Molecular Studies of a Patient with Complete Androgen Insensitivityand a 47,XXY Karyotype

C. M. Girardin, MD, C. Deal, PhD, MD, E. Lemyre, MD, J. Paquette, MSc, R. Lumbroso, MSc, L. K. Beitel, PhD,

M. A. Trifiro, MD, and G. Van Vliet, MD

A phenotypic female with complete androgen insensitivity from a maternally inherited mutation in the androgenreceptor had a 47,XXY karyotype. Partial maternal X isodisomy explained the expression of androgen insensitivitydespite the presence of 2 X chromosomes. (J Pediatr 2009;155:439-43)

In complete androgen insensitivity syndrome (CAIS,OMIM 300068), 46,XY individuals with 2 normally differ-entiated testes have normal female external genitalia but

a short blind-ending vagina with absence of Mullerian struc-tures. At puberty, they develop breasts but have primaryamenorrhea and no or very limited pubic and axillary hair de-velopment. The prevalence of CAIS is estimated to be between1 in 20 000 and 1 in 60 000 births. It is due to mutations in theandrogen receptor (AR) gene, which is located on the X chro-mosome on Xq12. These mutations may occur de novo orbe inherited, in which case the mothers are asymptomaticcarriers and the condition is transmitted as an X-linked disor-der.1On the other hand, Klinefelter syndrome (KS), describedin males with tall stature, eunuchoid proportions, small testes,and infertility and associated with the 47,XXY karyotype,occurs in approximately 1 in 1400 births.2

We report here a phenotypic female with CAIS who hada 47,XXY karyotype and review 6 previously reported cases. Inour patient, molecular studies established that partial maternalX isodisomy explained the expression of the CAIS phenotype.

Case Report

At surgery for bilateral inguinal hernias, two testes werefound in a 3-year-old girl. Pelvic examination showednormal female external genitalia, a very short vagina, andabsence of the uterine cervix.

The patient was the only child of unrelated Caucasian par-ents. There was no history of infertility or of ambiguous gen-italia in the family. The patient was born at 41 weeks after anuneventful pregnancy with a birth weight of 3.4 kg and nor-mal female genitalia. Her mother was 23 years old at delivery.The patient was described by her mother as an outgoing girlwho liked to play with her friends at daycare. Height was onthe 75th percentile of the female standards (for a target heighton the 40th percentile).3 There were no dysmorphic features.At 3 years of age, 5 days after 2900 U/m2 intramuscular of hu-man chorionic gonadotropin, plasma testosterone was 8.37

AR Androgen receptor

CAIS Complete androgen insensitivity syndrome

KS Klinefelter syndrome

nmol/L, androstenedione was 1.4 nmol/L, and dihydrotes-tosterone was 0.85 nmol/L, confirming the presence of func-tional Leydig cells and making an enzyme block unlikely.

CAIS was confirmed when sequencing of lymphocyteDNA (Figure 1, A) revealed a novel mutation in exon 5 ofthe androgen receptor gene, c.2560A > T (p.Gln733Leu)(nomenclature according to the Androgen Receptor GeneMutation Database, http://androgendb.mcgill.ca/). Themother was a carrier for this mutation (not shown).

The patient’s lymphocyte karyotype was 47,XXY (30 mito-ses), which is generally associated with KS. This wasconfirmed on a second blood sample (10 mitoses + Fluores-cent in situ hybridization).

A mild delay in language development prompted speechtherapy between ages 3 and 5 years. Because of recurrent her-nia, a left gonadectomy was performed at age 6 years. Theright gonad was identified but left in the abdomen. Pathologyconfirmed that the left gonad was a testis with normal archi-tecture and seminiferous tubules. The interstitial tissue wasdescribed as slightly edematous and fibrous.

When last seen at 11 years of age, school progression wasnormal, but attention deficit disorder was diagnosed. Heightwas at the 90th percentile for females and Tanner stage wasB3P1. The upper to lower segment ratio was 0.89 (N forage: 1.0).4 Plasma hormone levels were as follows (normsfor Breast/Genitalia Tanner stage 3 females/males): FSH,75.6 UI/L (1.5 to 12.8/1.2 to 5.8); LH, 28.7 UI/L (0.1 to12.0/0.2 to 5.0); testosterone, 28.4 nmol/L (0.5 to 1.2/3.5 to11.1) (http://www.esoterix.com/files/expected_values.pdf).Bone age was 11 years according to female standards.5

Molecular StudiesExon 1 of the AR is known to contain polyglutamine (pol-yGln) and polyglycine (polyGly) tracts that are polymorphic.To determine whether the patient’s two AR alleles were both

From the Endocrinology (C.G., C.D., J.P., G.V.) and Genetics (E.L.) Services, CHUSainte-Justine and Department of Pediatrics, University of Montreal; and Lady DavisInstitute SMBD-Jewish General Hospital and Department of Medicine (R.L., L.B.,M.T.), McGill University, Montreal, Quebec, Canada

The authors declare no conflicts of interest.

0022-3476/$ - see front matter. Copyright ª 2009 Mosby Inc.

All rights reserved. 10.1016/j.jpeds.2009.02.052

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Figure 1. A, Sequencing of patient’s exon 5 of AR showing mutation c.2560A > T; p.Gln733Leu. B, Sequencing of exon 1 of AR.Patient’s (B1) and mother’s (B2) polyglutamine tracts. Patient’s tract shows 25 glutamine codons, whereas both 25 and 26codons are seen in the mother. C, Patient’s (C1) and mother’s (C2) polyglycine tracts. Patient’s tract shows 24 glycine codons,whereas both 24 and 25 codons are seen in the mother.

of maternal origin and were identical (maternal isodisomy),as suggested by the sequence (Figure 1, A), we first deter-mined the length of these tracts in both the patient and hermother. Paternal DNA was not available.

PCR amplification was performed as previously described.6

Primer sequences used for the glutamine tract amplification

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were: 1.1A-forward: 5’GTG GAA GAT TCA GCC AAG CT 3’;1.1B-reverse: 5’TTG CTG TTC CTC ATC CAG GA 3’. Primersequences used for the glycine tract amplification were: 1.4A*-forward: 5’ CCA GAG TCG CGA CTA CTA CAA CTT TCC3’; 1.4B*-reverse: 5’ GGA CTG GGA TAG GGC ACT CTGCTC ACC 3’ (Invitrogen, Carlsbad, California).

Girardin et al

September 2009 CLINICAL AND LABORATORY OBSERVATIONS

Xp22.33

Xp22.22

Xp21.1Xp11.4Xp11.3

Xp11.22

Xq12

Xq13.1

Xq21.1

Xq22.3

Xq26.1

Xq27.1

Pseudo AutosomalRegion (PAR1)

PAR

Xq21.3

Markers Locations Heterozygosity GenotypeMarshfieldmap

(cM) Repeat

DXS7100

DXS1053

CYBB

DXS1003

AR

DXS981

DXS1047

DXS102

Xp11.3

Xp22.22

Xp22.33

Xp21.1

Xq12

Xq13.1

Xq26.1

Xq27.1

13,50

22,18

47,08

0,80

82,07

HETERO.

0, 68CA

CA

TG

CA

CA

GT

0,76

0,79

0,81

0,71

CAG

GATA

0.90

0,84

HETERO.

HETERO.

HETERO.

HOMO

HOMO

HOMO

HOMO

DXS1039

DXS1204

Xp11.23

Xp11.22

50,33

52,50 CA 0,80 HOMO

HETERO.0,61CA

Figure 2. Ideogram of human chromosome X showing the location of the informative microsatellite markers used in this study.The interval between makers DXS1039 and DXS1204 contains the point where transition from maternal heterodisomy to ho-mozygosity occurred in our subject.

PCR products were purified via a 1.2% agarose gel electro-phoresis. The DNA bands were cut out and purified using theQiagen Gel Purification Kit (Valencia, California). Sequenc-ing analysis of the purified PCR products was carried out atthe McGill University and Genome Quebec Innovation Cen-ter using the 1.1B-reverse and 1.4B*-reverse primers for theglutamine and glycine tracts, respectively.

The mother was found to have 2 different polymorphismsfor each tract (polyGln: 25 and 26 repeats; polyGly: 24 and 25repeats). By contrast, the patient had only one polyGln andpolyGly tract (25 glutamines and 24 glycines, respectively)(Figure 1, B and C). These results demonstrate that the pa-tient was homozygote for AR and likely inherited 2 identicalX chromosomes from her mother, whereas the mother had 2different AR alleles, as expected.

To confirm that the AR homozygosity was due to the inher-itance of 2 identical maternal X chromosomes (maternal iso-disomy), we performed an extensive X chromosome

Molecular Studies of a Patient with Complete Androgen Insensi

microsatellite analysis on peripheral blood leukocyte DNAfrom the patient and her mother. PCR conditions were opti-mized for 15 microsatellite markers (DXS7100, DXS1053,CYBB, DXS538, DXS1068, DXS1003, DXS1039, DXS1204,AR, DXS981, DXS1125, DXS986, DXS1120, DXS1047, andDXS102) chosen for their high degree of heterozygosity(mean = 78%) and their allele frequencies (#47%). Most mi-crosatellites were amplified with commercially availableprimers (MapPairs Human Markers) through InvitrogenCorp. (Burlington, Ontario, Canada), as previously described.7

Although there was indeed homozygosity of the entire longarm (Xq) as well as the proximal short arm, consistent withour hypothesis of a maternal isodisomy, it did not includethe entire X chromosome. All the Xp marker genotypes telo-meric to Xp11.22 were heterozygous and identical betweenmother and child. The crossing-over event occurred in theinterval between markers DXS1204 and DXS1039(Xp11.22–Xp11.23) (Figure 2).

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Table. Reported cases of AIS with 47,XXY karyotype

Genitalia

Ref (nb) Age (y) History External Internal Hormone levels AR sequencing Cytogenetic studies

(13) 34 Not reported. Normalintelligence

Tanner B1, scarcepubic hair,hypoplasia of labialfolds, clitoris andvagina

Echography andlaparoscopy:absence of uterus,ovaries andWolffian andMullerian ducts

FSH 36.24 mIU/mL,LH 10.31 mIU/ml,Testosterone 1.11nmol/L

AR gene notsequenced

No cytogenetic studiesto investigate Xmaternal isodisomy.Structurallyunaltered X and Ychromosomes witha normal SRY gene

(14) 30 Primary amenorrheaand infertility

Tanner 1. No sign ofvirilization

Echography: no uterus. FSH 46.3 mIU/mL 2 mutations in ARgene:

Microsatellite markerson Xq showed onlyone allele at each ofthe 5 loci examined.Results probablyreflect maternal non-disjunction inmeiosis 2 (parentalDNA not available)

Pathology: smalltestes, no Mullerianor Wolffianderivatives, fewdegeneratingseminiferous tubules

LH 19.2 mIU/mL,Exon 4: c.2280C >

T; p.Gln640Stop.Testosterone 3.46nmol/l Exon 5: c.2615 G >

A; pTrp751Stop

(15) 11 Clitoromegaly,bilateral inguinalhernias containingmasses since age 3mo (no surgery).Learning difficulties,language delay

Tanner I,clitoromegaly

Echography: no uterus,no ovaries.

Testosterone onceelevated at 3.71nmol/L, then normalat 0.87 nmol/L

AR gene notsequenced.

Southern blot of Xchromosomal DNA:double dosage ofa single maternalallele, probablyreflecting maternalnondisjunction inmeiosis 2

MRI: inguinal testes.Pathology: notavailable

(16) 35 Primary amenorrhea,operated for rightinguinal hernia atage 14 y

No sexual hair,breasts welldeveloped, no signof virilization

NA Urinary FSH >50IU/24 h,

AR gene notsequenced

None

Urinary LH >50IU /24 h,Plasma testosterone3.99 nmol/L

(18) 23 Primary amenorrhea Small breasts,clitoromegaly,blind-endingvagina

Laparoscopy: nouterus, no ovaries.

NA AR gene notsequenced

None

Pathology: bilateraltestes

(20) 12 Monozygotic twins,inguinal hernias atage 4

Normal femaleexternal genitalia

Surgery: no uterus. NA AR gene notsequenced

NonePathology:prepubertal testis(age 4 y)

Discussion

The patient reported here has 2 different medical conditions.She has CAIS, which was confirmed by the identification ofa point mutation in AR, and she also has a 47,XXY karyotype,which is associated with KS in males.

Our patient’s AR mutation (c.2560A > T; p.Gln733Leu,numbering according to Lubahn8) is not yet described inthe Androgen Receptor Gene Mutation Database.9 However,two different mutations of the same amino acid are de-scribed: p.Gln733STOP in a patient with CAIS andp.Gln733His in a patient with partial AIS.10

Aside from mild speech delay, relatively long legs, andattention deficit disorder, the patient did not appear tohave any of the physical or psychosocial characteristics thatare reportedly more frequent in 47,XXY males.2 At 11 yearsof age, the testosterone and LH levels were high, as expectedin androgen insensitivity. The high FSH level likely reflectstesticular dysgenesis in the context of the 47,XXY karyotype.

CAIS is an X-linked condition, and 46,XX females who arehemizygous for AR mutations are normal. Two differenthypotheses could explain a CAIS phenotype in a 47,XXY

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individual. First, homozygosity for the mutated AR geneimplies either complete or partial maternal isodisomy. Com-plete maternal isodisomy, meaning inheritance of 2 identicalX chromosomes, occurs either through a nondisjunction inmaternal meiosis 2 or an early postzygotic nondisjunctionof the X chromosome. These mechanisms are estimated tobe responsible for only 9% and 3% of cases of KS, respec-tively.11 Partial maternal isodisomy occurs when a recombi-nation process happens after a meiosis 1 nondisjunction. Thesecond hypothesis involves two different X chromosomeswith two different AR alleles but with skewed X inactivationof the nonmutated X. In our patient, this was less likely giventhat AR mutation analysis did not suggest the presence ofa normal allele on sequencing.

The analyses of the polymorphic regions of exon 1 of theAR gene showed that the patient has two identical AR alleles.The microsatellite analyses performed along the X chromo-somes surprisingly showed only partial X maternal isodis-omy. The power of discrimination of the analysis calculatedwith 5 of 14 microsatellites was estimated at 1.6 � 10�8.Thus, the chance of having a paternal X is null. The molecularmechanism leading to this event must include both

Girardin et al

September 2009 CLINICAL AND LABORATORY OBSERVATIONS

a nondisjunction in maternal meiosis 1, leading to maternalheterodisomy, followed by a recombination event betweenthe 2 homologous maternal chromosomes in mitosis, earlyafter fertilization. Interestingly, the crossing-over event,which occurred in a Mitelman breakpoint junction atXp11, is seen in a variety of tumors.12 The significance ofthis finding for the future health of our patient is unknown.

The estimated prevalence of the 47,XXY karyotype is ap-proximately 1 in 1400 births. On the other hand, the preva-lence of CAIS is estimated to be at most 1 in 20 000 births.Thus, the probability of having the 2 conditions by chanceis about 1 in 28 000 000 births. Surprisingly, when doinga PubMed search with the key words ‘‘47,XXY AND andro-gen insensitivity syndrome’’ and ‘‘47,XXY AND testicularfeminization,’’ 8 entries were found.13-20 However, 2 papersreport the same case.16,17 One other case had a mosaic46,XY/47,XXY karyotype.15 Thus, 6 cases (counting 1 pairof monozygotic twins as 1) similar to ours have been pub-lished since 1966 (Table). It is of note that in only 1 of thesewas AR sequenced. Interestingly, testosterone levels were of-ten lower than expected in androgen insensitivity. This couldbe explained by testicular dysgenesis due to the 47,XXY kar-yotype. In the patient reported here, particular attention willbe paid to the pathological analysis of the right gonad, whichwill have to be removed after puberty.

To conclude, we report a child with CAIS and a 47,XXYkaryotype and review 6 previously reported cases. In ourpatient, we show that partial maternal X isodisomy led tohomozygosity for the causative AR mutation. Whether theassociation of CAIS and a 47,XXY karyotype occurs moreoften than by chance cannot be determined with certainty.However, there is no known association between AR muta-tions and nondisjunction and, in addition, the AR genedoes not seem to be located in an important pairing zoneon the X chromosome.21

n

We thank Drs. Bruce Gottlieb and Scott D. Grosse for helpful discus-sions.

Submitted for publication Aug 11, 2008; last revision received Feb 2, 2009;

accepted Feb 25, 2009.

Reprint requests: Dr Guy Van Vliet, Research Center, CHU Sainte-Justine,

3175 Cote Sainte-Catherine, Montreal, H3 T 1C5, Quebec, Canada. E-mail:

guy.van-vliet@recherche-ste-justine.qc.ca.

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