Lyase Deficiency Due to P.R96W Mutation in the CYP17 Gene in a Brazilian Patient

clinical case report

Combined 17α-hydroxylase/17,20- lyase deficiency due to p.R96W mutation in the CYP17 gene in a Brazilian patient

Deficiência combinada de 17α-hidroxilase/17,20 liase devido à mutação p.R96W no gene CYP17 em um paciente brasileiro

Fabíola Costenaro1, Ticiana C. Rodrigues1, Claudio E. Kater2, Richard J. Auchus3, Mahboubeh Papari-Zareei3, Mauro A. Czepielewski1

SUMMARY 1 Division of Endocrinology, Congenital adrenal hyperplasia (CAH) resulting from 17α-hydroxylase/17,20-lyase deficiency is Hospital de Clínicas de Porto a rare autosomal recessive disease and the second most common form of CAH in Brazil. We Alegre, Universidade Federal do Rio Grande do Sul (UFRGS), describe the case of a Brazilian patient with CYP17 deficiency (17α-hydroxylase/17,20-lyase de- Porto Alegre, RS, Brazil ficiency) caused by a homozygous p.R96W mutation on exon 1 of the CYP17 gene, an unusual 2 Division of Endocrinology genotype in Brazilian patients with this form of CAH. The patient, raised as a normal female, and Metabolism, Department of Medicine, Escola Paulista sought medical care for lack of pubertal signs and primary amenorrhea at the age of 16 years. At de Medicina, Universidade evaluation, the presence of a 46,XY karyotype, hypertension and hypokalemia were observed. Federal de São Paulo (Unifesp/ We emphasize the recognition of CYP17 deficiency in the differential diagnosis of cases of hy- EPM), São Paulo, SP, Brazil 3 Division of Endocrinology and pergonadotrophic hypogonadism and hypertension in young patients who need specific treat- Metabolism, Department of Internal ment for both situations. Arq Bras Endocrinol Metab. 2010;54(8):744-8 Medicine, University of Texas Southwestern Medical Center, USA SUMÁRIO Correspondence to: A hiperplasia adrenal congênita (HAC), em razão da deficiência de 17α-hidroxilase/17,20-liase, Mauro A. Czepielewski Hospital de Clínicas de Porto é uma doença autossômica recessiva rara e a segunda causa mais comum de HAC no Brasil. Alegre, Serviço de Endocrinologia Descrevemos o caso de um paciente brasileiro portador da deficiência 17α-hidroxilase/17,20- Rua Ramiro Barcellos, 2350, liase (CYP17) em homozigose para a mutação p.R96W no éxon 1 do gene da CYP17A1, uma prédio 12, 4o andar 90035-003 − Porto Alegre, RS, mutação incomum entre os casos brasileiros descritos com essa forma de HAC. Esse paciente, Brazil criado como um indivíduo normal do sexo feminino, procurou atendimento por ausência de si- [email protected] nais puberais e amenorreia primária aos 16 anos de idade. Durante a avaliação, constataram-se Received on Jul/30/2010 um cariótipo 46,XY e a presença de hipertensão e hipocalemia. Enfatizamos o reconhecimento Accepted on Nov/3/2010 da deficiência da CYP17 dentre os possíveis diagnósticos em um paciente jovem com hipogo- nadismo hipergonadotrófico e hipertensão, os quais necessitam de tratamento particularizado para ambas as situações. Arq Bras Endocrinol Metab. 2010;54(8):744-8

Introduction gonads. Cytochrome P450 steroid 17α-hydroxylase ongenital adrenal hyperplasia (CAH) resulting (P450c17) catalyzes both the 17α-hydroxylation of Cfrom 17α-hydroxylase/17,20-lyase deficiency is pregnenolone and progesterone and also the 17,20-ly- an uncommon autosomal recessive disease. The hu- ase reaction of 17α-hydroxypregnenolone/17α- man 17α-hydroxylase (CYP17) gene is a single copy hydroxyprogesterone to produce the C-19 steroid gene located on chromosome 10q24.3-q25 (1) that precursors of androgens and estrogens, dehydroepi- consists of 8 exons spanning 6,569 bases and encod- androsterone (DHEA) and androstenedione. Genetic ABE&M todos os direitos reservados. os direitos ABE&M todos © ing a protein of 508 amino acids (2). This protein is abnormalities in the CYP17 gene affect both adrenal

744 Arq Bras Endocrinol Metab. 2010;54/8 the CYP17geneandfifthcaseinworldliterature. patient causedbyahomozygousp.R96Wmutationin deficiencyin aBrazilian 17α-hydroxylase/17,20-lyase (10). the lossofbothenzymaticactivitiesthisprotein defect andwithP450c17molecularmodelingpredicting of thisenzymatic with theclassicalclinicalpresentation was describedintwoBraziliansisters(46,XYkariotype), new mutationof25bpduplicationatexon5CYP17 inthissample(9).Recently, reported mutations were a andsevenCYP17gene reported were 24kindred from 30 Braziliansubjectswith17α intheworld (8). Previously,ethnically heterogeneous although theBrazilianpopulationisamongmost ofCYP17deficiencyinBrazil, to thehighprevalence mayalsocontribute in Brazil(4,7).Anfoundereffect ofCAH seems tobethesecondmostcommonform (DSD) orsexualinfantilismin46,XXfemales(3-6). ofsexdevelopment suchas46,XYdisorder normalities hypokalemia,andsexualab- acterized byhypertension, thisdeficiencyisclinicallychar velopment. Therefore, sexual de- leads to abnormal ofsexsteroids production hyperplasia.Inaddition,theimpaired bilateral adrenal hypokalemia,and inhypertension, resulting dosterone otherthanal- ofmineralocorticoids overproduction leadingtoanelevationofplasmaACTHand steroids and sex of cortisol production sociated withimpaired Arq Bras Metab. Endocrinol 2010;54/8 not andgonadswere was46,XYanduterus Karyotype logical agewas15years/9month-old (SD=9.23mo). - Her boneagewasdelayed(11 years),whilethechrono shownintable 1. are ity (<0.2ng/mL/h);other results activ um (3.3mEq/L)andundetectable plasmarenin potassi- lowserum sodium levels,BUNandcreatinine; showednormal and ablindvagina.Routinelabresults genitalia was B1P1,withaninfantilefemaleexternal 163 cm,83and68respectively. Tanner stage span,inferior,36,5 kg;arm andsuperiorsegmentswere 150 x115mmHg.Heightwas151cmandweight waselevatedat Recumbentbloodpressure her parents. andItalianancestry.Portuguese consanguineousfrom were Herparents amenorrhea. signsandprimary 16 yearsofagewithlackpubertal at A phenotypicallyfemaleBrazilianpatientpresented c ase REPOR Herein, we report thesecondcaseof wereport Herein, In fact,17α The 17α On physicalexaminationherskinwasdarkerthan -hydroxylase/17,20-lyase deficiencyisas- -hydroxylase/17,20-lyase T -hydroxylase/17,20-lyase deficiency deficiency -hydroxylase/17,20-lyase -hvdroxylase deficiency deficiency -hvdroxylase - -

17α-OHP: -hydroxyprogesterone;DOC: deoxycorticosterone;ND:notdone. T pressure control. pressure withblood diol (25mcg/day)withnointerference quently, estra- ontransdermic thepatient wasstarted excess (Table steroid levels and controlled 3). Subse- bloodpressure to7.5mg/daynormalized an increase and biochemical control; blood pressure appropriate (Table (DOC)andcorticosterone 2). corticosterone 17α and (21DF),11-deoxycortisol, sone, 21-deoxycortisol - andcorti levelsofcortisol detectable andunresponsive un- tion testaddedevidence to thediagnosisrevealing stimula- elevated.Acosyntropin were and progesterone low, were one andcortisol ACTH aldosterone, whereas although boneagewasdelayed). was belowcalculatedfamilytarget (172cmforaboy visualized onanabdominal-pelvicUS.Patient’sheight T 17α-OHP: -hydroxyprogesterone;PRA:plasmareninactivity. able 2. BasalandACTH–stimulatedadrenalsteroidvalues able 1. andhormonevaluesinplasma Baselinebiochemistry 21Deoxycortisol (ng/dL) Cortisone (ng/dL) Cortisol (µg/dL) 11Deoxycortisol (ng/dL) Steroids Corticosterone (ng/dL) DOC (ng/dL) 17α-OHP (ng/dL) Cortisol 8h(µg/dL) 17α-OHP (ng/dL) Estradiol (pg/mL) LH (mUI/mL) FSH (mUI/mL) Total testosterone(ng/mL) Progesterone (ng/mL) DHEAS (µg/dL) ACTH (pg/mL) Androstenedione (ng/dL) K (mEq/L) Bicarbonate (mEq/L) Aldosterone (ng/dL) PRA (ng/mL/h) Oral prednisolone was started at5mg/daywithout wasstarted Oral prednisolone levelsof17α Serum -OHP, ofsubstantiallyelevateddeoxy- inpresence Baseline 18, 377 527 ND ND 0.7 26 7 -OHP,- DHEA-S,androstenedi CYP17, congenital adrenal hyperplasia Results < 0.20 < 0.1 60.1 46.6 6.14 < 11 476 1.8 < 5 3.2 18 30 29 2 22, 030 ACTH Post- 612 ND ND 4.1 68 9

800

0.1-0.5 Normal rangevalues Normal rangevalues Basal 0-40 6-25 0-45 4-12 < 5 Upright: 0.98-4.18 Supine: 2.9-16.2 6.2-19.4 7.5-42.5 1.5-12.4 31-217 1.7-8.6 0.2-1.4 25-250 3.5-5.1 3.5-25 10-52 22-30 5-35 Post-ACTH 1.7-4.8 18-42 12-61 < 156 < 140 3500 <10 745

Copyright© ABE&M todos os direitos reservados. Copyright© ABE&M todos os direitos reservados. sodium and potassium were in the normal range. range. inthenormal sodium andpotassiumwere gonadotropin, steroids, sexandadrenal progesterone, ofserum profile andtheirhormonal hypertensive were nor herbrother gosis wasdetected.Neitherherparents, homozy in our patient;instead,anp.R96W(inexon1) notfoundin tions identifiedinBrazilianpatientswere fied byPCR(seebelow).Thetwomostcommon muta ampli - cytes andalleightexonsoftheCYP17genewere 746 identifiedbycomparison USA). Themutationswere of Texas MedicalCenter, Southwestern Dallas,TX, CenterSequencingFacilityatUniversity (McDermott tion methodonaPEApplied Biosystemsinstrument - DNAbythedyetermina 8 exonsandflankingintronic sequencingofthe submittedtodirect Amplicons were CA,USA). ing theQIAEXIIkit(Qiagen,Chatsworth, gelsus- tated withethanolandpurifiedon1%agarose - precipi were 72°C for5.5min.ThefinalPCRproducts geneandtheextensionparameterswere of theentire to1.5minforamplification nealing timewasincreased 3 minat94°C,165°C,and70°C.An- PCRparametersincluded40cyclesof to 4-kbproducts, and3%dimethylsulfoxide.Tomanufacturer amplify3- bythe anddeoxy-NTPsprovided actions usingbuffer merase (Takara- ShuzoCo.,Shiga,Japan)in100µLre µg genomic DNA using TaKaRa ExTaq DNApoly- 0.5-1 CYP17 genewasamplifiedinto1-4piecesfrom (11).The6.4-kb saltingoutprocedure standardized peripheralleukocytes bya DNA wasextractedfrom DNA prepara 17α-OHP: -hydroxyprogesterone;PRA:plasmareninactivity. T CYP17, congenital adrenal hyperplasia Biochemistry andhormonalvaluesafterprednisone7.5mg/day able 3.Biochemistry PRA (ng/mL/h) Aldosterone (ng/dL) Bicarbonate (mEq/L) K (mEq/L) ACTH (pg/mL) Androstenedione (ng/dL) DHEAS (µg/dL) Progesterone (ng/mL) 17α-OHP (pg/mL) Cortisol 8h(µg/dL) Genomic DNA was extracted from peripheral leuko- Genomic DNAwasextractedfrom tion, PCR,and seq uencing Prednisolone 7.5 mg/day < 0.11 1.15 < 10 0.28 0.18 8.2 5.6 1.9 2.8 28 25 µg/day Estradiol 22.8 1.31 1.13 990 4.5 28 - - - - range values 0.98-4.18 0.31-2.17 24.4-247 6.2-19.4 2.9-16.2 Normal Upright: Supine: 0.5-3.5 3.5-5.1 0.2-1.4 10-52 22-30 - - DNA was not available for investigation. DNA wasnotavailableforinvestigation. 1).Parental (Figure plification intheoppositedirection ofasecondPCRam- bysequencingtheproduct firmed con- Diego, CA, USA) (12). Identified mutations were M19489) usingMacVector San 6.5.3(AccelrysCorp. with theGenBanksequenceforCYP17(accessionno. (5,14-15). The purpose of replacement glucocorticoid glucocorticoid (5,14-15). Thepurposeof replacement steroid(s) and some other increased tween aldosterone be- oracross-reaction and/orregulation roidogenesis ofste- unclear and;possibilitiesinclude: adisorder ofwhichisstill thereason hyperaldosteronism, serum all (7).However, secretion sterone thisisnotthecasein aldo- in reduced synthesis, resulting for aldosterone stimulation renin doesnotreceive zona glomerulosa activity (6).Thus,the adrenal plasma renin suppressed hypokalemiaand hypertension, producing kaliuresis, ofsodiumand tubular resorption renal cess: increased ex- leads toclinicalmanifestationsofmineralocorticoid large amountsofDOCwhich stimulation toproduce andadrenal ACTHoversecretion insecondary results impairment crisis.Cortisol anadrenal tion andprevents - produc substitutes for low cortisol of corticosterone until adolescence orearlyadulthood: overproduction Veryremains undiagnosed oftenP450c17 deficiency elevated. levelswere cal examinationherbloodpressure andduringclini- fordelayedpuberty sought treatment another Brazilianpatient(14).Our16-yearold lyase deficiency(12),anItalianpatient(13)andalsoin withcombined17α-hydroxylase/17,20- presenting siblings CYP17: intwo46,XYDSDFrench-Canadian in rarely The p.R96Wmutationhasbeenreported Discussion in codonArg96(CGG)intoaTrp (TGG)inexon1. Figure 1. Electropherogramofp.R96WmutationwithatransitionC→T patients, and some, as our patient, present 17OHD patients, andsome,asourpatient,present Arq Bras Metab. Endocrinol 2010;54/8 Arq Bras Metab. Endocrinol 2010;54/8 andhypokalemia. hypertension controlling excess,and themineralocorticoid fasciculata reducing the ACTH stimulation ofzona therapy istosuppress ed in our patient with a transdermal 17b ed inourpatientwithatransdermal - replacement wasstart deficient patients(17).Estrogen inCYP17 syntheticroute mineralocorticoid’s altered thatmaystimulatethe cortex, intheadrenal receptors gravated; thisispossiblyduetoactivationofestrogen may be ag- essary, the risk that hypertension running isnec- therapy with oral estrogen Thus, replacement hypogonadisminbothgeneticsexes(7,16). nadotropic leadingtohypergo- impaired, are tion ofsexsteroids - andgonadalproduc boththeadrenal present, cies are er effect, since our patient was from southern Brazil,an southern sinceourpatientwasfrom er effect, (3,20). ity ofthemutantprotein 1),abolishingalmostcompletelytheactiv- (12) (Figure in codonArg 96(CGG)intoaTrp (TGG)inexon1 missense p.R96WmutationcausesatransitionC→T loss ofbothenzymaticP450c17activities(20).The gene, showingthatthesemutationscauseadramatic p.R96W and missense H373D mutation on the both Recently apatientwasdescribedwhocarried (7,15,19). ciency canhaveambiguousgenitaliaatbirth defi- 17α-hydroxylase (10,15). Patientswithpartial even amongpatientswiththesameCYP17mutations rateappeartovary, production mia, andaldosterone of hypokale- the degree age of onset of hypertension, catalyticactivity(7,19),butthe apartial that retain ity ofclinicaldiseasetendstobemilderwithmutations leading tocompleteenzymeinactivation(3).Thesever thisinteractionbetweenthe twodomains, disrupting by p.R96W mutation,appearstoinstabilizetheprotein whichoccursin moval ofthispositivelycharged group, A113andF114(18).Re- ofresidues carbonyl groups bondswith hydrogen of R96appearstoform group the flankingstrand2ofβ-sheet1,andguanidine levels. onbloodpressure but withnosignificanteffect ment schedulemayalsointeractwithsteroidogenesis, - levels.Thisreplace ofcortisol er withaslightreduction togeth- observed levelswere andaldosterone gesterone scheme;however,replacement - elevationofACTH,pro didnotchangeonthis fact, ourpatient’sbloodpressure In lower peakdosesandlessimpactondiseasecontrol. couldbeachievedat catabolism, thetherapeuticeffects reduced GI enzymatic first-pass hepatic metabolism and mulation (25µg/day).Asexpected,withthelackof When combined17a The p.R96Wmutationcanbe explainedbyafound- modelingsuggeststhatR96 lieswithin Structural -OH and17,20-lyasedeficien- -estradiol for CYP17 - - 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. References was reported. relevant tothisarticle nopotentialconflictofinterest Disclosure: a major German andItalianethnic extract. a majorGerman Brazil, ,especiallyinsouthern be higherthanreported ofthismutationmay it ispossiblethattheprevalence identified justforthesecondtimeinBrazil.However, of ourknowledge,thep.R96Wmutation,hasbeen ofCAHinBrazil(5,8),buttothebest form frequent ofinbr high coefficient alsosuggestsa ethnicheterogeneity withgreat country ity. canbemanifestedina Thefactthatfoundereffects ofaCaucasianethnic- and whosepopulationismore influence migratory traditionallyunderEuropean area

Interestingly, CYP17deficiencyisthesecondmost Clin EndocrinolMetab.2002;87(2):898-905. se deficiencycausedbyPhe93Cys mutationintheCYP17 gene.J gio A, Perona M,etal.Combined17 alpha-hydroxylase/17,20-lya- Di Cerbo A, Biason-Lauber A, Savino M,PiemonteseMR,DiGior dian patients.JClinEndocrinolMetab.1996;81(1):264-8. 17 alpha-hydroxylase/17-20-lyase deficiencyintwo French Cana- Mutation R96Wincytochrome P450c17 genecausescombined Laflamme N, Leblanc JF, Mailloux J, Faure N,Labrie F, Simard J. diabetes mellitus.ClinEndocrinol.(Oxf).2010;72(5):612-9. diabeticretinopathyassociated withproliferative inpatients AP, deSouza BM,etal.Polymorphisms oftheUCP2geneare Crispim D,Fagundes NJ, dosSantos KG,RheinheimerJ, Bouças lar modeling. Arq BrasEndocrinolMetabol.2008;52(8):1317-20. CYP17 resultinginaprematurestopcodonpredictedbymolecu- homozygous 25BPduplication(NT 4157-4181) atexon 5inthe Combined 17 alpha-hydroxylase/17,20-lyase deficiencyduetoa Martin PS,CostaEM, RM,Oliveira Arnhold IJ, MendoncaBB. 17-hydroxylase deficiency. 89(1):49-60. JClinEndocrinol Metab.2004; and genotype-phenotypecorrelations in24Brazilianpatientswith Santos MC, Kater C, Auchus RJ. Two prevalent CYP17 mutations ble from:www.etni-cidade.net/dados.htm p.226;RiodeJaneiro: IBGE[databaseonInternet]. Availa2000. - anosdepovoamento; Etni-cidade. EstatísticaE-IBGE.Brasil:500 Endocrinol Metab.1998;42(5):385-92. nital adrenalhyperplasia in Brazilianmedicalcenters. Arq Bras Santos MC,Kater CE.Group TBCMS. Prevalence studyofconge- 17alpha-hydroxylase deficiency. JClinInvest. 1970;49(10):1930-41. New MI,SuvannakulL.Malepseudohermaphroditismdueto man. JClinInvest. 1966;45(12):1946-54. Biglieri EG, Herron MA,BrustN.17-Hydroxylation deficiencyin deficiency. EndocrinolMetabClinNorth Am. 1994;23(2):341-57. Kater CE, Biglieri EG. of steroid17 alpha-hydroxylase Disorders 2001;30(1):101-19. human deficienciesofP450c17. EndocrinolMetabClinNorth Am. Auchus RJ. The genetics,pathophysiology, andmanagementof Acad Sci USA. 1987;84(2):407-11. indicates the same gene is expressed in both tissues. Proc Natl xylase/17,20 lyase):cloningofhumanadrenalandtestiscDNAs JE,etal.CytochromeShively P450c17 (steroid17 alpha-hydro - Chung BC,Picado-Leonard J, HaniuM,BienkowskiHallPF, larity withthegeneforP450c21. DNA.1987;6(5):439-48. gene forP450c17 (steroid17 alpha-hydroxylase/17,20 lyase):simi- Picado-Leonard J, Miller WL. Cloningandsequenceofthehuman eeding inlocalareas. CYP17, congenital adrenal hyperplasia 747 -

Copyright© ABE&M todos os direitos reservados. Copyright© ABE&M todos os direitos reservados. 748 16. 15. 14. CYP17, congenital adrenal hyperplasia 1996;81(10):3797-801. se patientswithmutationsofCYP17. JClinEndocrinolMetab. hyperaldosteronism:-responsive withareviewofJapane- Japanese patientpreviouslyreported ashavingglucocorticoid- et al.Mutationofcytochrome P-45017 alphagene(CYP17) ina Miura K, Yasuda K, Yanase T, Yamakita N, Sasano H, Nawata H, nition. EndocrRev. 1991;12(1):91-108. 20-lyase deficiency:fromclinicalinvestigation tomolecular defi- Yanase T, SimpsonER, Waterman MR.17 alpha-hydroxylase/17, J ClinEndocrinolMetab.2003;88(12):5739-46. sis throughprogesteronelevelsconfirmedbyCYP17 genotyping. al. P450c17 deficiencyinBrazilianpatients:biochemical diagno- Martin RM,LinCJ, CostaEMF, ML,Carrilho Oliveira A, Villar H,et 20. 19. 18. 7. 1

lyase deficiency. JClinEndocrinolMetab.2009;94(8):3089-92. mutation H373Dcausingcombined17alpha-hydroxylase/17,20- Sahakitrungruang T, Tee MK,SpeiserPW, Miller WL. Novel P450c17 1997;17(2):201-5. functional basisofisolated17, 20-lyasedeficiency. NatGenet. Geller DH, Auchus RJ, MendonçaBB,Miller WL. The geneticand Metab. 2006;91(6):2428-31. 17 alpha-hydroxylase/17,20 lyase deficiency. J Clin Endocrinol al. A novel pointmutationinP450c17 (CYP17) causingcombined Brooke AM, Taylor NF, Shepherd JH,GoreME, Ahmad T, Lin L, et tion ofsixpatients.JClinEndocrinolMetab.2007;92(3):1000-7. M, etal.P450c17 deficiency:clinicalandmolecular characteriza - Rosa S, Duff C, MeyerM, Lang-Muritano M, Balercia G, Boscaro Arq Bras Metab. Endocrinol 2010;54/8