I

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I MOLECULAR ANALYSIS of CYP21 GENE inPATIENTS PRESENTING with AMBIGUOUS

GENITALIA

MZ FUZIAH 1, KM YULIA2

, A. RUS ANIDA3, MR SIDEK2

, SFRAMLI2

, MN ISA4

1 Department ofPaediatrics,2 Human Genome Centre, .SchoolofMedicine, USM, 16150 Kubang ](erian, 3Department ofPaediatrics, Hospital [(ota Bharu, ](elantan, 4]nternational

Medical University, Putrajaya, Malaysia.

lema ..../\>1;ft.' , ., ~~",;

}i\r~'~:iL1~[~.;~;;· >: )\"I~f~;::~Congenital adrenal hyp~rpJasia (CAH) is a group ofaut9$_OJ1)~~{J~'c"': ; ,; :}:' ~;;'i~~iJ?' f

disorders of adrenal steroidogenesis. The genes of the st~~=-i~:'. 1enzymes and the mutations involved have been described. DetI~I~' fthe 21-hydroxylase (21-QH) enzyme is by far the most commpn, fQ, . ~.

CAH which arises as a result of deletions or deleterious. mutations 'iQ~ ....,~ Wactive gene (CYP21) located on chromosome 6p. Many different mutatipnsof the CYP21 gene cause varying degrees of impairment of 21-0H activitythat results in a spectrum of disease _expression. There is no sharp limitbetween the salt- wasting, the simple virilizing and the late onset forms.

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The objective of our study was to determine the 21-0H deficiency mutationdefects and correlate the genotype with their phenotypic _expression ofthe disease.

We performed mutational analysis using PCR-ASOH (Polymerase ChainReaction - Allele Specific Oligonucleotide Hybridization) technique on sixpatients who presented with ambiguous genitalia (AG) and or electrolytederangement as hyponatraemia and hyperkalaemia, suspected to haveCAH. The Val281 Leu and Pr030Leu mutations result in enzymes with 20­60% of normal activity and both are associated with the non-classical formof CAH. The Gln318stop mutation is categorized under the salt-wastingtype.

II

Among the six patients, three had Val281 Leu mutation, two had Pr030Leumutation and one had Gln318stop mutation. The three patients withVal281Leu mutation had presented with adrenal crises during infancy andwas classified as salt losers and treated with glucocorticoids andmineralocorticoids. These 3 patients could well be the other 40% who arecategorized as salt-losers. The two patients with Pro30Leu mutations havenormal male external genitalia and presented with hyponatraemia andhyperkalaemia. Only one patient required mineralocorticoid therapy thatwas given for about 5 months duration. Subsequently he had normalelectrolytes level even without mineralocorticoid therapy. The Gln318stopmutation was identified in one patient who presented with ambiguousgenitalia and adrenal crises.

Our study showed that the patients with genotype Val281 Leu, Pr030Leuand Gln318stop mutations correlated with their phenotype. The mutationanalysis of CYP21 gene proved to be a good complementary investigationand supportive to the diagnosis and management of our CAH patients.

Keywords: a1TJbiguou.s,9.fJ,.nitalia, congenital adrenal hyperplasia, CYl121

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Ambiguous genitalia (AG) are one of the clinical presentations ofcongenital adrenal hyperplasia (CAH). Congenital adrenalhyperplasia is an autosomal recessive disease caused by logsor severe decrease in the activity of 21-hydroxylase (21-0H).This enzyme is one of the five enzymes necessary for cortisolbiosynthesis. Deficiency of the 21-0H is the most common formof CAH accounting for 90-95% of all cases of CAH1

,6.

Congenital adrenal hyperplasia presents a wide spectrum ofclinical manifestations and patients are divided into 3 groups:salt wasting (SW), simple virilizing (SV) and non-classical (NC).Salt wasting patients manifest as neonatal electrolytedisturbances together with virilization of external genitalia atbirth in girls and early pseudoprecocious pUberty in boys, whileSV patients present the same manifestation as SW patients, butwithout electrolyte disturbances. Non- classical patients presentwith late onset symptoms of androgen excess, ranging fromprogressive virilization and pseudoprecoclous puberty inchildhood to menstrual disturbances, infertility and hirsutism inadult women2

•

It was reported that more than 90% of cases of CAH are causedby mutation of the CYP21 gene. This 21-0H (CYP21) gene islocated in the HLA class III gene region on chromosome 6p21.3and consist of 10 exons3

• Majority of the mutations on theCYP21 gene are Val281 Leu and Pr030Leu. These mutations werereported to result in 20-60% of normal enzyme activity and bothare associated with the non-classical form of CAH4,5.

To detect the presence of point mutations in Pr030Leu,lIe172Asn & Val281 Leu of CYP21 gene

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Figure 1: Presence of PCR product (873 bp) for CYP21 geneexon 1-3 using 2.00/0 agarose gel electrophoresis. Lane M : 100bp DNA ladder, lane N : negative control, lane 1, 2 : normalsamples, lane 3,4,5,6 : samples showing PCR amplificationproducts for CYP21 gene.

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Figure 2: Dot blotting was performed using 1 IJg genomic DNA.The blot was hybridized with 100 pmollml of the digoxigeninlabeled antiphosphatase (DIG-AP) specific probe from samplesof patient 1-7. ASOH of PCR product from CAH patients wasperformed with the probe corresponding to the site exon. Thestatus N: normal, P: patient.

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Figure 3: Pr030Leu hybridization DNA samples. Dot blottingwas performed using 1 IJg genomic DNA. The blot washybridized with 100 pmol Iml of the digoxigenin labeledantiphosphatase (DIG-AP) specific probe Pr030Leu. ASOH ofPCR product from CAH patients was performed with the probe(Pr030Leu) corresponding to the site exon 1. The status N:normal, P: patient.

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Table 1

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Neonataljaundice

IAmbiguousgenitaliaProminentphallusSingle orificeat its baseNo palpablegonadsRegistered asmaleReassigned asfemale

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92

10 weeks..---...--.._._-.-..

Failure tothriveAmbiguousgenitaliaPhallus 2 cmSingle orificeat its baseNo palpablegonads

At birth

131

Normal maleexternalgenitalia

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2 months

119

HaematuriaSalt lossNormal maleexternal

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110

81 daysI .---. -- ...--..

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Normal maleexternal

, genitaliai Both testesI descendedi Sepsis,: meningitis &

adrenalcrisis

120

AdrenalcrisisDefaultfollow upCPPMentalRetardation

32 days

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650(138-690)

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Majority of the mutations in the CYP21 gene in ourpatients are Val281 Leu and Pr030Leu. These mutationswere reported to result in 20-600/0 of normal enzymeactivity and both are associated with the non-classicalform of CAH4

,7.

In our study, we found that 3/52 patients (5.7%) haveVal281 Leu while 2/52 patients (3.8%) have Pr030Leurespectively. No mutations were observed in lIe172Asn.Patients with Val281 Leu presented with adrenal crisisduring infancy and were classified as salt wasting. Twopatients with Pr030Leu mutations have normal maleexternal genitalia and presented with hyponatraemiaand hyperkalaemia. We did not identify any patient with1172N mutation which is known to result in clearlyreduced enzymatic activity. About 1-2% of 1172Nmutation is usually associated with the SV form 8

,9.

Our findings showed that patients with Pr030Leumutations were associated with non-classical form ofCAH whereas Val281 Leu mutations were associatedwith salt wasting form of CAH.

This study suggested that phenotypes are not alwaysconcordant with the genotype in patients withVal281 Leu mutations dfagnosed with CAH.

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' .... " '. ~This project is supported by USM short-term grant304/PPSP/6131117

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1. M.Natividad Lobato et al. 1999: Mutation Analysis in-patients withcongenital adrenal hyperplasia in the Spanish Population: Identification ofputative novel steroid 21-hydroxylase deficiency alleles associated withthe classic form of the disease. Human Hered .49: 169-175.

2. Hughes lA, Clark AJLgeds, 2000: Adrenal Disease in Childhood. Clinicaland Molecular Aspects. Endocr Oev Basel} Karger, Vol 2, 93-111.

3. Phyllis W.Speiser et al. 1992: Disease expression and molecular genotypein congenital adrenal hyperplasia due to 21- hydroxylase deficiency. J ClinInvest 90: 584-595.

4. Anna Nordenstrom et a!. 1999: Genotyping Is Valuable DiagnosticComplement to Neonatal Screening for Congenital Adrenal Hyperplasiadue to 21- hydroxylase deficiency. J Clin Endocrinol Metab 84: 1505­1509.

5. Owerbach 0, Sherman L, Ballard AL, Azziz R. 1992.Mutation in CYP21 isassociated with nonclassical steroid 21-hydroxylase deficiency. MolEndocrinol 6 : 1211-5

6 A Wedell. 1998. Molecular genetics of congenital adrenal hyperplasia (21­hydroxylase deficiency): implications for aiagnosis, prognosis andtreatment. Acta Paediatr 87: 159-64.

7. Selma F. Witchel, Rhonda Smith, Carlo Enrico Crivellaro, Thais DellaManna, Vae Dichtchekenian, Nuvarte Setian, Durval Damiani. 2000.CYP21 mutations in Brazil patient with 21-hydroxylase deficiency. HumGenet 106:414-419,

- 8. Jarmo Jaaskelainen, Antti Levo, Raimo Voutilainen, Juka Pertanen, 1997.Population -Wide Evaluation of Disease Manifestation in relation toMolecular Genotype in Steroid 21-hydroxylase (CYP21) Deficiency: GoodCorrelation in well Defined Population. J. Clin. Endocrinol. Metab 82:3293-3297.

9. Catherine' Deneux, Veronique Tardy, Anne Dib, Etienne Mornet, LineBillaud, Daniel Charron, Yves Morel, Frederique Kuttenn.2001.Phenotype­Genotype Correlation in 56 Women with Nonclassical Congenital AdrenalHyperplasia due to 2.1-Hydroxylase Deficiency. Journal of Clinical.Endocrinology & Metabolism: 86;207-213.

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