Aberrant splicing and missense mutations cause steroid 21-hydroxylase [P-450(C21)] deficiency in humans: possible gene conversion products.

Higashi, Youichirou; Tanae, Ayako; Inoue, Hiroo; Hiromasa, Takako; Fujii‐Kuriyama, Yoshiaki

doi:10.1073/pnas.85.20.7486

Cited by 215 publications

(110 citation statements)

References 22 publications

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“…The effect for only one of these mutations (656 A/C<G) was proven from mRNA studies (Higashi et al 1988). By way of differential amplification of the CYP21 gene, we have established a reliable method to analyze the effect of mutations occurring at either splicing donor or acceptor sites of the CYP21 gene.…”

Section: Discussionmentioning

confidence: 99%

“…Four splicing site mutations in the CYP21 gene have been reported so far (Higashi et al 1988, Wedell & Luthman 1993, Lajic & Wedell 1996, Lee et al 1998. The effect for only one of these mutations (656 A/C<G) was proven from mRNA studies (Higashi et al 1988).…”

Section: Discussionmentioning

confidence: 99%

“…Finally, a common 655A/C<G mutation within intron 2 has been reported in CAH patients (Higashi et al 1988). S1 mapping and COS-7 cells transfection experiments have revealed that this mutation causes the introduction of an upstream acceptor site resulting in the synthesis of a truncated or an abnormal protein (Higashi et al 1988). In addition, transversion mutations at the splicing junction in intron 1 and intron 7 of the CYP21 gene have been reported in CAH patients (Wedell & Luthman 1993, Lajic & Wedell 1996.…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Multiple transcripts of the CYP21 gene are generated by the mutation of the splicing donor site in intron 2 from GT to AT in 21-hydroxylase deficiency

Hh¹,

Sf²

2001

Journal of Endocrinology

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Maturation of primary RNA transcripts of eukaryotic genes often involves the removal of introns and joining of exons. The fidelity of RNA splicing is dependent on the identity of the nucleotide (nt) sequences at exon/intron boundaries. Most importantly, the highly conserved intronic 5 GT and 3 AG sequences are essential for correct splicing. Substitution of GT by any other nt leads to incomplete mRNA and a disruption of protein structure. We describe here the results of our transfection experiments in COS-1 cells with a CYP21 genomic construct that contained an IVS 2+1G

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Multiple transcripts of the CYP21 gene are generated by the mutation of the splicing donor site in intron 2 from GT to AT in 21-hydroxylase deficiency

Hh¹,

Sf²

2001

Journal of Endocrinology

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“…The P-450 molecules expressed in COScells exhibited their correct cellular localizations and functional activities, although their activities seemed suboptimal. Our laboratory has reported a successful application of this system to the analysis of defective genes in congenital P-450c21 deficiency (4).…”

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confidence: 99%

Functional expression of microsomal and mitochondrial cytochrome P-450 (d and SCC) in COS-7 cells from cloned cDNA.

Minowa

Sogawa

Higashi

et al. 1990

Cell Struct. Funct.

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ABSTRACT. Using full length CDNAintroduced into COS-7 cells, two species of P-450 with entirely different physiological functions have been expressed in enzymatically active form. One is P-450d, which is known to reside in the microsomes of rat hepatocytes where it acts as a drug-metabolizing enzyme; the other is P-450(SCC), which catalyzes the conversion of cholesterol to pregnenolone in the rate-limiting reaction of steroidogenesis in mitochondria of adrenal cortex cells. Northern blot and immunoblot analyses revealed that the mRNA and protein of these P-450 species were efficiently produced in COS-7cells. The protein contents amounted to nearly 0.1% of the total cell protein as estimated from immunoblotting and low temperature CO difference spectra. The subcellular localization of the products indicated that they were correctly sorted to the microsomes and mitochondria, respectively. Wehave succeeded in eliciting most of the activity of the expressed microsomal P-450d by reconstruction with NADPH-cytochromeP-450 reductase, while the optimal conditions for the mitochondrial enzyme in the COScells remain to be studied. These results show the applicability of the COS-7expression system to investigations of the functions of members of the P-450 super family whose CDNAhas been newly isolated.Cytochrome P-450 constitutes a super family of hemoproteins which are believed to have evolved from a commonancestor. They catalyze the monooxygenase reaction of xenobiotics, such as drugs, carcinogens and environmental pollutants, as well as endobiotics, such as steroids, fatty acids, and prostaglandins (1, 2). This versatility of the enzyme system is probably due to the large number of P-450 species and their different and sometimes broad substrate specificities. The multiple molecular forms of P-450 have several properties in common,such as size, structure, and immunologicand biochemical characteristics, which makers it difficult to separate one form from another biochemically. Recent application of molecular cloning technology to the field of P-450 has overcome this difficulty and brought about clear-cut identification of many forms of P-450 (2, 3).However, there seem to remain a considerable number of uncharacterized P-450 species, especially the minor forms in higher organisms. Whena CDNA clone for a certain species of P-450 whose function is not knownis isolated by cross-hydridization with the CDNA of a * To whom correspondence and reprint requests should be addressed.known P-450, it is prerequisite for characterization of the newspecies to establish a suitable expression system for the CDNA information. The expression system may also be helpful for investigating the structure-function relationship in P-450 molecules by site-directed mutagenesis and for analyzing causative mutations in defective genes of genetic deseases related to the P-450 superfamily.In the present study we attempted to express two P- molecules expressed in COScells exhibited their correct cellular localizations and functional activities, although th...

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Who is a carrier? Detection of unsuspected mutations in 21-hydroxylase deficiency

1996

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Congenital adrenal hyperplasia due to 21‐hydroxylase deficiency is a common autosomal‐recessive disorder. During our routine genotyping of affected individuals and their relatives using allele‐specific oligonucleotide hybridization and single‐strand conformational polymorphism analysis, we identified two families each segregating three mutations. In both families, a mutation known to be associated with 21‐hydroxylase deficiency was identified in healthy individuals but was not detected in the propositus. The propositus in family 1 was shown to be a homozygous carrier for G at nucleotide 655, which alters the splice acceptor site at exon 3. The propositus in family 2 carried the same splicing mutation on the maternal allele and a gene deletion/conversion on the paternal allele. In both families, other clinically unaffected relatives carried the Q318X mutation in exon 8. If molecular diagnostic studies had been limited to the mutation carried by the propositi, relatives would have been misinformed regarding their status as carriers or mildly affected individuals. The findings in these two families emphasize the high frequency of alleles causing 21‐hydroxylase deficiency in the population. © 1996 Wiley‐Liss, Inc.

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Aberrant splicing and missense mutations cause steroid 21-hydroxylase [P-450(C21)] deficiency in humans: possible gene conversion products.

Cited by 215 publications

References 22 publications

Multiple transcripts of the CYP21 gene are generated by the mutation of the splicing donor site in intron 2 from GT to AT in 21-hydroxylase deficiency

Multiple transcripts of the CYP21 gene are generated by the mutation of the splicing donor site in intron 2 from GT to AT in 21-hydroxylase deficiency

Functional expression of microsomal and mitochondrial cytochrome P-450 (d and SCC) in COS-7 cells from cloned cDNA.

Who is a carrier? Detection of unsuspected mutations in 21-hydroxylase deficiency

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