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Centro de Biologia Molecular e Engenharia Genética (I.F.L., F.C.S., M.P.D.M.); Departamento de Pediatria/Centro de Investigação em Pediatria (S.H.V.L.-M., G.G.); and Disciplina de Endocrinologia-Faculdade de Ciências Médicas (M.T.M.B.), Universidade Estadual de Campinas, 13083-970 Campinas, São Paulo, Brasil
Address all correspondence and requests for reprints to: Maricilda Palandi de Mello, Ph.D., Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Caixa Postal 6010, CEP 13083-970, Campinas, SP Brasil. E-mail: mmello{at}obelix.unicamp.br
Abstract
In the classical form of 21-hydroxylase deficiency, CYP21- affected genes either carry mutations present in the CYP21P pseudogene (microconversions) or bear a chimeric gene that replaces the active gene as a result of large conversion or deletion mutational events. Previous genotyping of 41 Brazilian patients revealed 64% microconversion, whereas deletions and large gene conversions accounted for up to 21% of the molecular defect. The present paper describes a new mutation disclosed by sequencing an entire gene in which no pseudogene-originated mutation had been found. The patient with the classical form of 21-hydroxylase deficiency is the daughter of a consanguineous marriage, and she is homozygous for a novel frameshift H28+C within exon 1. The mutation causes a stop codon at amino acid 78. Both parents are heterozygous for the mutation as confirmed by allele-specific oligonucleotide PCR. The H28+C is not present in the published CYP21P sequences and is likely to result in an enzyme with no activity.
CONGENITAL ADRENAL HYPERPLASIA (CAH) due to 21-hydroxylase deficiency is one of the most common inborn errors of metabolism. The classical form of 21-hydroxylase deficiency may result in two distinct phenotypes: salt-wasting (SW) and simple virilizing (SV). Cortisol biosynthesis is impaired in both SW and SV forms (1). The main consequence is an increased production of androgens, generally causing ambiguous external genitalia at birth in females, precocious puberty in males, and acceleration of somatic growth in both males and females. The SW form also involves impairment of aldosterone production, causing failure to thrive and dehydration due to salt loss (1).
Gene deletions, gene conversions, and mutations normally present in the pseudogene account for 90–95% of the disease-causing alleles in all populations (1). Therefore, about 5–10% of 21-hydroxylase-deficient alleles do not bear any of the nine most frequent mutations related to the classical form. To date, a total of 52 CYP21 gene mutations have been deposited in the Cardiff Human Gene Mutation Database (2). More than 30 mutations are rare and have been described in specific families with SW- or SV-affected children (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). Although the great majority of rare mutations are considered to be present only in the CYP21 gene, some of them can be found in some CYP21P alleles, according to Wedell and Luthman (15).
In two previous papers (16, 17), the results of deletions, gene conversions, and microconversion analyses involving a total of 41 Brazilian families with children presenting the classical form of 21-hydroxylase deficiency were reported. Twenty-one percent of the affected alleles showed deletion or large gene conversion, 64% carried microconversions, and 15% remained undetermined. This paper describes one novel mutation found in a homozygous patient with no pseudogene-originated mutation.
Materials and Methods
The study was undertaken under an institutionally approved protocol, and informed consent was obtained from all subjects.
Patient
A Caucasian girl was delivered normally after the uneventful pregnancy of a 33-yr-old mother. Parents are first-degree cousins, and both are healthy. CAH was diagnosed in the patient’s second week of life. She had clitoromegaly, complete fusion of the labioscrotal folds (Prader grade III), no palpable gonads, hyponatremia (Na 126 mEq/liter), and high levels of urinary 17-ketosteroids, and she failed to thrive. Treatment with oral prednisone and fludrocortisone was started. Clitoroplasty and vaginoplasty were performed twice at the ages of 1 and 8 yr. In our first examination, at the age of 7, her bone age was 9 yr of age, according to Greulich and Pyle’s method; she weighed 20.5 kg (z = -0.07), her height was 113.5 cm (z = -0.82), and she had clitoral enlargement (3 cm) with posterior fusion of the labioscrotal folds but no acne or hirsutism. The karyotype was 46,XX. After treatment with prednisone and fludrocortisone, neither dehydration nor abnormal plasmatic renin activity has occurred. She had normal puberty (menarche at 12). At age 14, the patient decided to stop the treatment and went 4 months without medication. During that period she had no clinical symptoms of adrenal insufficiency, but when she returned for treatment she presented dark skin, irregular menses, and high 17-hydroxyprogesterone and androstenedione4 serum levels with normal plasmatic renin activity.
DNA analysis
CYP21 gene was amplified from genomic DNA into
two segments by PCR using selective primers (18) (Table 1
). Internal primers were used in the
sequencing reactions or in nested-PCR before the sequencing procedures
(Table 1). The amplified fragments were treated with the PCR Product
Pre-sequencing Kit (Amersham Pharmacia Biotech, Arlington
Heights, IL) and were directly sequenced with Thermo-Sequenase
Radiolabeled Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech). Sequencing data were compared with the
CYP21 gene sequence described by Higashi et
al. (19) (GenBank Accession no.
M12792).
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G polymorphism/mutation was carried out
as described by Wilson et al. (18), whereas the
ASO-PCR for H28+C mutation was performed using primers described in
Table 1
. The reaction mixture for the latter experiment consisted of: 1
µg genomic DNA, 1x Taq polymerase buffer (Life Technologies, Inc., Gaithersburg, MD), 1.5
mM MgCl2, 0.2
mM dNTP, 1% BSA, 20 pmol of each primer, 2.0 U
Taq DNA polymerase (Life Technologies, Inc.) in
a final volume of 30 µl. PCR steps were: 94 C for 5 min (1 cycle); 12
cycles of 94 C for 1 min, a touchdown annealing step at 72–65 C for 1
min (-0.5 C per step), 72 C for 1 min; 30 cycles of 94 C for 1 min, 65
C for 1 min, 72 C for 1 min; and a final cycle at 72 C for 5 min. Results
In the present study, the 21-hydroxylase disease-causing allele in a Brazilian girl with the classical form of 21-hydroxylase deficiency was analyzed.
Sequencing the entire CYP21 gene (10 exons and 9 introns,
655 nucleotides upstream to the transcription initiation codon
and 469 nucleotides downstream to the 3' end) revealed one single
sequence divergence when compared with the CYP21 gene
sequence published by Higashi et al. (19). The
insertion of one cytosine between nucleotides 82 and 83 in exon 1
modified the second nucleotide of codon 28, which normally codes for a
histidine (Fig. 1A, left sequencing gel).
The insertion of a C at this position changes the amino acid in codon
28 to a proline and causes a reading frame shift from this point on
leading to an in-frame stop codon at amino acid 78 (Fig. 1C).
Sequencing gel showed the patient to be homozygous for the mutation.
The patient’s mother is heterozygous for the mutation, because the
sequencing gel shows double bands for each nucleotide above the point
of the insertion, corresponding to the normal and the mutated allele,
which is one nucleotide longer (Fig. 1A, middle sequencing gel). The
father tested normal (Fig. 1A, right sequencing gel). Because the
father and the mother are first-degree cousins, they should be carriers
of the same mutation. To verify the presence of H28+C in the
disease-causing paternal allele, allele-specific primers for the normal
and mutated sequences were designed (Table 1), and an ASO-PCR was
performed. The correct segregation of the mutation was confirmed for
the whole family (Fig. 1B), indicating the occurrence of an allele
dropout either throughout the first CYP21 selective PCR or
throughout the sequencing procedures in the father-sequenced sample.
Further support for the father to be heterozygous other than normal
homozygous was given by the ASO-PCR analysis of the A/C656G most
frequent CYP21 mutation at nucleotide 656 in intron 2. This
experiment evaluates the occurrence of an A, C, or G nucleotide at the
656 position separately. The analysis of the family showed that the
father is heterozygous A/C, and both the mother and the patient are
homozygous C/C at this position (Fig. 2).
Therefore the C656 variant is associated with the H28+C mutation, and
this was not amplified in the A/C heterozygous father. The C656 allele
dropout in PCR procedures is not uncommon among A/C or G/C heterozygous
individuals (20).
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This paper describes the novel H28+C frameshift mutation found in
a homozygous patient with the classical form of 21-hydroxylase
deficiency. No other sequence divergence was found in the affected
allele. A CYP21 gene PCR allelic dropout artifact
(20) was observed in the study of the family. In
genotyping all individuals of the family for the A/CG nucleotide
variation/mutation at the 656 position, it was observed that the father
is A/C, whereas the mother and the affected child genotyped C/C (Fig. 2). Therefore, the H28+C allele bears the 656C
variant. Because the A/C heterozygous father always seemed to be a
normal homozygous for H28+C mutation in the sequencing gel, it could be
concluded that in this case the 656C variant
carrying the mutation was never amplified when Ex6na-5'21B1s or
Ex6na-Int2s primer pairs were used for the CYP21 gene
selective PCR performed before the sequencing experiments. Thereafter,
the mutation segregation in the family could only be determined by
ASO-PCR for the H28+C mutation (Fig. 2B). Because nucleotides A and G
are purines and C is a pyrimidine, it seems that DNA conformational
effects cause the 656A or
656G-bearing CYP21 allele preferential
amplification when the 656C allele is present in
the same genotype. A similar dropout effect is not observed in the
656A/G genotypes, probably because both
nucleotides are purines. When the PCR was performed with H28+C specific
primer that anneals only in the 656C-bearing
allele, the dropout effect did not occur, and the carrier status of the
father was verified. The observation of dropout artifacts in this study
confirms the data reported before by Day et al.
(20) and reinforces the idea that it is important to
always be aware of this common artifact when looking for sporadic
CYP21 mutations in families with CAH, especially in
procedures that use a selective PCR for the region comprising intron 2
prior sequencing.
The insertion of a C at the beginning of the gene sequence within exon 1 alters the protein structure due to a reading frameshift with the formation of a stop codon in position 78. The resulting protein is completely inactivated because it has 50 amino acid changes in addition to the premature termination of translation at codon 78. This will produce a severely truncated protein lacking most of the important residues for the enzyme activity and stability (21, 22). The mutation was not present in the published CYP21P sequence (19) (GenBank accession no. M12793). In addition, after an ASO-PCR screening, the H28+C mutation was not found in either CYP21 genes of any other patient or the genes of 20 normal controls. Therefore, it consists of a rare allele found only in this family.
Several CYP21 rare missense mutations associated with classical and nonclassical forms of the disease have been described. However, there are only 11 cases of rare CYP21 frameshift mutations reported so far (1, 2). Of those, seven are deletions, two are insertions, and two are combined insertion-deletions. The H28+C described here is the second CYP21 insertional mutation found in exon 1. Ezquieta et al. (23) described the W22+T mutation that consists of a T insertion between nucleotides 64 and 65. Both H28+C and W22+T mutations cause the appearance of a stop codon at amino acid 78.
Although the nucleotide insertion described here is different from the one reported by Ezquieta et al. (23), both might produce similarly truncated proteins leading to severe salt loss symptoms, once both mutations cause a stop codon at the 78 amino acid position. Their patient was a homozygous boy presenting severe SW symptoms 10 d after birth who, with continuous treatment, developed well. Our female patient showed less severe clinical features. She did not experience clinical signs or symptoms of salt loss even after discontinuing the treatment, except failure to thrive in her second week of life. Certainly, the genital ambiguity at birth led to an early diagnosis and treatment, which prevented SW crises.
Another rare mutation, G424S, has been reported in several SV Brazilian patients (24). It was always associated with C4A+CYP21P gene deletions and with the human leukocyte antigen DR17 on the same haplotype, suggesting linkage disequilibrium and representing a probable founder effect for a rare affected allele. The H28+C mutation is the second rare mutation to be reported in a patient with the classical form of 21-hydroxylase deficiency in Brazil, and it seems to be an isolated case because it was not found in any other SV or SW patient.
Acknowledgments
We are grateful to Maria Madalena V. Rosa for technical assistance.
Footnotes
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, São Paulo, SP, Brasil, (proc. n° 97/07622-2). I.F.L. received a personal grant from Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.
Abbreviations: ASO, Allele-specific oligonucleotide; CAH, congenital adrenal hyperplasia; SV, simple virilizing; SW, salt-wasting.
Received June 5, 2001.
Accepted August 31, 2001.
References
This article has been cited by other articles:
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  F. C. Soardi, M. Barbaro, I. F. Lau, S. H. V. Lemos-Marini, M. T. M. Baptista, G. Guerra-Junior, A. Wedell, S. Lajic, and M. P. de Mello Inhibition of CYP21A2 Enzyme Activity Caused by Novel Missense Mutations Identified in Brazilian and Scandinavian Patients J. Clin. Endocrinol. Metab., June 1, 2008; 93(6): 2416 - 2420. [Abstract] [Full Text] [PDF]
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 M. Janner, A. V Pandey, P. E Mullis, and C. E Fluck Clinical and biochemical description of a novel CYP21A2 gene mutation 962_963insA using a new 3D model for the P450c21 protein. Eur. J. Endocrinol., July 1, 2006; 155(1): 143 - 151. [Abstract] [Full Text] [PDF]
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