Hereditary isolated glucocorticoid deficiency is associated with abnormalities of the adrenocorticotropin receptor gene

Hereditary isolated glucocorticoid deficiency is

associated with abnormalities of the

adrenocorticotropin receptor gene.

C Tsigos, … , W Hung, G P Chrousos

J Clin Invest. 1993;92(5):2458-2461. https://doi.org/10.1172/JCI116853.

Research Article

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Hereditary Isolated Glucocorticoid

Deficiency

Is Associated

with Abnormalities

of the Adrenocorticotropin

Receptor Gene

Constantine Tsigos,*Keiko Arai,*WellingtonHung,t and GeorgeP.Chrousos*

*Developmental Endocrinology Branch, NationalInstituteof ChildHealthandHumanDevelopment, Bethesda,Maryland 20892; andtDepartmentofPediatrics, Georgetown University School ofMedicine, Washington, DC 20007

Abstract

Isolatedglucocorticoid deficiency (IGD) is an autosomal reces-sive disorder characterized byprogressive primary adrenal in-sufficiency, without mineralocorticoid deficiency. The cDNA and gene of the human ACTH receptorwererecently cloned. The geneencodes a297-amino acidprotein that belongstothe Gprotein-coupledsuperfamily of membrane receptors. We

hy-pothesizedthat theACIH receptor genemightbedefective in IGD. To examinethis, we studied itsgenomicstructureby PCR anddirectsequencing ina5-yr-old proband with thedisease,

his parents, and grandparents. Theproband was acompound heterozygote fortwodifferent point mutations,onein each al-lele:

(a)

asubstitution

(C

--

T),

also found in_oneallele of the mother and maternalgrandmother, which introduceda

prema-turestop codon(TGA)atposition201of theprotein;this

mu-tantreceptorlacksits entirecarboxy-terminalthirdand,if

ex-pressed, should be unableto transduce thesignal; and (b)a

substitution(C- G),also found inoneofthepaternal alleles,

which changed neutral serine " _{in theapolar third}

transmem-brane domain of the receptortoapositively charged arginine,

probablydisrupting theligand-bindingsite.Standard ovine

cor-ticotropinreleasinghormone(oCRH)testintheheterozygote

parents and maternal grandmotherrevealed exaggerated and prolonged ACTH responses, suggestive of subclinical

resis-tance toACTH.Weconclude that IGD in this

family

appears tobe duetodefects of the ACTH receptor gene.TheoCRHtest

appears tobeuseful inascertaining

heterozygosity

inthis

syn-drome.(J. Clin.Invest.

1993.92:2458-2461.)

Keywords: adre-nalinsufficiency* ACTH receptor * CRHtest* G

protein-cou-pled receptors

Introduction

Hereditaryisolatedglucocorticoid deficiencyisa rare

autoso-malrecessive disorder characterizedby

primary

adrenal

insuf-ficiencywithout mineralocorticoid

deficiency (1-3).

Affected

childrenusuallyhaverecurrent

hypoglycemia, convulsions,

or

coma, which may result in death within thefirst 2 yr of life.

Unlessrecognizedand treatedearly,this conditionmay leadto

chronic asthenia and failuretothrive.Thedisorder is occasion-ally associated with alacrima and achalasia of theesophagus, suggesting potential heterogeneity in its etiology (4, 5). Af-fected individualshavenocortisoloraldosterone responsesto

Addresscorrespondenceto Dr.C.Tsigos, Developmental Endocrinol-ogyBranch,Building10,RoomlON262,National Institutes ofHealth,

9000RockvillePike, Bethesda,MD20892.

Receivedfor publication27July1993.

exogenous ACTH but retainanormal aldosterone responseto

activation of therenin-angiotensin axis by salt restriction,

or-thostasis, and furosemide-induced diuresis (6-8). In keeping with thebiochemical studies, in thesepatients the angiotensin II-dependent zonae fasciculataand reticularisare extremely atrophic, reducedto a narrowband of fibroustissue,while the

zona glomerulosa is relatively well preserved ( 1, 2, 6). The generalized adrenal unresponsivenesstoACTH thatunderlies thepathogenesis ofisolatedglucocorticoid deficiencysuggests

that thedefect responsiblecould be anywhere in thesignaling cascade, from the membrane-bound G protein-coupled ACTH receptortothekinases stimulating steroidogenesis. A defect in the Gproteinoradenylyl cyclase, both of which are

ubiquitousmolecules whosedysfunctionwould be expected to

affectmultipleorgans, would bearatherunlikelyexplanation for thesyndromeofACTHresistance, which is limitedtothe adrenalcortex.

Recently, the human ACTH receptor gene was cloned and itssequencedetermined (9).It encodes a297-aminoacid

pro-tein,whichtogetherwiththe melanocyte-stimulating hormone receptors, constitute a class of G protein-coupled receptors whosegenes areintronless.To test thehypothesisthat

abnor-malitiesof the ACTH receptor gene might lead to hereditary isolatedglucocorticoid deficiency, we examined this gene in a boy with this condition, his parents, and grandparents. We used standard ovine corticotropin releasing factor (oCRH)' stimulationtestingtoascertainsubclinicalACTH resistance in asymptomatic members ofthefamily found to be

heterozy-gotesfor defects of the ACTHreceptor gene.

Methods

Patients.Thepedigreestudied is summarized in Fig. 1 A. The proband, a 5-yr-old black boy, wasborn at full-termby vaginal deliveryto healthy 21-yr-oldnonconsanguineousparents.Generalized hyperpig-mentationwaspresentfrom birth.He was in goodhealthuntil9 moof

age, when hewasfound in themorningtobeunconsciousand

unre-sponsivetostimuli. Hewastakento anemergencyroomandhisblood glucosewas20mg/dl.Hehadagood response tointravenousglucose butnodefinitivediagnosiswasmade. Thepatienthadanother docu-mentedhypoglycemic episodeat12moof age. He wasseenbyoneof us(W.Hung)at 14 moof age because ofunresponsivenessand blood glucose of 25mg/dl.Atthistime,hisserumcortisolwas <1 ug/dl and plasma ACTHwas426pg/ml (normal, 9-52 pg/ml). Further work-up revealed normalplasmareninactivityandaldosterone concentra-tion. Therewas noincrease inserumcortisol levelsafter intravenous

injection ofACTH 1-24. Adiagnosis ofisolatedglucocorticoid

defi-ciencywasmade and thepatienthassince been maintainedon

hydro-cortisonereplacement,onwhich he has had normalgrowthand intel-lectualdevelopment. Thepatienthasnosiblings,and his parents and

TheJournal of ClinicalInvestigation,Inc. Volume92,November 1993,2458-2461

1.Abbreviationusedinthispaper:oCRH,ovinecorticotropin releasing

hormone.

grandparents have beenhealthy and have not had any symptoms or

signs suggestiveof adrenal dysfunction.

DNA preparationand analysis.Genomic DNA was prepared from whole bloodfrom the proband, his parents, and three grandparents, and usedas atemplate foramplification oftheintronless ACTH recep-tor geneby the PCR. Thefollowing pair of flankingprimers were used: sense 5'-GTCCAAGTAACATCCCCGCCTTAACCAC-3' and anti-sense 5'-GGATTCTAAAACCAGGGATCAGCCATTC-3'. Amplifi-cationwasallowed for30 cycles in a Gene Amp PCR system (9600; Perkin Elmer Cetus, Norwalk, CT). Exceptforthefirst and last, all cycles were30 s at980C,30 s at550C,and 3 min at730C,in a100-Ml

volume with Pyrococcusfuriosus DNA polymerase (Stratagene, La Jolla, CA). In thefirst cycle, all temperature steps were 5min,while in the last cycle theextensionstep at730Cwas15min.The PCRproduct was used to producesingle-stranded DNA by asymmetric PCR, using

either primeralone. Thiswascarried outfor20 cycles in a I00-,u1 volume, asdescribed above. The amplified single-stranded DNA was

purifiedbyfiltration throughamilliporemembrane andwasdirectly sequenced bythedideoxynucleotide chain termination method, as modified by Kadowaki et al. (10), usingmodifiedT7-DNA polymer-ase(Sequenase; UnitedStatesBiochem. Corp., Cleveland, OH)in the presence of"S-dATP. Innerprimers that span the ACTH receptor domainwereusedforsequencingand thereactionproductswere run on6%acrylamidegel.

oCRHstimulation testing. Weperformed astandard 3-h oCRH stimulationtestin theobligate heterozygote parents ofthe proband and his carrier maternal grandmother in an attempt to detect any subclini-cal abnormalities of the hypothalamic-pituitary-adrenal axis that might be associated with their heterozygote status for the mutations

identified.Anintravenous catheterwasinserted 1 h before thetestinan antecubital vein. oCRH (1 ,g/kg) was given at 1900 h as an intrave-nousbolusinjectionand bloodwasdrawnat-15,0, 5, 15, 30, 60, 90, 120, 150, and 180min,aspreviouslydescribed ( 1). Plasma ACTH and cortisolweremeasured by previously described radioimmunoas-says(11).

Results

Two different point mutationswereidentifiedin the genomic DNAof the ACTH receptorofthe proband(Fig. 1). The first mutation was a substitution of cytosine by thymidine at the first position of codon 201, normally encoding arginine,

intro-ducing thestopcodon TGA (Fig. 1 B). Thesite ofthis

muta-tioncorresponds to the beginning ofthe third intracellular loop ofthe ACTH receptorprotein. Thus, the mutant receptor lacks theentirecarboxy-terminal thirdandretains only 3 of the 20

B

i

B 3rd

cytosolic

loop

G A T C

..%..i.

MM-|-4f

I-Normal

G A T C

-0-_of_

_O.m

Mutant

C

Transmembrane

dom,

G ATC

Mutant

G ATC

*._

-_

-_

-..^.,

Mutant

G AT C

_of-..Om

Muan

I

)Leu198

G)

L.u199

'C

_I

_Mam

GA

I

Arp*'

Stop

C }SePo

C )Thr203

r)

)

ain III

/

A/T)

1&.118

G ATC / C

T

_IT

Phe119

*'a,_[

~G)

SeI012

Arg12

F

~~~~~)

L,.12

>;e_ ~~G

_

-

~~~T

Normal \

_G)

\T V61123

GJ

Truncated Receptor

NH2 l

It

II

_gIII%

I V _X

Celle

Arg24

Stop

Normal Length Receptor

,11B

9

§

OUT

I II ₁₂₀

WIIIV

V VI

Cell

V membrane

Ser2-

Argl20

Figure1. DNAsequencing of the ACTHreceptorgeneintheproband and his family.(A)Family pedigree.The proband(smallarrow)isamixed

heterozygoteforastopcodonatposition201(solid)andanonconservative amino acid substitution from SertoArgatposition 120(hatched). Thestopcodonmutation is alsocarriedby his mother and maternal grandmother, and the nonconservative substitution by his father. (B)DNA sequencingoftheareaofthegenecarryingthestopcodon within the thirdcytosolicloop,which resultsinthetruncated ACTHreceptor, is

shownontheright. (C)DNAsequencingof theareaof thegenecarryingthe nonconservative substitution withintransmembrane domainIII,

which results inanACTHreceptorcarryingabasicArgin this domain, is shownontheright.N.T.,nottested.

AdrenocorticotropinResistance 2459 COOH OUT

*Ia

aminoacids of the third intracellularloop. The second

muta-tionwas asubstitution ofcytocine by guanineatthethird posi-tion ofcodon 120causinganonconservative amino acid

sub-stitution from serinetoarginine (Fig. 1 C).This mutation is in transmembrane domainIIIof thereceptor.

Eachof themutationsfound in the probandwerealso pres-entinhis mother andfather,whowere,respectively,

heterozy-gotes for the stop codon mutation and the nonconservative aminoacid substitution (Fig. 1). The maternal grandmother

was also a heterozygoteforthe stop codonmutation.

The oCRH stimulationtest revealed abnormalities inall

three heterozygote carriers determined by gene sequencing, with all ofthem having exaggerated and prolonged plasma ACTH responses tooCRH (Fig. 2). Plasma cortisolresponses

ranged between normal and slightly elevated.

Discussion

It appears thatthe combination of the two point mutations

identifiedin eachoftheallelesof the ACTHreceptor gene of the proband is the cause ofhis isolated glucocorticoid

defi-130

100

E

I0CD

30

n 20

.0

0 ₁₀

s l

... . . . . .. ... -... ..****.-..*******...

_s~~~~.. . .. .... _* **_*** *,_*i_

A*

..

i_ . **_*_...**** ** ***i

V.

Bv*...

_ _. ...*.

*. .. . ... . . . .

-1501530 60 90 120 150 180 Time(min)

Figure 2. Plasma ACTH (top) and cortisol(bottom) responses to oCRH(1 ug/kg i.v.) in the carrierparentsandmaternalgrandmother of the proband. All carriers had exaggerated and prolonged ACTH responsesbut normalorslightlyelevatedcortisolresponses. Shaded

areasrepresentthemean±2 SD of normalcontrols. To convertvalues of ACTHtopicomolesperliter and ofcortisoltonanomolesperliter,

multiplyby 0.22 and 27.6, respectively.

ciency.The prematuretermination introduced byoneof these mutationswouldeliminatea large partofthe receptor

mole-cule that interacts with the Gprotein. Theprotein product of thismutantallele, if expressed, should betruncated and unable totransduceasignal. Recently, similar mutations, introducing

stopcodons in the third cytosolic loop of the vasopressinV2 receptor, were associated with vasopressin resistance and

nephrogenic diabetes insipidus(12-14). Null mutations inthe

rhodopsin gene were also associated with rod photoreceptor

dysfunctionand autosomalrecessiveretinitis pigmentosa(15). The nonconservative amino acid substitution introduced by the other mutation in transmembrane domain III of the ACTHreceptorwould also be expectedtoresultina dysfunc-tionalreceptorprotein.Gprotein-coupledreceptors have

sev-eral highly conserved, negatively charged aspartate residues

withinthe apolartransmembrane domainsII andIII,whichare

apparently crucial in the formation oftheligand bindingsite of thereceptor(16, 17). Substitution of suchahighlyconserved

aspartate at thebeginningof transmembrane domainIIIwith asparagine causedareduction in theaffinity ofmutant

musca-rinic anda2- andf2-adrenoreceptors in the order of 10,000-fold( 18,

19).

Evenaconservative

change

of this aspartate resi-duetoglutamate causeda 1,000-foldreduction in thebinding affinityof the

f-adrenoreceptor

(19). Theclinicalimportance ofthisregion inreceptorfunction is underscored bytworecent reports of nephrogenic diabetesinsipidus, in which the gene

abnormalityresponsibleforthediseasewas apointmutation in transmembrane domainIIIofthevasopressinV2 receptor( 12, 13). Thus, we believe that the substitution of the neutrally charged serine 120 in the overall apolar transmembrane domain

III by arginine might interfere with the formation of the

ACTH-binding pocket, rendering the mutant receptor dys-functional. By virtue of itsposition, this mutationmightalso

interferewith theconformationof the secondcytosolicloopof

thereceptor,whichcontributestothe Gprotein bindingsite(20).

The inappropriately exaggerated and prolonged plasma ACTH responses to oCRH in the heterozygote parents and maternalgrandmother ofourpatientsuggestthat

heterozygo-sityfor the mutations identifiedrepresents acarrierstate char-acterized by subclinical resistance to ACTH. These findings

provide in vivo evidence insupportofacause-effect

relation-shipbetween the mutations identified and the isolated gluco-corticoiddeficiencyinthisfamily,andindicate that the oCRH

test might be appropriate for ascertaining heterozygosity in other families with thissyndrome. Interestingly,similar

hetero-zygosityinkindrends withnephrogenicdiabetesinsipidusand retinitispigmentosa results in subtle but detectable abnormali-ties in osmoregulation and retinal electrical activity,

respec-tively ( 14, 15).

So far, there has been a singlereport ofa differentpoint

mutationoftheACTHreceptor gene in anotherkindred with

hereditaryisolatedglucocorticoid deficiency (21 ).Itis ofnote

that theprobandinthisfamilywashomozygoticfora

conserva-tive substitution ofaseine by isoleucine in transmembrane domain II. No biochemical ascertainment ofheterozygosity

wasattempted.

Acknowledgments

WethankDr.Roger Cone (Vollum Institute, Portland, OR) for send-ingusthe DNA sequenceof the ACTHreceptor andDr. Nicholas Vamvakopoulos for his assistance inthe early phasesof this project.

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