aged up to
6 months. Results could be obtained within approximately 14 h of
receiving the sample, with sufficient DNA remaining to allow a number
of repeat analyses if necessary. Among the 603 New Zealand neonates
analyzed, 17 (2.8%) individuals were carriers of 1 or more classic
mutant 21-OHD alleles; 12 (2.0%) carried a nonclassic mutation (Table 1). DNA sequencing confirmed the accuracy of LDR typing in all cases.
For the 5 individuals with more than 1 mutation (subjects NZ 9–13,
Table 1), the mutant alleles were identified as being in cis
(i.e. on the same chromosome) by selective amplification of
the mutant and normal genes due to mutation of the PCR priming site on
the affected haplotype only (8). Thus, in these 5 heterozygous
individuals, their several mutations were part of a large gene
conversion (25) contiguous on one chromosome. None of the randomly
selected New Zealand infants was identified as a CAH patient with CYP21
mutations on both chromosomes.
Each mutant haplotype detected was designated either classic (salt
wasting or simple virilizing) or nonclassic (7). CYP21 mutations
associated with the classic form of CAH and screened in this study
include the splice mutation at nucleotide (nt) 656, where G is
substituted for A (nt 656 A/G indicates heterozygosity for this
particular mutant allele, and nt 656 G/G indicates homozygosity);
deletion of 8 bp in the third exon (8 bp); isoleucine at codon 172
substituted for by asparagine (I172N, most often associated with simple
virilizing CAH, indicates heterozygosity for this mutation); a cluster
of mutations in exon 6; a single base (cluster); T insertion in exon 7
(Phe plus T); glutamine at codon 318 substituted for by a stop codon
(Q318X was the single most commonly identified heterozygous mutation
among New Zealanders); and arginine at codon 356 substituted for by
tryptophan (R356W). These mutations represent so-called gene
conversions, i.e. transfer of segments of deleterious
sequence from the CYP21 pseudogene to the active CYP21. The presence of
several mutations on the same chromosome is designated gene conversion
(GC) in Table 1. Most of these mutations ablate 21-hydroxylase
activity. Mutations screened for and known to be associated with
nonclassic CAH included the less deleterious substitutions of leucine
for proline or valine, respectively, at codons 30 (proline 30 to
leucine, P30L) or 281 (valine 218 to leucine V281L). Thus,
approximately 4.8% (1 in 21) New Zealanders were found to be carriers
of mutations associated with all forms of CAH, of which 2.8% (1 in 36)
carried classic salt-wasting or simple virilizing mutations, and 2.0%
(1 in 50) carried nonclassic mutations. The only nonclassic mutation
detected in the heterozygous state in the New Zealand population was
V281L.
Statistical comparison of our genetic heterozygote detection rate for classic CAH (1 in 36) with those calculated from hormonal screening studies (1 in 63 worldwide to 1 in 77 for New Zealand) (3, 11) indicates that they are significantly different.
Microsatellite linkage analysis
We genotyped the 29 individuals identified as carriers of CYP21
mutations and 51 CYP21 wild-type New Zealand infants for comparison at
the designated 6 loci on chromosome 6p flanking CYP21 (Fig. 1A and
Table 1). The microsatellite allele frequencies for the normal subjects
were similar to those determined for Northern European and Danish
populations (26, 27). The classic CYP21 allele Q318X was in genetic
linkage disequilibrium with 1 specific upstream microsatellite
haplotype (TNF-5; D6S273–7), a combination present in only 1 of 53
infants who did not carry a CYP21 mutation. D6S299–5 was associated
with this same haplotype in 4 of 8 CAH heterozygotes. The nonclassic
CYP21 allele V281L appeared in all 12 carriers in genetic linkage
disequilibrium with a separate upstream microsatellite haplotype
(TNF-2; D6S273–5), a combination found in only 4 of 50 control
infants. D6S299–1 was associated with the nonclassic mutation in 50%
of cases. There were no other microsatellite haplotypes that appeared
distinctively in the CAH heterozygotes compared with the normal
controls. Sex determination by amelogenin analysis agreed with
clinically assigned sex in all cases; there was no sex bias
detected.
Hormonal-genetic correlations
Among 50 samples of Swiss or North American origin, 21 were sent
for genotyping because of abnormal 17-OHP levels detected by newborn
screening. Among these infants, 10 proved to be affected (Table 2); genotype was in agreement with the
ultimate clinical diagnosis in each case. There were a total of 8
classically affected CAH patients (3 African-American and 5 Caucasian)
and two nonclassic Caucasians. Among all affected infants, serum
equivalent 17-OHP levels ranged from a normal low value of 15 nmol/L
for the first screen to a high of 1400 nmol/L, characteristic of severe
CAH. Of the 2 infants with normal 17-OHP levels, 1 carried the L30
nonclassic allele (patient 6). The other infant with low basal 17-OHP
on days 1 and 6 of life had a classic CYP21 null genotype (patient 8);
ACTH stimulation caused a rise in 17-OHP to approximately 600 nmol/L,
confirming the diagnosis of 21-OHD. Two of the 5 affected female
infants with classic CAH mutations had clitoromegaly, but not complete
genital ambiguity. Three males showed signs of hyponatremia and
hyperkalemia characteristic of salt wasting. Note that 1 of the 2
nonclassic infants was detected in the newborn screen with high 17-OHP
levels of 65.7 and 75 nmol/L during the neonatal period; this male
infant was full term and healthy.
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. Genotypes
designated homozygous could also represent compound heterozygotes with
deletions (e.g. intron 2, nt 656 G/intron 2, nt 656 G might
actually be intron 2, nt 656 G/deletion), as PCR-based methods
generally do not identify heterozygous deletions. Homozygous CYP21
deletions would be identified based on failure to detect any
gene-specific PCR product with normal amplification of CYP21 in control
samples and normal amplification of flanking loci in the same
individual. No individuals with homozygous CYP21 deletion were
identified in this study, and they presumably represent 4% or fewer of
CAH patients (7).
Among 50 infants for whom both 17-OHP measurement and CYP21 genotype
data were available, 2 full-term infants who were tested while in the
neonatal intensive care unit showed initially moderately high 17-OHP
levels, but no mutations (patients 11 and 12; Table 2). Two other
full-term newborns screened on the first day of life showed rather
markedly elevated 17-OHP levels (patients 13 and 14). Seven preterm
infants showed a broad range of elevated 17-OHP levels (from 37.4–183
nmol/L). All of these proved to have normal genotypes. Three other
full-term infants with normal 17-OHP levels were heterozygous for CYP21
mutations (data not shown for full-term wild-type and heterozygous
infants with normal 17-OHP levels).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All manipulations were performed manually in this study; however, the DNA isolation, gene-specific PCR amplification, and ligase detection reaction are simple pipetting steps that can be readily performed by an automated pipetting robot, allowing a high throughput of samples. Polyacrylamide gels used for analyzing LDR products (36 samples at a time) were often reused (in some instances 3 or 4 times) after the previous products had been analyzed, without loss of resolution. This significantly increased the number of samples that could be processed in a day and reduced the lag time to obtaining results. We estimate that the reagents’ cost for each sample analyzed was about $5 U.S. ($10 New Zealand); this figure could be significantly reduced if the procedure were automated. Also, more rapid data analysis is possible if LDR fragments are sized by capillary electrophoresis (31) or MALDI TOF (matrix assisted laser desorption ionization time of flight) mass spectrophotometry (32) rather than by using polyacrylamide gels. MALDI TOF mass spectroscopy is a technique for obtaining rapid and accurate mass and size determinations on biological samples such as DNA (33).
Lee et al. (34) described direct molecular diagnosis of CYP21 mutations in which the whole CYP21 gene is amplified in one fragment using a CYP21-specific forward primer that anneals about 100 bp upstream of the TATA box and a CYP21-specific reverse primer that anneals about 20 bp upstream from the polyadenylation signal. Although this approach may seem simpler than amplification of two overlapping gene fragments, any chimeric CYP21/CYP21P (pseudogene) or CYP21P/CYP21 (17% in this study) genes will not be amplified if the gene conversions extends to the PCR priming sites. Thus, allele dropout in such an individual would not be detected. Indeed, we have previously reported the occurrence of allele dropout as a potential pitfall in PCR-based diagnosis of CAH, probably accounting for many cases of so-called asymptomatic homozygotes for the intron 2 splice mutation, nt 656G found in CAH pedigrees (17, 35). Allele dropout may have significant implications for PCR-based genetic analysis and is the principal reason for employing confirmatory microsatellite typing in the setting of pedigree analysis, especially when used for diagnostic or forensic purposes.
This is the largest study of CYP21 genotypes among randomly selected individuals, as opposed to known CAH families. Owerbach et al. (36) genotyped 81 randomly selected Texan blood donors at nt 656 and identified a single carrier (nt 656 G). Stochastic variation makes it impossible to draw significant conclusions from such a small sample size. In this much larger sample, we found a 2.8% carrier rate for all classic CYP21 mutations and a 4.8% carrier rate if nonclassic alleles were included. This study, however, was unable to distinguish hemizygous individuals (i.e. heterozygous deletions) from normal individuals with two copies of CYP21, so the true carrier frequency for classic 21-OHD is probably even higher than that estimated in this study. DNA analysis also identifies CAH males with unusually low initial 17-OHP levels (e.g. samples 3 and 10) who may manifest delayed onset of salt wasting (37).
Our data demonstrate that carriers of classic CAH genes are more prevalent in New Zealand and probably more prevalent in the worldwide population than hitherto estimated. We speculate that the higher genetic detection rate for classic heterozygotes compared with that predicted by clinical and hormonal case detection might be due to incomplete ascertainment by the latter method. Thus, infants who escaped clinical detection, either because of low initial 17OHP levels or because of death before diagnosis, would not be included in the earlier case detection reports; therefore, an erroneously low heterozygote rate would be calculated. Alternatively, one could postulate that haplotypes bearing CYP21 mutations, when present in the homozygous state, are partial embryonic lethals. Fetal deaths early in gestation could also account for missing affected haplotypes in population screening.
The classic mutant CYP21 allele frequencies for the non-New Zealand samples in this study were similar to those observed in other studies (9, 10, 38, 39, 40), with nt 656G being the most prevalent. In contrast, in the New Zealand population, a termination codon at position 318 was the most prevalent, followed by multiple contiguous gene conversions. The relatively high incidence of heterozygosity for CYP21 mutations in the New Zealand population is unlikely to be due to Maori or Pacific Island people, because the New Zealand population is mainly Caucasian (78%). Our microsatellite analysis suggests that founder effects may have influenced the frequencies of the various CYP21 mutant alleles.
This study represents the largest unbiased population screening to date for nonclassic CYP21 mutations. Our random genetic screening of New Zealanders shows a 2% carrier rate for V281L, which is in good agreement with the calculated estimate of 1 in 17 in a mixed ethnic population based on pedigree analysis, ACTH stimulation testing, and HLA linkage (4). Further, we have demonstrated linkage disequilibrium between the Q318X and V281L mutations and flanking microsatellite markers not previously been reported.
Genetic analysis allowed unequivocal identification of individuals affected with mild, nonclassic CAH who were phenotypically normal at birth. Interestingly, one of these infants had a normal screening 17-OHP level, and another had an elevated hormonal level. Both were compound heterozygotes for a mild (P30L) and severe (either intron 2, nt 656 A to G or Q318X). The fact that the girl with nonclassic CAH presented in later childhood with clitoromegaly is consistent with earlier observations about nonclassic P30L-carrying patients manifesting more signs of virilization compared with the more common nonclassic allele, V281L (41). Nonclassic CAH is most often diagnosed in children with precocious pubarche and in girls and young women with symptoms of moderate androgen excess, such as hirsutism and oligo- or amenorrhea. It is appropriate to administer glucocorticoid replacement therapy only in children showing physical signs of sex steroid excess. Early genetic identification and prospective careful monitoring of such affected individuals, however, could lead to better anticipatory management and reduced morbidities, particularly for the affected females. In contrast, genetic identification of nonclassic males might prevent long term unnecessary steroid treatment.
The accuracy of hormonal screening can be enhanced by performing a second neonatal screen. Therell et al. (42) found that 14% of all classic CAH and 87% of nonclassic infants were missed on the first screen (often performed during the first day of life) and were detected by later 17-OHP measurements at 1–2 weeks of life. Routine screening by genetic analysis could obviate performing a second routine hormonal screening for CAH and the additional waiting intervals involved. Although DNA testing of all newborns is not currently practical for economic reasons, this may change as the cost of molecular biology reagents decrease, and multiplexed analysis of other loci becomes possible.
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Acknowledgments |
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Footnotes |
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Received January 12, 1999.
Revised February 23, 1999.
Accepted February 23, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxyprogesterone
radioimmunoassay: its application for the rapid screening for
congenital adrenal hyperplasia. J Clin Endocrinol Metab. 50:572–577.-hydroxyprogesterone
radioimmunoassay: its application for rapid screening for congenital
adrenal hyperplasia. J Clin Endocrinol Metab. 45:1003–1008.This article has been cited by other articles:
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