Nonsense Mutation Causing Steroid 21 -Hydroxylase DeficiencyHadas Globerman, Mounira Amor, Keith L. Parker,* Maria 1. New, and Perrin C. WhiteDivision of Pediatric Endocrinology, Cornell University Medical College, New York, New York 10021; and *Howard Hughes MedicalInstitute and Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710

Abstract

Wedetermined the sequence of a mutant CYP21Bgene iso-lated from a patient with the severe, "salt-wasting" form ofcongenital adrenal hyperplasia due to steroid 21-hydroxylasedeficiency. Codon 318 in this gene is changed from CAG, en-

coding glutamine, to TAG, a nonsense codon. This is predictedto result in a completely nonfunctional enzyme due to prema-

ture termination of translation. In addition, when the clonedmutant gene was transfected into mouse VI adrenal cells, theresulting mRNAlevels were decreased compared with trans-fected normal CYP21Bgenes. This mutation was carried by 3of 20 unrelated patients with 21-hydroxylase deficiency allelesas determined by hybridization with a specific oligonucleotideprobe. This mutation is also seen in the normal CYP21Apseudogone, so that its presence in the abnormal CYP21Bgene may be the result of a gene conversion event.

Introduction

Human 2 1-hydroxylase deficiency is the leading cause of im-paired cortisol synthesis in congenital adrenal hyperplasia (1).The incidence of classical 21-hydroxylase deficiency is 1 in5-10,000 live births; a milder "nonclassical" form of the dis-ease occurs in 0.3% of Caucasians and in 1-3% of EuropeanJews. Deficient cortisol synthesis causes increased secretion ofcorticotropin (ACTH), resulting in hyperplasia of the adrenalcortex, excessive production of androgens, and consequentvirilization. In two-thirds of patients with the classical disease,2 1-hydroxylase deficiency also impairs aldosterone synthesis,resulting in urinary "salt wasting" with the risk of shock anddeath in the neonatal period. Steroid 21-hydroxylase defi-ciency is inherited as an autosomal recessive trait linked to theHLA MHCon chromosome 6. This disorder is caused by a

defect in the structural gene that encodes an adrenal micro-somal cytochrome P-450, which is specific for steroid 21-hy-droxylation (P450c21) (2). There are two 21 -hydroxylasegenes, CYP21A and B (also termed CA21HA and B, or

P45OC21Aand B), respectively, adjacent to the C4A and C4Bgenes, which encode the fourth component of serum comple-ment (3, 4) (Fig. 1). Individuals with homozygous deletions ofCYP21A demonstrate normal cortisol synthesizing capacity,

Address all correspondence to Dr. Perrin C. White, Laboratory ofMolecular Endocrinology, Department of Pediatrics, Rm. N236, Cor-nell University Medical College, 525 East 68th Street, NewYork, NY10021.

Receivedfor publication 10 November 1987 and in revisedform 2February 1988.

whereas homozygous deletion of CYP21Bcauses 2 1-hydroxy-lase deficiency. Nucleotide sequence analysis has confirmedthat the active enzyme is encoded by the CYP21Bgene. TheCYP21A gene is 97% homologous to the B gene, but it isrendered nonfunctional by several critical mutations in thecoding region, including an 8-bp deletion in exon 3, a l-bpinsertion in exon 7, and a nonsense mutation in exon 8 (5, 6)(Fig. 2).

Deletions of CYP21B account for about one-fourth ofclassical 21-hydroxylase deficiency alleles (7, 8). These alleleswere presumably generated by unequal crossing-over duringmeiosis due to the presence of the adjacent CYP21A pseudo-gene. Two nondeleted mutant CYP21Balleles have been char-acterized thus far. One (9) contains an amino acid substitution(Ser-269 to Thr) of unknown functional significance and un-known frequency; the other (10) carries a nonconservativeamino acid substitution (Ile- 172 to Asn) and is found in about15%of patients. This latter mutation is normally present in theCYP21A pseudogene, which suggests that it may have beentransferred to the CYP21Bgene by a smaller recombinationalevent, termed a "gene conversion." This paper presents furtherevidence that gene conversion events are a frequent cause of21 -hydroxylase deficiency alleles: a nonsense mutation nor-mally present in CYP21A was detected in the CYP21Bgenesof several patients. In addition to affecting protein synthesis,this mutation may adversely affect mRNAlevels.

Methods

Enzymes and related reagents were purchased from International Bio-technologies, Inc. (New Haven, CT) and were used according to themanufacturer's instructions.

Southern blot hybridization. DNAsamples were prepared as de-scribed ( 11) from peripheral blood leukocytes of 20 unrelated patientswith classical 21-hydroxylase deficiency (Table I). Samples were di-gested with restriction endonuclease Taq I, subjected to electrophoresisin agarose, and blotted (12) to MSI nylon membranes (Fisher ScientificCo., Springfield, NJ). Blots were hybridized as described (7) to ra-dioactively labeled pC2 1/3c, which contains a nearly full-length cDNAclone encoding human steroid 2 1-hydroxylase (6). Diminished inten-sity of the 3.7-kb Taq I band in certain patients, which suggestedheterozygous deletion of the CYP21Bgene, was confirmed by scan-ning densitometry and by analysis with additional restriction endo-nucleases. Many of these patients were previously studied in thismanner (10).

Construction of a partial genomic library. A patient with severe,salt-wasting 21-hydroxylase deficiency was selected who appeared tohave a heterozygous deletion of CYP21Bon the basis of Southern blothybridization. Any 2 1-hydroxylase enzyme remaining in this patientwould have to be enpoded by the single nondeleted CYP21B gene.DNAfrom this patient was digested with BamHI and was size frac-tionated by electrophoresis in agarose. CYP21A and B genes are bothcarried on 14-kb BamHI fragments (Fig. 1 b); thus, fractions contain-ing DNAof about this size were electroeluted from the gel (13) andassayed for the presence of the CYP21 genes by Southern blotting.Appropriate fractions were ligated to digested XEMBL3arms (Strata-

Nonsense Mutation Causing Steroid 21-Hydroxylase Deficiency 139

J. Clin. Invest.©The American Society for Clinical Investigation, Inc.0021-9738/88/07/0139/06 $2.00Volume 82, July 1988, 139-144

21 C4 21 C4 TNF

OP D0 DR .B B A A BI C2 a b B C A

II III Ib C4A CYP21A 0C48 YP21B

B X EE B

TT T

B X E B

T T

Figure 1. Cloning of CYP21 genes encoding P45c2 1. (a) The HLAcomplex on the short arm of chromosome 6. The centromere is indi-cated by the circle at left. HLA Class II, III, and I genes are grouped.Class I and II genes encode transplantation antigens; there are moreClass II genes than are diagrammed here. TNFa and b encodetumor necrosis factor alpha and beta. C2, Bf, C4A and C4B encodeserum complement components; 21A and 21B are the CYP21 genes.(b) Strategy for cloning of the CYP21 genes. The relative sizes andlocations of the C4 and CYP21 genes are shown. The genes are alltranscribed left to right. Relevant restriction sites are indicated: B,BamHI; E, Eco RI; T, Taq I; X, Xho I.

gene, La Jolla, CA) (14). The ligated material was packaged (usingextracts obtained from Stratagene) into bacteriophage lambda particles(15), which were then used to infect Escherichia coli strain C600. Thelibrary was plated at a density of about 20,000 plaques/ 1 50-mm plate.

Isolation of a mutant CYP21Bgene. Nitrocellulose filter replicas ofthe library were screened with radioactively labeled pC21/3c as de-scribed (16). Clones carrying CYP21genes were identified by autoradi-ography, purified by replating, and grown in large cultures. Bacterio-phage DNAwas prepared (17); clones were identified as carrying eitherthe CYP21Aor B genes by the presence of a 3.2- or 3.7-kb Taq I band,respectively, on blot hybridization with labeled pC21/3c plasmid (3).

DNAsequence analysis. The 3.7-kb Taq I fragment from a clonedCYP21Bgene was subcloned into the Acc I site of the bacteriophageM13mp8 (18). The recombinant phage were used to transform E. coliMV1 190, and clones with the insert in each orientation were selected.

SILENT MUTATIONu AMINOACIDSUBSTITUTION] NONSENSEORFRAMESHIFT

2 3 14569 121A

H > > >

21 <AN MEN < I<

JIM< F

l~ ~ ~ ~ ~~---- f----- F---- 4F---- 4

Figure 2. Organization of the CYP21genes. Numbered bars repre-sent sequences expressed in mRNA,or exons, while the spaces be-tween bars correspond to introns. The shorter bars at the ends of thediagram represent 5' and 3' untranslated regions. Types of mutationswithin the CYP21Apseudogene are indicated by the shading ofexons asnoted. A line represents locations of possible gene conver-sions that have resulted in the mutant CYP21Bgene, which is dia-

grammed below. Arrows at the bottom of the diagram indicate posi-tions and orientations of primers used for sequencing. The bracketindicates the fragment used in the RNase protection assay presentedin Fig. 5.

Sequence analysis was performed by the chain termination method(19) using `S-dATP as described (20) except that reactions were car-ried out at 370C. In addition to the M13 universal sequencing primer(International Biotechnologies, Inc.), specific 17-bp oligonucleotideprimers (synthesized by the Department of Microbiology at CornellUniversity Medical College) were used, which correspbnded to thenormal CYP21Bsequence (5, 6). 10 primers were used in each direc-tion to cover the 3. 1-kb length of the gene (Fig. 2). A previously charac-terized normal CYP21Bgene was also analyzed with each primer toeliminate the possibility of artifacts arising from the use of specificprimers.

Hybridization studies using oligonucleotides. DNAsamples from20 patients were digested with Taq I and subjected to electrophoresis in3-mm-thick; horizontal 0.8% agarose gels. Gels were alkalinized, neu-tralized, and dried as described (21).

Oligonucleotides were end labeled using [32P]ATP and polynucleo-tide kinase to a specific activity of I09 dpm/Mg, and purified by electro-phoresis in polyacrylamide. Gels were hybridized overnight at 530Cwith labeled oligonucleotides (4 X 106 dpm/ml) in 0.9 MNaCl, 90 mMNa citrate (pH 7), 0.5% SDS, and 0.2 mg/ml salmon sperm DNA. Gelswere washed in 3 Mtetramethylammonium chloride as described (22,23), with the modification that the first three washes were at roomtemperature and the fourth was at 630C for 10 min.

Transfection into YJ cells. Cultured Y1 mouse adrenocorticaltumor cells were cotransfected as described (24) with pSV2-neo (2 Mg)and plasmids carrying normal or mutant human CYP21Bgenes ( 15Mig). After selection with G418 (Gibco Laboratories, Grand Island, NY)at 400 Mg/ml, resistant clones were pooled and maintained in selectivemedium.

Analysis of expression of transfected CYP21Bgenes. The expres-sion of the transfected genes was assessed by RNase protection assaysas described (25). The probe consisted of a 316-bp Pst I-Eco RI frag-ment of the normal human CYP21Bgene, which contained the entirethird exon (155 bp), the third intron, and 48 bp from the fourth exon,cloned into pIBI31. A radioactively labeled antisense transcript wassynthesized using T7 polymerase (Boehringer Mannheim Diagnostics,Inc., Indianapolis, IN) and [32P]UTP. Total cellular RNAthat wasprepared (26) from a human adrenal gland or from pools of transfec-tants was hybridized to the radiolabeled probe for 12 h at 50°C underthe described conditions. Single-stranded RNAwas then digested witha mixture of RNase A and RNase T1. The protected fragments wereresolved by electrophoresis on an 8 Murea, 6% polyacrylamide gel,and the dried gel was autoradiographed.

Results

A nonsense mutation causing 21-hydroxylase deficiency. Apartial genomic library of 200,000 clones was prepared fromDNA from a patient with salt-wasting 2 1-hydroxylase defi-ciency. Seven clones were isolated that carried CYP21 genes,of which one carried the single nondeleted CYP2JBgene.

Several mutations were observed in this gene (Fig. 3).These included a 3-bp insertion between codons 9 and 10 inexon 1 (which inserts an extra leucine residue into a series offour successive leucines), a silent third-position change incodon 248 (numbering of codons does not include the 3-bpinsert) from CTCto CTG, and a change in codon 281 fromGTG, encoding valine, to T TG, leucine. A cluster of severalmutations was also observed within the second intron (intronb). Most importantly, codon 318 in exon 8 is changed fromCAG, encoding glutamine, to TAG, which is a nonsensecodon. This is predicted to cause premature termination oftranslation of the mRNAbefore the conserved "heme-bind-ing" region of the P450 polypeptide, which results in a com-pletely nonfunctional enzyme. All of these mutations are

140 H. Globerman, M. Amor, K L. Parker, M. I. New, and P. C. White

a

Met TI LeI Ieu GI Ieu Ie TI me IM Pro m mi Ala Gl Ala AM ImTMAsn Tt1rPSj LY5 uI Ara Ser lia His li 29ATIG CIG CM CI d dIGCMI CIM GM GOCCIG CIG dIA C 13 AIG CIG AAC AGCMCCAC

A: ...

B:-Pro Pro Lau Ala Pro Gly Ie m His la Im Gln Pro Asp 1I Pro Ile Tyr Ie Leu Gly Iu Thr Gln Ly'sR e Gly Pro Ile Tyr 59OG CCI dTI OX oG GOC =TI TI CAC CIG CIG CG OCc GAC CIC cOc AIV TAT CIG CIT C cIG ACr CAG AMA TIV COM ATCTACT

A q iHiSm G1 Lu Gin A (67) sp Val Val Val Im Asn Ser I1s AmIlr Ile Glu Glu Ala Net Val LYSLV TipAla Asp 87AGG CrI CAd CIT GGCIG CAA G gt. .ag AT GIG GI GIG CIG AMC MGAM ACC ATT GAGGAA GOCAIG GIC A T GA

e Ala G1YArq Pro Glu Pro I Thr T (97) yr LYS Ie Val Ser 101TTGCTGGC OCT GAG OCACIT ACC T gtaac :...ogttxt ca ctcta AC MGCMGGmTC:taa~q~jpI. . .cW.t .. g g

B: t a q caHer T ITp Ala His IWLy Hnwrq Ser Ala Im Ieu Im Gl Ile 131AGAACTCOGACICGTCC TIG GGA GA CTOCCIG CTC TMGAAGCCCAGAGCAACG C C I CTG GZ ATC

A:G - - TB:.

Ser Met Glu Pro Val Val Glu Ginmr 1w Gin GluRe Glu 148-9 AmMet AA Ala Gln P Gl r Pro Val Ala 159CICC AEG GAG CCA GIG GIG GAGCAG CdG ACC CYAG TTCTIC GAGgt. A A CAG CCCG, ACC CCTGIG GC

Ile Glu Glu Glu nhe Her Im LIu mr Cys Her Ile Ile Cvs Tyr Im Thr RI Gly ASp Lvs Ile AIS 182-3 AS ASP As Lau Net 187ATT GAGGAGGAA TIC TCV CTC CIc ACC TIC AGC ATC ATC 'Ir TAC CIV ACC TTC GACAG RIV AAG gt... ag GAC G AAC TIA AIG

A: A A:GB: . B:.

Pro Ala TYr TYrLy s Cys Ile Gln Glu Val LIu LYS Thr p Ser His Trp Ser Ile Gln Ile Val Asp Val Ile Pro Ee Ieu Arg 216-CCr GOC TAT TAC MIATGIRC CAG GAGGIG TTA MAACC T&G AGC CAC T= TCC RVC CAA ART GMGGA GIG ATT COC TIT CIC AGGgt.

217 Rhe Ee Pro Asn Pro Gl rL qArq I y Gln Ala Ile Glu HwArq MHis Ile Val Glu Met Gln Ie Arq Gln His L 245.ag TIC TIC CCCANT OCAGT CTCOGAGCTGm ^ CAG GCCATA GAGARG A;G GA CAC ATC GTG GAGATG CAG CTG AC;G CAG CAC G

A: C A A A

245-6 Glu Ser Ieu Val Ala Gln Not Not MtIy GlnGGi Val Ala Gin Pro Ser Met Glu Gu Giy Ser 272gt...ag GAGAGC CIG GIG GCA GGCCAG AMtRGGGARAGATGGCTG CM GG£ GIG GOGC COOGAGCATG GAA GAG G TCTET

A:.B:C

mGlneu G Gly His Il His Met Ala Ala Val leu lIu Ile iy GyY hr Glu wr Thr Ala Asn Thr Um Ser TAla 302GGAx CAG CIc CIG GAA GGCAC TIG CAC ATG GCT GCAGG CIVc CGATC Gr G ACT GAG ACC ACA GCA AAC ACC CIC TICC M GC

A:.B:G

Val Val Ehe Leu Lu His His Pro Glu 312-3 Ile Gln Gln Arq m ID Glu Glu [eu Asp His Glu Ieu Gly Pro Gly Ala Ser Ser 331GIG GET'rrT TIG CrT CAC CAC CCT GAG gt.. .ag ATT CUI CAG CGA CIG AG GAG CTA GAC CAC GAA CIG GG OCT GOT GOCTVC AGC

A:T AllB:. B (C

Ser Arq Val Pro Tyr Lys Asp Arq Ala Arq Leu Pro Lm Lm Asn Ala 1 Ile Ala Glu Val I AArqmI rq Pro Val Val Pro Leu 361TCC CGGTC! TAC AAG GAd Cof GCA O CGGCOCTTG Cic AAT GCCACC ATC GoC GAG GIG CG CX;C CTG od OC GIT GIG OCC TA

A:TB:.

Ala Lm Pro His Arm ¶ffw thw Aru Pro Ser Se (372) r Ile Ser Glyvyr ASp Ile Pro Glu Gly Ihr Val Ile Ile Pro Asn ei Gln 389GCCTG OCC CAC GC ACC ACA OGGCCC AGC AG gt. .ag C REVC W GGCTACGAC RTC CCT GAG GGCACA GTC ATC ATT COGAAC C CAA

GI Ala His EI Asp Glu ffhr Val Tip Glu AZG Pro His Glu Ihe Tip Pro A (407) sP A hle Leu Glu Pro Glv LEs Asn Ser Arq 417G OXGC CAC CIG GAT GAGACG G1C EGG GAGRIG lCA CAT GAGTIC TGG CC G gt..ag RI GIC TIC CIG GAG CCA GGCAAG AAC I=CC AAAla Leu Ala Phe Gy Cys Gly Ala Arq Val Cys Leu Gly Glu Pro Leu Ala Arq Leu Glu LeI [he Val Val euI Ara Le Leu Gln 447GCI CGXTTV GGTGCGG CC CGCGIG TIC GIG G GCGAG COX TIGOG GIG GCAG GI TIC GIG GIG GIG ACC &GA CIG CIG CAG

Ala [he Tr leu LIc Pro Ser Gly AsP Ala Leu Pro Ser Lau Gln Pr eMI Pro His Cys Her Val Ile eui LAs Net Gin Pr Ihe Gln 477GOC TIC AOE CIG CIG COC TCC GGGA GCO CGIG COCTCC CI C GC CCGI OX CAC EGC AGT GTC ARE CIC AAG ATG CAG CC TIC CAA

494Val Arq Leu Gln Pr Arq Gly Met Gly Ala His Ser Pro Gly Gln Asn Gln DidGM COG CI CX OCX OC O ATG GO GOC CAC AGCOXG O CAG AAC CAG ETA

A:AB:.

present in the normal CYP21A pseudogene. However, othermutations present in the CYP21A gene were not observed inthis mutant CYP21Bgene.

Oligonucleotide hybridization studies ofpatients. A 2 1-meroligonucleotide corresponding to codons 315-321 of the mu-tant gene on the antisense strand (5'-TAG CTC CTC CTACAGTCGCTG-3') was hybridized to Taq I digests of DNA

Figure 3. Sequence of themutant CYP21Bgene.Only coding sequencesand intron junctions are

shown; codons are num-bered at the introns.Numbering does not in-clude the 3-bp insert inexon 1; numbers in pa-rentheses indicate an in-tron that is located withina codon. Where the se-

quence differs from se-

quences of the normalCYP21Apseudogene orthe CYP21Bgene, thosesequences are also dis-played and are labeled A:and B: (dots signify nodifference between themutant CYP21Bgeneand the normal A or Bgene, as indicated).Dashes represent missingbases. The critical muta-tion in codon 318 isboxed; the location of thecorresponding oligonucle-otide is underlined.

samples from patients with 2 1-hydroxylase deficiency (Fig. 4).Because this mutation is normally present in the CYP21Apseudogene, it was expected that DNAfrom normal individ-uals would contain a hybridizing Taq I fragment of 3.2 kb,corresponding to the CYP21A genes, but no signal at 3.7 kb,which is the size of the Taq I fragments from the CYP21Bgenes. In fact, 19 of 20 samples from patients contained a

IL IL 4L.LJ4 "4, 4

p-ea A-- >w w -w w* u

A B C D E F G H J K L M N 0 P O R S T

.1 1~~~~~~It~L~

4 I

A B C D E F G H I J K L M N 0 P 0 R S T

Figure 4. Hybridization to Taq I di-gests of DNAsamples from 20 pa-tients with classical 2 1-hydroxylasedeficiency. Numbers to the right ofeach line indicate fragment sizes inkilobases. (1) Southern blot usingradioactively labeled pC21/3c, a

human cDNAclone, as a probe.Arrows indicate patients with pre-

-3.7 sumed heterozygous deletions ofCYP21B. (2) Hybridization with an

-32 oligonucleotide probe that corre-

sponds to the mutation in codon318. All patients carry a 3.2-kb TaqI fragment that hybridizes with eachprobe and corresponds to theCYP21Apseudogenes that normallycarry this mutation. Three patients(arrows) carry a 3.7-kb Taq I frag-

-3.7 ment that also hybridizes with the

-3.2 probe, presumably signifying a mu-tant CYP21Bgene. The mutant

gene was first isolated from pa-tient C.

Nonsense Mutation Causing Steroid 21-Hydroxylase Deficiency 141

2.-K:

'W"I .111&

hybridizing band of 3.2 kb, while 3 of 20 patients (patients C,0, and P; the mutant gene was isolated from DNAof patientC) carried a 3.7-kb Taq I fragment that hybridized with thisprobe. These three patients who have CYP2JBgenes with thecodon 318 mutation suffer from the salt-wasting form of 21-hydroxylase deficiency. They all have a heterozygous deletionof CYP2JB. Two of them carry the HLA-Bw47;DR7 haplo-type known to carry such a deletion; no other HLA-B antigenis shared by any of these patients (Table I).

Expression of human CYP21B genes in transfected YJcells. To determine if mRNAtranscribed from this mutantgene was present in normal amounts, plasmids carrying thenormal and mutant CYP2JB genes were transfected into Ylmouse adrenocortical tumor cells. Pools of transfectant cloneswere prepared to minimize the effect of clonal variation on21-hydroxylase (CYP21B) expression. RNase protectionassays (which are more sensitive than Northern blots) wereused to determine steady-state mRNAlevels in transfectants.The results (Fig. 5) show that Yl cells transfected with eitherthe normal or the mutant gene contain CYP2JBtranscripts, asdocumented by their protection of a 155-nucleotide fragment(corresponding to all of exon 3) that is identical in size to thefragment protected by authentic human adrenal mRNA. Inaddition to the protected fragment of 155 nucleotides, thesesamples also contained a 48-nucleotide fragment protected bysequences contained in exon 4, which was poorly resolvedunder the gel conditions used (not shown). As previouslynoted (24), parental Y1 cells do not express their own 2 1-hy-droxylase genes. This technique is only semiquantitative, asevidenced by the substantial variation in mRNAlevels in dif-

Table I. Patients Used in Hybridization Studies Shown in Fig. 4

HLA

Patient Diagnosis B DR B DR

A SV 18 6 44 5B SW 49 8 44 6C* SW 47 7 57 7D SW 39 2 47 7E SW 52 2 52 2F SW 39 9 51 5G SW 14 1 35 5H SW 44 4 51 4I SW 22 4 39 9J SW 35 5 47 7K SW 8 3 35 4L SW 44 7 47 7M SW 7 10 7 10N SV 7 1 40 10* SW 7 2 47 7P* SW 35 5 40 7Q SV 51 6 44 1R SW 45 5 49 6S SV 44 7 51 4T SW 18 1 18 5

Patients are "salt-wasters" (SW), who are unable to synthesize aldo-sterone normally, or "simple virilizers" (SV), who are able to synthe-size aldosterone. HLA-B and -DR haplotypes are shown.* Patients with a mutation in codon 318.

isas4 .~l

ADRENAL

YI

NORMAL-1

NORMAL-2

NORMAL-3

NORMAL-4

MUTANT-I

MUTANT-2

Figure 5. Levels of 21 -hydroxylase (CYP2 I B) mRNAin the normalhuman adrenal gland, in parental Y1 adrenocortical cells, and in Y1cells transfected with cloned normal or mutant CYP21Bgenes. TheRNase protection assays were performed using 2 (human adrenalgland) or 20 ,ug (all other lanes) of each RNA. The position of the155-nucleotide fragment that is protected by sequences from thethird exon is indicated.

ferent pools of cells transfected with the normal CYP21Bgene.

Nevertheless, 2 1-hydroxylase mRNAlevels in cells transfectedwith the codon 318 mutant appear to be markedly lower thanthe levels seen with transfection of the normal humanCYP21Bgene.

Discussion

These data identify a genetic basis for certain cases of thesevere, salt-wasting form of 2 1-hydroxylase deficiency. Saltwasting results from impaired synthesis not only of cortisol,but also of aldosterone, and it is to be expected that this formof the disease should be caused by a (nearly) complete absenceof the 2 1-hydroxylase enzyme. It was previously known thatthe genetic basis for salt wasting in a few patients was a totaldeletion of the CYP21B gene (2, 8). Wehave now shown inthree patients, all of whomalso have a heterozygous deletionof CYP21B, that salt-wasting disease also results from a non-

sense mutation that precludes synthesis of the P450c21 en-

zyme. As patients without salt wasting demonstrate the capac-ity to synthesize aldosterone, which requires 21-hydroxylaseactivity, it is presumed that these individuals carry milder mu-

tations that are compatible with the production of some en-

zyme, however abnormal. However, epigenetic or nongeneticfactors may also influence the ability of patients to synthesizealdosterone (27).

The results also suggest a mechanism whereby the muta-tions in the sequenced CYP21Bgene have occurred. Relativelysmall nonreciprocal recombination events, or "gene conver-

sions," could account for identity between the mutationsfound on the CYP21B gene and on the corresponding posi-tions on the CYP21Apseudogene. In the case of the codon 318nonsense mutation, an independent point mutation cannot beruled out. The precise borders of the putative gene conversion

142 H. Globerman, M. Amor, K L. Parker, M. I. New, and P. C. White

cannot be firmly established because of the high degree ofhomology between the CYP21A and B genes; the maximumsize, based on the absence of other mutations from CYP21A,would extend from the 3' end of intron g to codon 355 in exon8. The two other possible gene conversions in exons of thismutant gene (the 3-bp insertion in exon 1 and the mutations inexon 7 affecting codons 248 and 281) and the cluster of muta-tions in intron b are separated in CYP21A from the codon 318mutation by intervening mutations that are not present in themutant CYP21Bgene. Thus, if they are gene conversions, theymust have been transferred to CYP21Bin independent events.The mutations in intron b and the extra leucine resulting fromthe 3-bp insertion in exon 1 have no obvious functional signifi-cance and might exist as polymorphisms of the CYP21Bgenein the normal population. The extra leucine is present inP45Oc21 of mice (28) and cattle (29) and has been documentedin a cloned normal CYP21Bgene (9). The codon 281 muta-tion is associated with a very commonnonclassical 21 -hydrox-ylase deficiency allele (30) and therefore heterozygous carriersof this mutation should also occur frequently in the generalpopulation. Thus, the existence of these multiple gene conver-sions in one mutant CYP21Bgene is not surprising.

In addition to containing a mutation that prevents synthe-sis of a functional protein, the steady-state levels of mRNAtranscribed from this mutant gene in Yl adrenal cells are sub-stantially decreased from the levels observed when normalCYP21B genes are transfected. This finding might be ex-plained by a difference in the rate of integration of normal andmutant CYP21Bgenes into the Yl cell genome or by an addi-tional undetected mutation in the promoter region that affectstranscription of the mutant gene. It is more likely that thenonsense mutation itself affects mRNAstability. This mayoccur because the mRNAdownstream of the nonsense muta-tion does not carry ribosomes and may be more susceptible tonucleases. The same phenomenon has previously been notedin a nonsense mutation, which causes #0-thalassemia (31), andin the murine 21-hydroxylase pseudogene (28). It will be nec-essary to assess the transcriptional activity of the transfectedgenes in order to definitively answer this question.

While this study has identified 3 out of 20 patients withpossible gene conversions causing 21-hydroxylase deficiency,there are 10 other mutations in the CYP21A pseudogene thatterminate protein synthesis or change amino acid sequences,and which therefore might result in 2 1-hydroxylase deficiencyif they were transferred to the CYP21Bgene. Indeed, a muta-tion in codon 172 which is normally found in the CYP21Agene has been documented in the B genes of three patientswith 21-hydroxylase deficiency (10). Thus, gene conversionsmight prove to be as commonas deletions as a cause of 21 -hy-droxylase deficiency alleles.

Humans, mice (32), and cattle (33) all have two 21-hydrox-ylase genes, which suggests that the duplication occurred be-fore mammalian speciation. It has been slightly puzzling that,in mice and men, the two 21 -hydroxylase genes are muchmore homologous to each other than they are to the corre-sponding gene in the other species. The high frequency ofapparent gene conversions provides an explanation for thisfinding. It is likely that sequences from CYP21B may occa-sionally be transferred to CYP21A by this mechanism. Itshould be noted that DNAfrom patient K in this study (whohas a heterozygous deletion of CYP21A) yields no signal at 3.2kb when probed with the oligonucleotide that corresponds to

the codon 318 mutation, although the identical gel displays astrong signal at this position when probed with additionalCYP21A-specific oligonucleotides (not shown). This CYP21Agene may thus have reverted to the CYP21Bsequence in thisregion. Gene conversions have also been documented as afactor in the evolution of other cytochrome P450 gene fami-lies (34).

Oligonucleotide hybridization might improve prenataldiagnosis of 21-hydroxylase deficiency. At present, prenataldiagnosis is based on HLA typing of fibroblasts and measure-ment of hormone levels in amniotic fluid obtained by amnio-centesis at about the 16th wk of gestation (35), or HLA typingcan be performed by Southern blot hybridization using DNAprepared from a chorionic villus sample (36). Prenatal diag-nosis using oligonucleotide probes has two potential advan-tages over HLA typing: it obviates the need for a DNAsamplefrom the index case and it excludes possible misdiagnosis dueto recombinations within the HLAcomplex. The efficiency ofthis approach would be improved by prior identification of theinvolved mutations in the parents. Southern blot analysis ofseveral restriction digests using a 2 1-hydroxylase cDNAprobewould be necessary to detect deletions and to rule out geneconversions involving the restriction sites used to distinguishthe CYP21Aand CYP21Bgenes.

Oligonucleotide hybridization using DNAobtained bychorionic villus biopsy would enable a diagnosis before thedifferentiation of the fetal external genitalia. Virilization of theexternal genitalia in an affected female fetus might be pre-vented by the administration of glucocorticoids to the motherbefore this critical time of development (37). To be practical,this method of prenatal diagnosis will require identification ofadditional mutations that cause 2 1-hydroxylase deficiency.

Acknowledgments

Wethank Alena Vitek for technical assistance and Dr. R. Bruce Wal-lace for helpful discussions. HLA genotyping of patients was per-formed by Dr. B. Dupont. Oligonucleotides were synthesized by theDepartment of Microbiology, Cornell University Medical College.

H. Globerman is the recipient of a Charles H. Revson Foundationfellowship in biomedical research, and her work has been aided by aDaland Fellowship from the American Philosophical Society. M.Amor is supported by a fellowship from Centre National de la Re-search Scientifique (France). This work is supported by National Insti-tutes of Health grants DK-37867 (P. C. White), CA-22507 (B. Du-pont), and HD-00072 (M. I. New), and by the Horace GoldsmithFoundation. P. C. White is an Andrew W. Mellon Teacher-Scientist.

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144 H. Globerman, M. Amor, K L. Parker, M. I. New, and P. C. White