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Endocrinological Research Center (A.T., N.K., T.S., I.D., G.K., V.P.), Engelhardt Institute of Molecular Biology, Russian Academy of Sciences (P.S., P.R.), and Research Center for Medical Genetics (A.P.), 117036 Moscow, Russian Federation
Address all correspondence and requests for reprints to: Anatoly Tiulpakov, M.D., Ph.D., Institute of Pediatric Endocrinology, Endocrinological Research Center, Ulitsa Dmitriya Ulianova, 11, 117036, Moscow, Russian Federation. E-mail: ant{at}endocrincentr.ru.
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Abstract |
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Objective: The objective was to study the molecular basis of AES in a kindred with 16 affected subjects, both males and females.
Patients: The propositus, currently a 17-year-old boy, presented with breast enlargement in the first year of life, which persisted thereafter. Investigations at the age of 7.5 yr revealed growth acceleration (height SD score, 2.8), puberty staging Tanner P1B3, testicular volume 6 ml, and bone age 13 yr. The hormonal data were compatible with increased conversion of androgens to estrogens, which was independent of gonadotropin secretion. In the affected adults, there were short stature (height SD score ranged from –3.7 to –2), gynecomastia in males, and macromastia in females.
Design: Linkage analysis was performed using a polymorphic tetranucleotide (TTTA) repeat marker at nucleotide position 682 of CYP gene, as well as two additional STS markers, D15S123 (CA)n and D15S209 (CA)n, located within genetic distance of less than 5 cM from CYP19 gene. Using RNA extracted from the breast tissue of the propositus, a 5'-rapid amplification of cDNA ends (RACE) was performed with gene-specific primers corresponding to exon 2 of CYP19 gene.
Results: Linkage analysis with (TTTA)n, D15S123 (CA)n, and D15S209 (CA)n markers produced LOD scores 0.85, 1.5, and 1.17, respectively. 5'-RACE revealed a novel untranslated exon 1 composed by exon 1 of TRPM7 gene (Transient Receptor Potential Cation Channel, Subfamily M, member 7), which has ubiquitous expression.
Conclusions: 5'-RACE finding points to a potential rearrangement between CYP19 and TRPM7 genes on chromosome 15q21.2 as a cause of AES.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Although increased activity of aromatase in patients with AES has been postulated years ago, the pathogenesis of this disorder until recently has not been clarified. Linkage of the AES with the CYP19 has been evaluated in one study, in which a weak segregation with CYP19 gene (LOD score of 0.6) was found (8). P450 aromatase, the product of the CYP19 gene on chromosome 15q21.2, catalyzes the key step in the estrogen biosynthesis pathway, conversion of C19 steroids androstenedione, and testosterone (T) to C18 steroids estrone and estradiol (E2), respectively. The CYP19 gene is composed of nine coding exons (II–X) and alternative untranslated exons I distributed over a large 93 kb fragment upstream of its coding region (9). Aromatase mRNA is expressed in various human tissues, such as ovaries, brain, skin fibroblasts, adipocytes, liver, breast, and testes. The level of expression is dependent on alternative use of multiple promoters, which regulate transcription of tissue-specific mRNA species produced by splicing of each of the first exon onto a common splice acceptor site in the exon II.
This complex organization of the CYP19 gene led to a hypothesis that the disease is caused by abnormal expression of aromatase, possibly due to some defect in the 5'-untranslated region of the gene. Shozu et al. (10) have reported recently two families with AES in which they identified two distinct chimeric CYP19 transcripts containing untranslated exons 1 of genes TMOD3 and FLJ14957 These transcripts are believed to originate from heterozygous inversions at 15q21.2–15q21.3, which in turn resulted in expression of CYP19 from ubiquitous promoters of the above genes.
Here we describe a large kindred, in which AES was reported in 16 subjects in five generations, in both males and females. From the breast tissue of the proband, we obtained a novel chimeric CYP19 transcript containing exon 1 of another gene at chromosome 15q21.2, TRPM7 (transient receptor potential cation channel, subfamily M, member 7) (or LTRPC7, for long TRP channels) (11), also known as CHAK1 (channel-kinase 1) (12, 13).
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Subjects and Methods |
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The propositus, currently a 17-yr-old boy, had presented at our center at the age of 7.5 yr with heterosexual precocious puberty. According to his parents, breast tissue was present in his first year of life; it was initially recognized as postnatal influence of maternal estrogens but then progressed slowly. At the age of 5 yr, he was referred to a local pediatric endocrinologist. At that time, his height was 120 cm [height SD score (HtSDSCA), 2.5], puberty staging Tanner G1P1B3, testicular volume (Te) 4 ml, and bone age 10 yr (HtSDSBA, –2.7) (Table 1
). At the time of the referral to the Endocrinological Research Center at the age of 7.5 yr, his height was 139 cm (HtSDSCA, 2.8), weight 33.2 kg (SDSCA, 2.1), puberty staging Tanner G1P1B3, Te 6 ml, and bone age 13 yr (HtSDSBA, –1.8). His karyotype was 46, XY. The adrenals appeared to have normal size and structure at ultrasound and magnetic resonance imaging (MRI). The structure of testes, as evaluated by ultrasound, was normal. There were no signs of Peutz-Jeghers syndrome. Hormonal investigations revealed prepubertal basal level of T [T, 16.7 ng/dl (0.6 nmol/liter)] increasing to Tanner 2–3 level [T, 153 ng/dl (5.5 nmol/liter)] (14) after human chorionic gonadotropin (hCG) test. Basal concentration of E2 was elevated [E2, 13.6 pg/ml (50 pmol/liter)], and it rose dramatically after hCG stimulation [E2, 163 pg/ml (598 pmol/liter)], which indicated increased conversion of androgens to estrogens. Basal levels of gonadotropins were low. Analysis of the family history showed other cases of gynecomastia in male members of the family, as well as precocious puberty in females (Fig. 1). Diagnosis of familial gynecomastia due to increased aromatase activity, or AES, was made. Therapy with aromatase inhibitor testolactone has been considered but not performed due to social and economical reasons.
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). There was appearance of scarce pubic hair at the age of 10 yr. Basal and hCG-stimulated concentrations of E2 remained elevated. There was persistent suppression of basal and GnRH-stimulated levels of gonadotropins. At the age of 11 yr, he was reexamined at our center. His height was 157 cm (HtSDSCA, 2.3), puberty staging G2P2B3, Te 12 ml, bone age 17 yr (HtSDSBA, –2.6). The gonadotropin secretion was still suppressed. At the age of 11 yr, reductive mammoplasty was performed.
At the last follow-up visit at chronological age of 17.3 yr, his height was 163 cm (HtSDSCA, –1.7), the bone age was 18 yr (HtSDSBA, –1.8), the weight was 66 kg, and the body mass index was 24.8 kg/m2. There were classical "feminine" body proportions with hip circumference exceeding those for waist and breast (103, 74, and 86 cm, respectively). His pubertal development corresponded to Tanner stage G4P4, with distinct female type of pubic hair growth. There was scarce axillary hair and no facial hair. His Te was 25 ml, and penile length was 10 cm. He reported to be able to produce minute amounts of ejaculate during intercourse but refused semen evaluation. Ultrasound of adrenal and testes revealed no abnormalities.
His bone mineral density (BMD), evaluated by dual-energy x-ray absorptiometry (Lunar; GE Medical Systems, Milwaukee, WI) compared with age- and sex-matched reference population, was above average: Z-score for total body BMD was 0.8, and Z-score for BMD of lumbar spine ranged from 1.6 to 2.1.
Hormonal investigations showed decreased basal level of T [T, 61.2 ng/dl (2.2 nmol/liter)] that increased significantly 48 h after hCG test [T, 981 ng/dl (35.3 nmol/liter)]. Basal concentration of E2 was elevated [E2, 228 pg/ml (835 pmol/liter)], and it rose dramatically 48 h after hCG stimulation [E2, 966 pg/ml (3545 pmol/liter)]. His basal serum gonadotropins were in the low normal range. After GnRH test, LH rose significantly, whereas FSH response was negligible (Table 1).
Family history
Sixteen family members in five sibships of five generations were reported to be affected (Fig. 1). Males presented with gynecomastia and short stature. In subject III-8 (age 38 yr), who consented for physical examination, there were clinical signs of hypogonadism (scarce pubic hair, testes 10 ml, penis length 7 cm, phimosis). His height was 152 cm (–3.4 SD). He underwent reductive mammoplasty at 24 yr.
Females presented with precocious puberty (age at menarche, ranged from 9 to 10 yr), short stature (140–150 cm), and macromastia (Fig. 1). Mother of the proband (subject IV-9, age 40 yr) reported to have breast enlargement at the age of 6 yr and menarche between 9 and 10 yr, her height is 146 cm (–2.7 SD), and she has irregular and frequent menses since the age of 35 yr. At examination at the age of 40 yr, thickness of endometrium, as measured by ultrasound, was 19 mm; serum LH was 2.8 mIU/ml (normal, 2.5–12), FSH was 4.0 mIU/ml (normal, 1.9–11.6), and E2 was 321 pg/ml [1180 pmol/liter (normal, 26–161 pg/ml)]. Maternal grandmother (subject III-12), who is currently 70 yr, reported to have episodical menstrual-like bleedings. Her height is 140 cm (–3.7 SD); serum LH was 5.4 mIU/ml (normal postmenopausal, 40–120); FSH was 9.9 mIU/ml (normal postmenopausal, 40–120), and E2 was 88 pg/ml [1180 pmol/liter (normal postmenopausal, 7–44 pg/ml)].
The following information was available about other family members (Fig. 1): subject I-1, reported to have short stature and macromastia; subject II-3, gynecomastia, died in combat; subject II-5, gynecomastia, cause of death is unknown; subject II-7, gynecomastia, died in combat; subject II-8, currently 87 yr, final height 144 cm (–3.0 SD), macromastia, no serious health problem; subject II-11, final height 142 cm (–3.4 SD), macromastia, died of endometrial cancer at 62 yr; subject III-1, menarche at 13–14 yr, final height 156 cm (–1.0 SD); subject III-2, menarche at 13–14 yr, final height 160 cm (–0.4 SD); subject III-4, gynecomastia, short stature; subject III-6, macromastia, final height 150 cm (–2.0 SD), surgery for endometrial leiomyoma; subject III-7, gynecomastia, short stature, cause of death is unknown; subject III-9, menarche at 13–14 yr, "average" height, death of leukemia; subject III-11, gynecomastia, final height 156 cm (–2.8 SD); subject IV-2, menarche at 12 yr, final height 164 cm (0.3 SD); subject IV-4, menarche at 14 yr, final height 161 cm (–0.2 SD); subject IV-5, menarche at 10–11 yr, final height about 150 cm (–2.0 SD), endometrial leiomyoma; subject IV-7, menarche at 13 yr, final height 168 cm (1.0 SD); subject IV-8, final height 189 cm (2.2 SD); subject IV-10, menarche between 9 and 10 yr, final height 145 cm, (–2.9 SD), macromastia, irregular and frequent menses; subject V-1, Tanner 1 at 8 yr, height 130 cm (0.7 SD); subject V-2, Tanner 1 at 11 yr, height 147 cm (0.8 SD); subject V-3, Tanner 1 at 9 yr, height 138 cm (1.2 SD); subject V-4, height 178 cm at 16 yr (0.9 SD); and subject V-6, menarche at 13 yr, height 170 cm at 15 yr (1.3 SD).
All studies were approved by the review board at the Endocrinological Research Center, and written informed consent was obtained from the subjects and (or) the parents.
Methods
Endocrine testing. LH and FSH levels were measured before and 30, 60, and 90 min after iv GnRH stimulation (Triptorelin, 100 mg). T and E2 were measured at baseline 48 and 72 h after hCG test (1500 U/m2, im). Dexamethasone suppression test was performed using a standard dose (2 mg, four times a day). Bone age was evaluated by the Greulich and Pyle method (15).
Hormone assay. LH, FSH, T, and E2 were assayed by RIA using World Health Organization-matched reagents (16), as described previously (17). Sensitivity and interassay and intra-assay coefficients of variation were as follows: for LH 0.5, mIU/ml, 7.9 and 9.4%; for FSH, 0.5 mIU/ml, 6.4 and 9.2%; for T, 0.09 ng/ml, 7.8 and 9.8%; and for E2, 5.45 pg/ml, 6.8 and 8.1%, respectively.
DNA and RNA extraction. Genomic DNA was extracted from peripheral leukocytes by standard procedure. RNA from breast tissue was isolated by acid phenol-guanidinium thiocyanate extraction method (18).
Linkage analysis. Genomic DNA samples were amplified by PCR with the following primers: 5'-TTTGTCTATGAATGTGCC-3', CYP19 (TTTA)n (forward); 5'-GTGATAGAGTCAGAGCCT-3', CYP19 (TTTA)n (reverse); 5'-GCACCTAAACAAATCCTA-3', D15S123 (forward); 5'-TTCATGCCACCAACAAA-3', D15S123 (reverse); 5'-AAACATAGTGCTCTGGAGGCTTAG-5', D15S209 (forward); 5'-GGGCTAACAACAGTGTCTGC-3', D15S209 (reverse).
Forward oligonucleotide in each pair was 5'-labeled with [
-32P]-ATP by T4 polynucleotide kinase (MBI Fermentas, Hanover, MD). PCR was performed with Taq DNA polymerase (Syntol, Moscow, Russia). The amplification reactions were subjected to PAGE and then analyzed by autoradiography. Linkage analysis was performed using the LINKAGE version 5.1 software (ftp://linkage.rockefeller.edu/software/linkage), assuming a dominant model of inheritance and 100% penetrance in both sexes. Population-specific allele frequencies were determined by analysis of genomic DNA samples obtained after informed consent from unrelated healthy Russian subjects: CYP19 (TTTA)n (46 alleles) (19), D15S123 (76 alleles), and D15S209 (64 alleles).
Initial amplification and cloning of 5'-terminal sequences of aromatase transcripts.
To isolate 5'-terminal sequences of aromatase coding transcripts, cDNA synthesis on total RNA extracted from breast tissue of the propositus was performed with SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) as per the instructions of the manufacturer. The synthesis was primed by a 5'-biotinylated reverse primer complementary to exon 2 of CYP19 (Bio-Aro-1R): 5'-Bio-TGTTATAATGTATCGGGTTCAGC-3' (Fig. 2) obtained from Syntol (Moscow, Russia). Double-stranded cDNA was produced with random hexanucleotide primers and purified using streptavidin-coated paramagnetic particles (Streptavidin MagneSphere; Promega, Madison, WI). Then the double-stranded adaptor oligonucleotide (5'-AATTTAATACGACTCACTATAGGGATATC-3'; 3'-NH2-TTAAATTATGCTGAGTGATATCCCTATAG-5') was ligated to the 5'-ends of cDNA. To ensure directional joining of the adaptor top strand (used further as anchor) to cDNA, the 3'-end of the adaptor bottom strand was blocked with amino group. The PCR was performed with anchor primer and another CYP19 exon 2-specific primer (Aro-2R): 5'-CAAAACCATCTTGTGTTCCTTGA-3' (Fig. 2). PCR products were cloned to pGEM-T Easy vector (Promega). The obtained clones were screened by colony hybridization using the third CYP19 exon 2-specific 5'-32P-labeled oligonucleotide (Aro-CDS-For): 5'-GACTCTAAATTGCCCCCTCTG-3' (Fig. 2) as a probe. The vast majority of clones hybridized with the probe, thus confirming the high specificity of the approach. Positive clones were further characterized by one-letter sequencing (G-tracking) using Cycle Reader DNA Sequencing Kit (MBI Fermentas) and Aro-2R primer. All clones derived from patient breast tissue showed identical pattern of G residues, distinct from any 5'-untranslated known aromatase exons 1. Several longest inserts were completely sequenced with universal primers.
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) were used as the outer and the nested reverse primers, respectively. For 3'-RACE, the outer and the nested forward primers corresponding to exon 1 of TRPM7 were TRPM7-1, F 5'- CGGAGACGCTTCCCGATAGATGGCTACA-3'; and TRPM7-2, F 5'-CTTCCGGAGTCCGCCCCGTGAGGAGAA-3', respectively (Fig. 2). PCR products were cloned to pGEM-T Easy vector (Promega) and then sequenced using the universal primers. Genome walking. Genome walking was performed using The Universal GenomeWalker Kit (Clontech). PCR was performed in 5'-3' direction and 3'-5' direction using for initial reactions the outer and the nested gene-specific primers corresponding to TRPM7 exon 1 (primers TRPM7-1 F and TRPM7-2 F) and CYP19 exon 2 (primers Aro-1R and Aro-2R), respectively.
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Results |
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To investigate whether the AES in this kindred is linked to the CYP19 gene, we performed genetic linkage analysis. Genomic DNA was obtained from 20 members of the family, nine of whom had symptoms of AES and 11 of whom were unaffected (Fig. 1). Initially, a polymorphic tetranucleotide [(TTTA)] repeat marker at nucleotide position 682 of CYP19 gene was used (19). Most of members of this family had homozygocity for seven (TTTA) repeats (allele A5) (Fig. 1 and Table 2). The frequency of this variant in our population, as determined by analysis of 46 unrelated alleles, was 45.7% (Table 2). This resulted in a low LOD score for (TTTA) marker (LOD score of 0.85; 
= 0.00). We also used two additional sequence tagged site markers, D15S123 (CA)n and D15S209 (CA)n, located within genetic distance of about 5 cM from CYP19 gene (Fig. 2 and Table 2). Maximum LOD scores for markers D15S123 and D15S209 were 1.5 and 1.17, respectively, without observed recombinations ( = 0.00). All affected subjects in the family had the same haplotype, A5B4C7.
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Initial amplification and cloning of 5'-terminal sequences of aromatase encoding transcripts were done using RNA isolated from the propositus’s breast tissue as a template as described above in Subjects and Methods. All clones were shown to contain the novel chimeric cDNA composed of exon 1 of TRPM7 gene spliced to the common acceptor spliced site of CYP19 exon 2. The sequence of the longest insert with 251 bp derived from TRPM7 gene is shown in Fig. 2. No clones containing any 5'-untranslated known aromatase exons 1 were detected among the analyzed samples in the library of 5'-RACE products derived from the propositus’s RNA, which could be explained by much more active transcription from the promoter of ubiquitously expressed TRPM7 gene compared with CYP19 promoter. The control experiments done on normal human placental RNA, to the contrary, produced only clones harboring CYP19 exon 2 sequence spliced to alternative 5'-untranslated aromatase exons identified previously in placental mRNA (GenBank accession nos. S52793, S96437, and M74714).
Thus, the expression of CYP19 gene in breast tissue of the propositus appeared to be driven predominantly, if not exclusively, by promoter of ubiquitously expressed TRPM7 (CHAK1) gene.
To confirm this, RACE was performed using SMART RACE cDNA Amplification Kit (Clontech).
5'-RACE
Using total RNA extracted from the propositus’s breast tissue as a template we performed 5'-RACE with gene-specific primers corresponding to exon 2 of CYP19 gene. The nested PCR produced a single band of approximately 330 bp. TA cloning and subsequent sequencing revealed a hybrid sequence containing, 3' to 5', first 73 bps of exon 2 of the CYP19 and the whole exon 1 (241 bp) of the TRPM7 (Fig. 2).
3'-RACE
To verify results of the 5'-RACE experiment, an outer and a nested gene-specific primers corresponding to exon 1 of TRPM7 gene were designed. The nested PCR produced a major band of approximately 3 kb. TA cloning and subsequent sequencing verified that the PCR product contained, 5' to 3', exon 1 of TRPM7 followed by CYP19 sequence starting from exon 2. No wild-type TRPM7 sequence was detected, which is most likely explained by the method limitations for the full-length cDNA of approximately 7.2 kb.
Genome walking
To test whether observed TRPM7-CYP19 hybrid transcript could result from the rearrangements in the proximity of exon 1 of TRPM7 or exon 2 of CYP19, the genome-walking approach was used. Series of overlapping PCR failed to reveal any changes in the restriction pattern in the approximate 15 kb region 3'-ward of exon 1 of TRPM7 and approximate 6 kb region 5'-ward of exon 2 of CYP19.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In females, several subjects in the family presented with gynecological disease that could be attributed to estrogen excess (endometrial cancer, leiomyoma, and menstrual irregularities) (Fig. 1). Remarkably, one female (subject III-12) was shown to have elevated E2 level and persistent menses in the beginning of her eighth decade of life.
In the males, there were distinct signs of hypogonadism in a 38-yr-old male (subject III-8), the only adult male in the family available for physical examination. Although pubertal development of the proband at the age of 17.3 yr could be considered as appropriate for age, it might be expected that persistent estrogen excess and suppressed secretion of gonadotropins would lead to development of hypogonadism, similar to that observed in his elder male relative. Nevertheless, according to the pedigree, in two male members of the family (subjects II-5 and II-7), fertility apparently was preserved.
The propositus presented with enlargement of breast tissue already in the first months of life, and, at 11 yr, his bone age was advanced to 17 yr. It is not clear what was the source of sex steroids causing that early feminization and acceleration of growth. It might be speculated that physiological activation of gonadal function in the first months of life as well as estrogens received from breast milk could play a role. Later adrenal cortex could provide a substrate for aromatization. Usually, a rise in adrenal androgens is observed in boys not earlier than at 8 yr, at the time of adrenarche. It might be possible, however, that even a small amount of adrenal androgen substrate was sufficient to produce estrogens under conditions of ubiquitous aromatase expression. It also cannot be excluded that aberrant expression of aromatase itself could affect adrenal function. In AROM+ transgenic mouse, animal model expressing human P450 aromatase under the human ubiquitin C promoter, enlargement of adrenals associated with hyperplasia of adrenal cortex and high serum concentration of corticosterone have been found (21). We were unable, however, to prove that estrogen secretion in our case was ACTH-dependent. This was in contrast with the observation of Leiberman and Zachmann (22) who described a dexamethasone-suppressible form of familial prepubertal gynecomastia they termed "familial adrenal feminization." Unlike our observation, familial adrenal feminization was characterized by clinical signs of androgen excess and absence of estrogen response after stimulation with hCG (22), which clearly points to the difference in pathogenesis of these two conditions.
Another potential substrate for estrogen synthesis was T. In the period of the follow-up from 5 to 11 yr, Te of the patient progressed from 4 to 12 ml (Table 1), despite sustained suppression of basal and LHRH-stimulated levels of gonadotropins, which was suggestive of autonomous function of the testes due to aberrant expression of aromatase. This might to some extent resemble observations in male AROM+ transgenic mice who show enlargement of testicular interstitium and hyperplastic Leydig cell exhibiting active steroidogenesis (21). In fact, the size of the testes in the proband was in the range usually seen in a human model of gonadotropin-independent Leydig cell hyperplasia, e.g. testotoxicosis, and clearly exceeded that observed in conditions with increased production of adrenal androgens, e.g. congenital adrenal hyperplasia. It is of interest that, even at the last follow-up visit at the age of 17.3 yr, the increase in testes volume to 25 ml could not be completely explained by pubertal rise of gonadotropins, because FSH levels remained low after GnRH test (Table 1). The ability of the proband’s testes to produce androgens is illustrated by results of hCG tests, pointing to the significant increase in T concentration despite the immense rate of its conversion to E2 (Table 1).
The 5'-RACE result from the breast tissue of the proband revealed a novel aromatase transcript containing exon 1 of the gene TRPM7. The presence of this hybrid, transcript was also confirmed by 3'-RACE showing the full-length coding sequence of CYP19 preceded by exon 1 of TRPM7. TRPM7 is ubiquitously expressed in the vertebrate cells. The gene encodes a polypeptide with intrinsic ion channel and protein kinase domains whose targeted deletion causes cells to experience growth arrest within 24 h and eventually die. Moreover, recent evidences show that TRPM7 plays a central role in the Mg2+ homeostasis (11, 23). Both CYP19 and TRPM7 genes are located on 15q21.2 at the distance approximately 0.6 Mb (UCSC Genome Bioinformatics, http://genome.ucsc.edu/). Two other chimeric transcripts recently described in AES families by Shozu et al. (10) contained untranslated exons 1 of genes TMOD3 and FLJ14957located on 15q21.2 and 15q21.3, respectively. It is not clear what makes this chromosomal region so susceptible for rearrangements causing the aberrant expression of aromatase. It might be speculated that, in addition to TRPM7, TMOD3, and FLJ14957 defects involving other genes will be found in the future. It is likely that rearrangements on 15q21.2–15q21.3 associated with chimeric CYP19 transcripts are responsible for other variants of hereditary gynecomastia. In such a chimera, expression pattern of its 5'-untranslated component would determine the clinical phenotype of the syndrome, e.g. age of presentation, rate of progression, ACTH dependence, etc. Investigation of CYP19 expression in patients with AES, therefore, might provide in some instances a clue for targeted therapeutic intervention.
The mechanism of the chromosomal defect in our case is apparently different from the rearrangements recently described by Shozu et al. (10), which most likely were the results of heterozygous inversions. Both CYP19 and TRPM7 genes have the same direction of transcription from telomere to centromere, TPRM7 lying 3'-ward (downstream) of CYP19 (UCSC Genome Bioinformatics, http://genome.ucsc.edu/). Thus, rearrangement bringing CYP19 under the control of TRPM7 promoter could not be the simple inversion of the 15q21.2 portion. A more complex heterozygous rearrangement such as partial duplication of 15q21.2 with placing of TRPM7 5'-regulatory regions in front of CYP19 coding exons is required to produce chimeric transcripts discovered in this study. It must be also added that the exact breakpoint(s) in this and two other cases of AES with chimeric CYP19 transcripts are still not known. Our attempts to find any alterations within chromosomal regions in proximity of exon 1 of TRPM7 and exon 2 of CYP19 using genome walking were not successful.
Last, it was shown in this study that a polymorphic tetranucleotide [(TTTA)] repeat marker at nucleotide position 682 of CYP19 gene that have been used previously for assessment of association between AES and CYP19 (8) could not be universally used for linkage studies. In a control Russian population we studied, 21 of 46 chromosomes (frequency, 0.46) contained allele A5 (seven repeats) (Table 2), which was almost two times more frequent than in the original report by Polymeropoulos et al. (19), who performed the study in the North American population.
In summary, we reported a follow-up of a pediatric case of AES caused by a potential rearrangement on chromosome 15q21.2, resulting in aromatase aberrant expression driven by promoter of the TRPM7 gene. Symptoms of increased aromatase activity have been reported in 15 other members of the proband’s family.
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Footnotes |
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Abbreviations: AES, Aromatase excess syndrome; BMD, bone mineral density; hCG, human chorionic gonadotropin; HtSDS, height SD score; RACE, rapid amplification of cDNA ends; T, testosterone; Te, testicular volume.
Received November 4, 2004.
Accepted March 30, 2005.
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