· .:;, .

MOLECULAR DETECTION

OF GENETIC DEFECTS IN AMBIGUOUS GENITALIA (AG)

AND

CONGENITAL ADRENAL HYPERPLASIA (CAH)

DUE TO 21-HYDROXYLASE DEFICIENCY

by

Dr. Fuziah Md.Zain

Prof. Mohd.Nizam Isa

Dr. Rus Anida Awang

YuHa Kcsuma Muhamad

I Human (ieno!ll!! ('enlJ'(!,

]Department ofPaediutrics, Scho%j'!'v1edicine,

Universiti Sains Malaysia, 16150 Klibang Kerian, Ke/antan, Malaysia,

JD'epartment ofPaediatrics, Hospi/u/ Kula Bhorll, Ke/anlan, Malaysia.

Introduction:CAH due to 21-(OH) deficiency

.;. Group of recessively inherited diseases"" More than 95% of all cases of CAH"" CAH exist in a very wide severity:

i. Salt Wastingii. Simple Virilizingiii. Non Classical

} Classical

.;. Incidence 1: 10,000-17,000 in Western Europe & USA.World wide 1: 14,000 births.

Adrenal and Gonadal Steroidogenesis

Ctlolcsterol

\ .

II.

. ',1 ';"!_

Androsh~rH!·<t,olDHEA

..; ./." ~•.•• ,.( ..•. if";)

... Androstenedione .. TcstoSl~l olle.. J1-0H P-rQflestcron..~

C--... ~~-:l

Pre9n(~nolone

. .P.LQ.Ql-!stt;!!ro"e

;: "_.__ .~.~_) lOe~) .. 'I C ()n I(;(JS{f)rone

Corticosterone

11 ·Dcoxycortlsol

:•Cortisol

18·()H CortIcosterone"

Aldosterone

1

.-, ~~ ....... '.

---- -- -- ------ ------._--------- ~-

CAH due to 21-(OH) deficiency:

... Arises as a result of deletions ordeleterious mutations in the active gene(CYP21) located on chromosome Gp.

• Many different mutations of the CYP21gene have been identified causing varyingdegrees of impairment of 21-hydroxylaseactivity that result in a spectrum ofdisease expression.

Jntroduction: Clinical Picture

i. Sa[t Wasting

• The most severe form of CAH

.• Salt wasting crisis in the first 2 weeks of life

~~~<bt~i •

..13&.u,,odSl)'ll;

&,21

..22

• First sign of the disease:Girls - born with ambiguous genitaliaBoys - hypovolemia, acidosis, hyponatremia, and

hyperkalemia (adrenal crisis)

,

2

.J. ... ''-'''.,.•

-- -- --------- --------- -- -- - - - -- -

Introduction: Clinical Picture

ii. SimpCe 'Virifizing

.;. Diagnosed as virilization at 3-7 yr

.;. Female - pubic hair, phallic enlargement, increasemuscle mass, and advanced bone age.

Boys - testicular size remains pre pubertal in CAH but

increases in central precocious puberty.

Introduction: Clinical Picture

iii. :Non C[assica[

• The mild NC form of 21-0HD.

• Females - diagnosed at or after adolescene- present with hirsutism, acne,

irregular menses, infertility

• Male not recognized.

3

~ ~_.. ~. ~~. ~··~T~~~·~~"F'1~~'ii!i~;~.~«:::*~"f.-;T::·,~,,~, T·-j~"--;·f'~~:~~~i!~'_~·!hW~a22p!!SS;~~q.21~~" ... '".' ,',',,~ .. ,-. ,.~," , . , , "" ' -, ""=''iim!:~fy':~:~'~.,.j!~':~~'" , ,r • -. ,,'~

I",'~' , '. ' , ,".. ; .r'" , -,.:" ,.,. ,t., ,'''' ," " " ' ,., • ,

.,

I

'" Phenotype graded accordingto clinical severity

.... ;.,~.:' . .. .

OBJECTIVE:

• To determine the presence of point mutations inpatients affected with conge'nital adrenal

hyperplasia

"r-:".•~~),..... ~.,",••••

4

Genotype and Phenotype Relationships

'" CYP21 mutations can be grouped into 3 categories according toenzyme activity

'" The relationship between genotype and phenotype in the commonmutation of the CYP21 gene.

Genotype Phenotype Activity enzyme(%)

Arg 356Trp • Salt wasting ~2

Gin 318StopIlel172Asn Simple virilizing 18~9

Pro30LeuVal281 Leu Non-classic 30-60

Normal

Genotyping can be used to predict the d«;gree of

disease severity patients affected with CAH

a. P30L

'" The mutations in exon 1

'" e~G (Proline) -7 to eIG (Leucine)

'" Associated with nonclassical 21-hydroxylase deficiency

... ~.;....... .,..-' .. -

- ---- - -----------------

.. '- ~ -~ '!f .~ :" .. 'l". ,"'.: .;~

5

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b. V281L

olo The mutations in exon 7

olo A change in codon 281 fromGTG (Valine) ~ TTG (Leucine)

olo The codon 281 mutation is associatedwith nonclassical 21-hydroxylasedeficiency

c. Q318X

olo The mutations in exon 8

olo Codon 318 in this gene is changed from

CAG ( glutamine) ~ lAG stop codon

olo The codon 318 mutation is associated

with salt wasting form in CAH

6

- - - ----------

Simple virilizing

A

Nonclassic

1 2

••I

P30L

3 45 6• •••L

1172N

7

•.,V281L

8 9 10•••Salt wasting

F306+1nt

R356W

031PX

B» c:

Exons »1-3,3-7

Exons ]I c:

6-101 kb

Schematic representation of 21-hydroxylase deficiency (CYP21) genestructure and location of the common mutations of the gene. The numbersoutside the box indicate exon number.

METHODS

Blood samples were obtained from patients referred to Hospital UniversitiSains Malaysia, Kelantan, Malaysia during 1995·2002. A thorough clinical

examination and hormonal analyses were performed. A total of 52samples included patients with external genitalia or electrolyte

'- --"i""mb~_. _

DNA extraction (non-phenol chloroformstandard procedure)

•

Polymerase Chain Reaction (peR). L-----,--------'

PCR·ASOH (Allele Specific OligonucleotideHybridization) mutational analysis using peR·

ASOH technique

DNA sequencing I

. ,:~. ~~.~. "

7

I

.1

RESULTS

1000 bp800bpSOObp

Figure 1: Presence of PCR product for CYP21 geneexon 1-3 using 2.0% agarose gel electrophoresis.Lane M : 100 bp. DNA ladder, lane N : negative control,lane 1: normal samples, lane 2·6 : samples showingPCR amplification products for CYP21 gene.

RESULTS

1000 bp _

800 bp

500 bp

Figure 2: Presence of PCR product for CYP21 geneexon 4-7 using 2.0% agarose gel electrophoresis. Lane M :100 bp. DNA ladder. lane 1 . negative control. lane 2:normal samples, lane 3.4.5,6.7 samples showing PCRamplification products for CYP21 gene.

8

RESULTS:

p • ii ~, .•

Figure 3: Dot blotting was performed using 1 ~g genomic DNA The blot,was hybridized with 100 pmol/ml of the digoxigenin labeledantiphosphatase (DIG-AP) specific probe from samples of patient 1-7,ASOH of PCR product from CAH patients was performed with theprobe V281L corresponding to the site exon, The status N: normal, P,patient.

RESULTS

3 4

NP •

•

----------

Figure 4: Pr030Leu hybridization DNA samples, Dot blotting wasperformed uSing 1 IJg genomic DNA, The blot was hybridized with100 pmol Iml of the digoxigenin labeled antiphosphatase (DIG-AP)specific probe Pr030Leu, ASOH of PCR product from CAH patientswas performed with the probe (Pr030Leu) corresponding to the siteexon 1, The status N: normal, P: patient.

9

RESULTS

\,?~~""f.)'~"""~;'ii''''liii~.. !~ • "'f< ~~ ~ .. ~, .... ~-~.1" '"., ... ;.

. '.",. .; . _.

p .. ,~ .

I .

Figure 5: 0318X hybridization DNA samples. Dot blotting wasperformed using 1 I-Ig genomic DNA. The blot was hybridized with 100pmol Iml of the digoxigenin labeled antiphosphatase (DIG-AP) specificprobe 0318X. ASOH of PCR product from CAH patients wasperformed with the probe 0318X corresponding to the site exon 8.The status N· normal, P: patient.

RESULTS

Table 1 : Identified mutations in samples analysed

Exon Codon Nucleotide Amino acid No. ofalteration changes samples

1 30 C~G-CIG Prol;ne- 2Leusine

7 281 CQT-CIT Valine - 3Leusine

8 318 G~A-GIA Glutamin - 1Stop codon

10

RESULTS

R356W0%"\ IIc172N

\0%

F306+t

Q3;~~t0e9:. -':;,::",,"-' 33'%

Val2811eu _ '50% .

Figure 6 : A pie chart showing percentage of CYP21 genemutation according to each exon.

RESULTS

Table 2: Frequency of CYP21 gene mutation in patients diagnosedwith congenital adrenal hyperplasia (CAH).

Diagnosis of CAH Mutation I Percentage (%l

Pro30Leu 2/52 ! 3.85!

Val281Leu 3/52 I 5.77I

Q318Stop 1/52 I 1.92

TOTAL 6/52

I11.54

----- - -------- -- ------ -- -----------------

11

RESULTS

, 0""'0/r-- "'2~{.

VaI281t.eU, .........~~S.77~ .~.__.~

'~~~,:~-~,~~=:,--~ProJOLeu, \

J."';; \.

._-~

no mulallon,8846"/.

./

/

__________..J

Figure 7 : A pie chart showing percentage of frequency for CYP21gene mutation in patients diagnosed with CAH.

RESULTS

Table 3: Relations between genotype &phenotype

Genotype Salt Simple Non-wasting virilizing classical

(SW) (SV) (NC)

Pro30Leu 0 0 2

Val281 Leu 3 0 0

Q318stop 1 0 0

Total 4 (66.67%) 0 2 (33.33%)

P)OL

f,

~rQJt8X

12

Frequency of common mutationsamong 21-hydroxylase deficiency alleles in different populations

1- -- -iotalno. patients P30L

-,- 394 i- - -2

:------- --1<:l218_~ ~35~~_J

4 4

Nationality

USA

Sweden

England

France

Finland

Italy

Italy (south)

Malaysia(USM,Kelantan)

Spain

Japan

China

Chile

Mexico

: Brazil

Argentina

400

220

258

102

146

50

52

58"

102

40

126

94

74

72

2

NA

3

o2

2

o

9

1172N

10

20

14

9

29

6

6

o

2

13

28

12

19

15

V281L

9

6

7

17

3

11

6

3

17

9

4

2

4

2

8

o8

9

4

11

14

3

NA

4- •• 0 •• __• _

o

3

13

10

11

7

8

6

CONCLUSION

'" Some variability in clinical expression can occa;ionally be seenamong patients with the same genotype but in the majority ofcases good relationships between CYP21 genotype and CAH

phenotype are found,

'" Therefore we find genotyplng of CYP21 to be very useful forprediction of clinical outcome in CAH patients

'" The possibility to predict disease outcome in CAH patients bymutations analyses has had several implications for treatment.

'" Our findings showed that patients with Pr030Leu mutations wereassociated with non-classical form of CAH whereas Val281 Leu

mutations were associated with salt wasting form of CAH,

--------- --- ---

13

PRESENTATION / PUBLICATION

1) Molecular Analysis of CYP21 Gene in Patients Presenting with AmbiguousGenitalia. MZ Fuziah, KM Yulia, A Rus Anida, MR Sidek, SF Ramli SF, MN Isa. Posterpresentation at European Society for Paediatric Endocrinology 41" Annual Meeting,Madrid on 25-28 September 2002

Publication Hormone Research 2002, 58 (suppl 2): 1-197 (International Journal ofExperimental and Clinical Endocrinology) P1-160, page 48.ISSN 0301-0163

2) Mutations of Pro30Leu and Val281 Leu of the CYP21 Gene in Patients Diagnosedwith Ambiguous Genitalia.

MN Isa. YK Muhamad, Fuziah MZ, Rus Anida A, M. Ros Sidek, S.F Ramli.Poster presentation at 4'" HUGO Pacific Meeting and 5'" Asia- Pacific Conference onHuman Genetics on 27-30.10 02 at Pattaya. Thailand.

Publication: Poster DY 16. abstract book 41n HUGO Pacific Meeting and 51h Asia- PacificConference on Human GeneticsISBN: 974-05-0173-7

PRESENTATION / PUBLICATION

3) Molecular Analysis in the Management of Congenital Adrenal Hyperplasia(CAH) and Ambiguous Genitalia.MN Isa, Y.K. Muhamad, Fuzlah MZ. Rus Anlsa A, M. Ros Sidek, S.F. Ramli, N. Adam.Journal of the Asean Federation of Endocrine Societies, Vol 20, No 1/2, Jan/July2002 (12-18). ISSN0857-1074

4) Detection of Point Mutation (Pr030Leu) in Exon 1 of the 21-hydroxylasegene(CYP21) in patient with Congenital Adrenal Hyperplasia using Digoxigeninsystem.Y.K. Muhamad, Fuziah MZ. Rus Anda A. M.R. Sidek , S.F., Ramli . NA Adam, M. N.Isa. Poster presentation at 13th National B;oatechnology Seminar ,1 0-13th November2001, Penang.

5) Molecular Analysis in Gender Assignment and Management ofAmbiguous Genitalia. Y.K Muhamad . Fuziah MZ , Rus Anda A" M.R. Sidek , S.F.,

Ramli, NA Adam, M. N. IsaPoster presentation at First Asean Conference On Medical Sciences, , 18-21 May2001 ,Kota Bharu, Kelantan.

14

" Suppkmcnt kIf SubliCnhcl's ti"cc ofchargt>. ".

58 IS21 02Released Augnst 2002"tSSN 0301':'01635~(Sllppl 2) I-IlJl3 (20t)2)

International Journal ofExperimental and

Clinical Endocrinology

\. Kalgcl

~tedjLal and '·icn(ifit.PUH1"h,~r"

Ba~,,:l ~ Fr..nburgPari ... • L,lrh!\,!l'\c'-\ Y", ~ • :-,,,,, Delhi

Hangk\lJ,.: • Sin·gapon.:r;lb.,). ,\ dn,~\

41 st Annual Meet ing of the

Ellropean Society forPaediatric Endocrinology(ESPE)Madrid. SpJin, 25-28 September. 2002

Guest Editor:Jesl....s Argente, MJdrid (Sptlin)

-------- ------ -- --- ----

I III

i I

I

IIIII

I

Supplement for subscribers free of charge

58152102Released August 2002ISSN 0301-016358(suppI2) 1-198 (2002)

International Journal ofExperimental and

Clinical EndocrinologV

S, K<lrgcrMedical and ScientilicI'uhli,hcr,Basel, Freiburg "Paris· LondonNew York· New DelhifJangkok'Singapore'lokyo • Sydney

41st Annual Meeting of the

European Society forPaediatric Endocrinology(ESPE)

•Madrid, Spain, 25-28 September, 2002

Guest Editor:

Jeslls Argente, Madrid (Spain)

------------- - ----- - -

iII

Ii I

II

P1-160 Poster Session 1

MOLECULAR ANALYSIS OF CYP21 GENE IN PATIENTSPRESENTING WITH AMBIGUOUS GENITALIAF. Md. Zain 1; Y KM2; R. A3; M. Sidek2; S. RamlP; M. Isa2

lPaediatrics, School of Medicine, Health Campus, Universiti SainsMalaysia, Kelantan, Malaysia; 2Human Genome Centre, School ofMedicine, Health Campus, Universiti Sains Malaysia, Kelantan,Malaysia; 3Paediatrics, Hospital Kota Bharu, Kelantan, Malaysia

Introduction: Congenital adrenal hypcrplasia (CAH) is a group of autosomalrecessive disorders of adrenal stcroidogcncsis. The genes of the steroidogenicenzymes and the Illutations involvcd havc bccn described. Deficiency of the 21­hydroxylase (2 I-OH) enzymc is hy I'm thc most common form of CA H whicharises as a result of dcletions or dcletcrious mutations in the active gene (CYP21)located on chrolllosome 6p. Many difrcrent mutations of the CYP21 gene causevarying degrees in impairment or 2 I-OH activity that results in a spectrum ofdisease expression. There is no sharp limit between the salt-wasting, the simplevirilizing and the late onset forms. Objective: To determine the 21-0Hdeficiency mutation defects and correlate the genotype with their phenotypicexpression of the disease. Patients/Material and Methods: We performedmutational analysis using Polymerase Chain Reaction - Allele SpecificOligonucleotide Hybridization (PCR-ASOH) technique on 6 pat"ients whopresented with ambiguous genitalia (AG) and or electrolyte rl~nmgement ashyponatraemia and hyperkalaemia, suspected to have CAH. The Val28 I Leu andPr030Leu mutations result in enzymes with 20-60% of normal activity and bothare associated with the non-classical form of CAH. The Gln3 I8stop mutation iscategorized under the salt-wasting typc. Results: Among the 6 patients, 3 hadVal28 ILeu mutation, 2 had Pro]OLcu Illutation and I had Gin] 18stop mutation.The 3 patients with Val2S ILcu Illutation had prescnted with adrcnal crises duringinfaricy and was classified as salt loscrs and treated with glucocorticoids andmineralocorticoids. Thcsc ] paticnts eould well be the other 400/0 who arecategorized as salt losers. The 2 pat icnts wi th Pro30Leu mutations have normalmale external genitalia and prcscntcd with hyponatraemia and hyperkalaemia.Only I of them rcquired Illincralocorticoids that was given for about 5 monthsduration. Subscqucntly hc had normal clcctrolytcs evcn withoutmineralocorticoid therapy. Thc Gln318stop mutation was identified in onepatient who presented with AG (ll1d adren,t1 crises. Conclusions: Our studyshowed that the patients with genotypc Val2S ILeu, Pr030Leu and Gln318stopmutations correlated with their phcl1otype. Thc mutation analysis of CYP21 geneproved to be a good complementary investigation and supportive to the diagnosisand management of our CAH paticnts.

P1-161 Poster Session 1

PROPOSAL FOR PRENATAL MANAGEMENT OF_"""llI~C'lI-IIJloll.L _lLJIrl .... IU&.a1 ..,.". ...... 1'4:_1 U_I._" 1.-." .... ' 11"1'!l' _ _ _ _ _ _ _ • _. _ _ •• • • _... __.-. '" ....... ,""'". 1/ •• ,.

EUROPE BASED ON THE EXPECTED NUMBER OFPATIENTS ----_.- --------

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