ANDROGEN SYNTHESIS DEFECTS Drs. Marco Aurelio Rivarola and Alicia Belgorosky, Hospital de Pediatria Garrahan, Buenos Aires, Argentina Adapted from the Conference presented during the Symposium “Recent Advances in Sex Diferentiation and Related Disorders” (SERONO SYMPOSIA INTERATIONAL FOUNDATION, Scientific Comité: I. Bergadá, S. Gottlieb, R. Rey), Buenos Aires (Sheraton Hotel), April 19-21, 2007, organized by Centro de Investigaciones Endocrinológicas and División de Endocrinología del Hospital de Niños R. Gutierrez, Buenos Aires, Argentina. Disorders of prenatal sexual development (DSD) are “congenital conditions in which development of chromosomal, gonadal, or anatomical sex is atypical”. They are secondary to multiple causes. Frequently, the condition is detected at birth because of ambiguity of external genitalia. Defining a specific diagnosis is usually helpful for medical management of the patient. It is not the purpose of this review to provide a diagnostic guide, but to revise the current bibliography on the subject related to one of the important causes of DSD, that one secondary to failure of the embryo 46,XY testis to adequately masculinized external (or internal) genitalia. For this purpose we will systematically review the main steps of testosterone biosynthesis and secretion carried out by fetal Leydig cells. We will also comment on some patients followed by us in our Hospital. FIGURE 1

FETAL LEYDIG CELL

Mitoch.

Loss-of-function mutations of genes involved in androgen biosynthesis

In Figure 1, a cartoon of human fetal Leydig cell is shown. A mitochondrium, rather than the nucleus is depicted. This cell is the star of our review. Its function is essential for our species. This cartoon will be used for a brief review of loss-of-function mutations of genes involved in androgen biosynthesis. In Figure 2 we have added the LH receptor (LHR) to the membrane. In fetal life, LHR is bound by two ligands, placental hCG, during the first part, and pituitary LH, during the last part of gestation. FIGURA 2

Inactivating mutations of the LHR are one of the causes of the syndrome of Leydig cell hypoplasia. LHR is a 7 transmembrane receptor. It is located in chromosome 2p21 in humans, close to the FSH receptor. It has 11 exons. Most of the long extracellular domain is transcribed by the first 10 exons. The rest of the protein is transcribed by Exon 11. The gene is similar to the gene for the FSH receptor and the TSH receptor. The LHR consists of 674 amino acids and has a molecular mass of about 85-95 kDA, based on the extent of glycolization. LHR is a protein G-coupled receptor. Upon hCG/LH binding, the receptor indergoes a conformational change activating trimeric protein Gs s (( � , � , � subunits). After binding of LHR to protein G� s, this protein interchanges GDP for GTP, releases the receptor and binds to adenylcyclase to activate cAMP production. Next, cAMP activates cAMP-dependent protein kinase A. This a tetramer of two regulatory and two catalytic subunits. cAMP bids to regulatory

LHR

FETAL LEYDIG CELL

Mitoch.

hCG/LH

sub-units, releasing the catalytic subunits to phosphorylate several proteins. Some of these P-proteins migrate to the nucleus and are bound to responsive elements in the promoter zones of certain genes to modulate transcription. This process is modified by prostaglandis and other intra-cellular regulators. The syndrome of Leydig cell hypoplasia has variable phenotypes. A typical one is a 46,XY DSD subject with female external genitalia with slight masculinization, gonads in the inguinal region, no uterus, and absence of sexual development at puberty (1). A less frequent phenotype is a 46,XY, male with micropenis and absence of, or poor sexual development at puberty (1). Hormonal profile. There is absence of gonadal steroid synthesis. In children, this is ususally put in evidence by determination of serum testosterone, androstenedione, 17-hydroxy-progesterone, progesterone and DHEA after a hCG stimulation test. Serum gonadotropins, particularly LH, are elevated, depending on the age of the patient. Adrenal function is normal. Molecular studies. Inactivated mutations have been described in the 3 domains (extra, trans and intramembrane) of the protein (2). The function is disrupted by several mechanisms (ligand binding, interaction with G protein, transport to the cell membrane (3). Good correlation between in vitro activity and clinical phenotype. There is an autosomal recessive mode of transmission. Leydig cell hypoplasia is genetically heterogeneous. Other genes have been proposed also to play a role in Leydig cell differentiation and proliferation. Figure 3. Subcellular localización of GFP (green fluorescent protein) labeled LHR showing differences between the wild type and the V144F mutation.

Copyright ©2004 The Endocrine Society

Richter-Unruh, A. et al. J Clin Endocrinol Metab 2004;89:5161-5167

Figure 3 shows an elegant study of LHR wild type normal localization in the plasma membrane in a cell line with induced LHR expression (Panel A) contrasting with absent membrane localization in the V144F mutated LHR cell line. Ligands bind LHR at the membrane level. In Figure 4 a new step of testosterone biosynthesis has been added, the uptake of cholesterol from the extra cellular space and/or the endogenous synthesis of cholesterol. In any instance, the action of 7-dehydrosterol reductase is needed for cholesterol synthesis from 7-dehydrocholesterol. Figure 4

Loss-of-function mutations in 7-dehydrocholesterol reductase, have been reported in the

Syndrome of Smith-Lemli-Opitz (4). The gene for 7-dehydrocholesterol reductase is localized in chromosome 11q13. It has 9 exons and spans ~14 kilobases. The protein has 475 amino acids. DHCR7 is located in the endoplasmic reticulum of cholesterol synthesizing cells. Nine putative transmembrane segments have been identified in the amino acid sequences.

The Smith–Lemli–Opitz syndrome (SLOS) is an autosomal recessive multiple congenital anomaly/mental retardation disorder caused by an inborn error of post-squalene cholesterol biosynthesis. The SLOS phenotypic spectrum is broad and variable, from early embryonic non-viability to varying levels of severity postnatally, including distinctive facial appearance, growth and mental retardation, autistic behaviour, hypotonia, failure to feed, decreased lifespan and variable structural anomalies of the heart, the lungs, the brain, the gastrointestinal tract, the limbs, the genitalia and the kidneys.

Cholesterol deficiency and abnormal increase of pre-defect sterols might be involved in the multiple anomalies reported (5). 46,XY DSD is one of possible phenotypes. It seems to be present in

LHR

FETAL LEYDIG CELL

Mitoch.

Chol.

7-dehydrocholesterol

7-dehydro- sterol-Δ7-red.

Chol.

CG/LH

severe defects. The key morphogen, Sonic hedgehog (and its related proteins Indian and Desert hedgehog), is affected, as this protein needs covalently attached cholesterol for regulated short and long-range signalling processes. Prenatal diagnosis. Low to undetectable levels of E3 have been observed in the urine, amniotic fluid and serum of pregnant women carrying foetuses affected with SLOS. The inability to synthesize cholesterol leads to predictable deficiency in adrenal steroid synthesis and may require steroid replacement (6). Adrenal insufficiency should be considered when faced with a child with a prolonged iones.

CASE PRESENTATION. An 8-day-old 46,XY SDS baby, born in 1997, was admitted to Hospital de Pediatria Garrahan

because of multiple congenital anomalies typical of the SLO syndrome. Baby had mostly feminized external genitalia: a 1 cm long and thin phallus, labioscrotal folds and a single perineal orifice. The two gonads were palpable in the inguinal canals. Karyotype was 46,XY. The female gender was assigned.

Serum cholesterol was 50 mg/dl (N: 125-200), serum 7-dehydrocholesterol was 546 � g/ml (N: <0.08). Post-natal serum testosterone: 1.5 ng/ml (N: 1-3), but no response to hCG. Bilateral orchidectomy at 33 months of age. Testicular histology was normal for age. Testicular cell culture: adequate testosterone secretion but no response to hLH (7).

In conclusion, the mechanisms of undervirilization of fetal external genitalia in 46,XX DSD patients might be: a) Decrease testosterone synthesis by the fetal testis secondary to lack of precursors because of cholesterol deficiency?, b) Abnormal plasma LH receptor response to CG stimulation because of abnormal plasma membrane fluidity secondary to low cholesterol levels or excessive cholesterol precursors?. Indeed, cholesterol is a known important component of membrane lipid rafts.

Figures 5 and 6. Function of StAR proteín.

LHR

FETAL LEYDIG CELL

Mitoch.

StAR

Chol.

7-dehydro chol.

7-dehydro- sterol-Δ7-red.

Chol.

CG/LH

Figure 5

Figure 5 incorporates the function of the StAR (steroidogenic acute regulatory) protein in steroid biosynthesis, i. e., the transport of cholesterol from the external to the internal mitochondrial membrane.

StAR mRNA expression is determined by the balance between transcription and mRNA turnover, each of which is regulated by multiple factors. Promoter elements and mRNA sequence elements are subject to regulation by physiological changes, such as hormonal stimulation (which increases cAMP levels) and cholesterol depletion (which activates SREBP). cAMP and oxidant-activated MAP kinases exert negative and positive effects respectively on mRNA stability, possibly via the StAR AU-rich element (AURE). StAR mRNA stability is also regulated by suppression of transcription or translation. Stabilization of an otherwise rapidly degraded mRNA is a regulatory mechanism that allows for extremely rapid and sensitive control of gene expression.

In Figure 6, a model for StAR localization and cholesterol transfer into the mitochondrion is

depicted (8). "N" indicates the amino-terminus of the cytoplasmic p37 form of StAR; the circled "N" indicates the amino-terminus of the processed mitochondrial form of StAR, pp30. (a) StAR localization. Newly synthesized p37, shown here being translated by a cytoplasmic ribosome, becomes associated through its N-terminal sequence with the OMM surface protein TOM20. Effective phosphorylation of p37 by the catalytic unit of PKA (Cat) is facilitated by interaction of the regulatory subunit (R) with the adapter protein PAP7. This protein, in turn, binds to the PBR in the OMM, a partner with porin (Por). Interactions between the StAR mRNA 3'-untranslated region (3'UTR) and OMM proteins may also help target nascent StAR to the mitochondrion. (b) Transfer of StAR to the inner mitochondrion and delivery of OMM cholesterol to cytochrome P450scc. In step 1, following interaction with TOM20, pp37is denatured with assistance from cytosolic HSP70 complex and then extruded across the OMM and IMM. This process requires the proton gradient across the IMM and the action of mitochondrial matrix HSP70 (HSP70M). A protease cleaves pp37 to pp30, which is then integrated into the IMM. In step 2, cholesterol (C) transfers to pp30 from outer membrane domains formed by PBR, which is activated by acyl-CoA–binding protein (AcCoABP). In step 3, activated membrane regions, which contain high levels of FFAs, accept the transfer

Copyright ©2002 American Society for Clinical Investigation

Jefcoate, C. J. Clin. Invest. 2002;110:881-890

FIGURE 6

of cholesterol from the OMM cholesterol-rich domains generated around PBR. AcCoABP may also participate in the transfer of acyl-CoA to the IMM, where mitochondrial thioesterase (MTE) hydrolyzes this compound to FFA. In step 4, pp30 facilitates the relocation of cholesterol to p450ssc, which is distributed throughout the highly extended IMM, and possibly separate matrix vesicles. Mechanisms of cholesterol transfer to adrenal mitochondria: cholesterol is taken up from both LDL receptors and apoA/HDL receptors (SR-BI) in caveolin-rich (Cav-rich) domains. Late endosomes mediate this transfer to the mitochondria via the activities of NPC-1 (which is inhibited by U18666A) and possibly the StAR-like protein MLN64. Acyl-CoA:cholesterol acyltransferase (ACAT) converts free cholesterol derived from organelles (e.g., endosomes and endoplasmic reticulum (ER)) to the CEs that represent the predominant components of lipid droplets. Blue arrows indicate the routing of cholesterol into and through the cell. StAR is represented at the OMM and IMM, respectively, in its p37 and p30.

The gene for StAR is located on chromosome 8p11.2. The protein has 285 amino acids and undergoes truncation when it performs its transfer function. StAR is located in the mitochondria of the adrenal and gonadal glands. However, it has not been found in placenta and brain.

In lipoid congenital adrenal hyperplasia there is a mutation of the gene for StAR resulting in deficient steroidogenesis and in 46,XY DSD. Problems caused to persons with lipoid CAH can be divided into: a) mineralocorticoid deficiency, b) glucocorticoid deficiency, c) sex steroid deficiency, d) damage to gonads (and adrenals) caused by lipid accumulation. Adrenal sex steroid deficiency is present during pregnancy resulting in a low steroid production by the feto-placental unit. In 46,XY individuals, fetal Sertoli cell function, however, is normal resulting in inhibition of müllerian structures. ACTH stimulates growth of the adrenal cells, and increases LDL receptors to amplify transport of cholesterol into the adrenal cells, where it accumulates because little is transferred into the mitochondria. The adrenals become markedly enlarged by the combination of ACTH-induced hyperplasia and accumulated lipid (9). Lipid accumulation is thought to damage the cells further (“second hit hypothesis”). Because the StAR protein is also involved in cholesterol transport into testicularand ovarian cells for sex steroid synthesis, testicular production of testosterone and ovarian production of estrogen are also impaired. Lipid accumulation damages the Leydig cells of the testes more completely than the granulosa cells of the ovaries.

The usual phenotypes during pre-puberty and puberty are the following (10): 46,XY DSD. Female external genitalia: Neonatal adrenal insufficiency. Gonads in the inguinal region or intra-abdominal. No uterus. Absence of sexual development at puberty. 46,XX DSD. Normal female external and internal genitalia. Neonatal adrenal insufficiency. Partial sexual development at puberty. In this patient, a molecular study of the StAR gene was carried out in the Laboratory of Molecular Biology of our Hospital (Drs. S. Baquedano y A. Belgorosky). A heterozygote mutation at the level of the first base of the splicing acceptor site in intron 1 (IVS-2G>A) was found. It is expected that this nucleotide change will affect slicing. However, no second inactivating mutation was found, as expected in autosomic recessive mutations. No mutation of DAX-1 was found either. Additional studies are required to define the functional significance of the reported mutation.

In the next step of steroid biosynthesis (Figure 7), intra-mitochondrial cholesterol is converted into pregnenolone by P450scc. cP450scc has cholesterol monooxygenase (side-chain-cleaving) activity. The gene, CYP11A1, is located on chromosome 15q23-24. The protein has 521 aminoacids. P450scc is located in the mitochondria of the adrenal and gonadal glands.

Type I P450 enzymes, found in mitochondria, receive electrons from (NADPH) via the intermediacy of two proteins, ferredoxin reductase (a flavoprotein) and ferredoxin (an iron/sulfur protein). Type I P450 enzymes include P450scc, the two isozymes of 11-hydroxylase (P450c11beta and P450c11AS), and several vitamin D-metabolizing enzymes (11).

FIGURE 7

Deleterious mutations of the CYP11A1 gene were thought to be incompatible with fetal survival

because of impaired progesterone production by the fetoplacental unit. However, loss of function of cP450scc can be compatible with survival in rare instances, as shown by a few reported patients with these mutations. The phenotype is similar to that observed in StAR protein loss-of-function mutations (12)

The next step in the biosynthesis is the conversion of pregnenolone into 17-hydroxypregnenolone and further down into dehydroepiandrosterone by P450c17 which has 17-hydroxylase and 17, 20-desmolase activities (Figure 8). The gene (CYP17A1) is located on chromosome 10q24.3. It encodes eight exons over 6.4 kb DNA .The protein has 509 amino acids. cP450c17 is located in the endoplasmic reticulum of the adrenal and gonadal glands.

Type II P450 enzymes, found in the endoplasmic reticulum, receive electrons from NADPH via P450 oxidoreductase (POR), which contains two flavin moieties. Steroidogenic Type II P450 enzymes include 17alpha-hydroxylase/17,20 lyase (P450c17), 21-hydroxylase (P450c21), and aromatase (P450aro). All P450 enzymes catalyze multiple reactions, but P450c17 appears to be unique in that the ratio of its activities is regulated at a posttranslational level.

LHR

FETAL LEYDIG CELL

Mitoch.

StAR

Chol.

P5

P450scc

P5

7-dehydro chol.

7-dehydro- sterol-Δ7-red.

Chol.

CG/LH

FIGURE 8

In P450c17 mutations, ACTH stimulates overproduction of 11-deoxycorticosterone (DOC), a

mineralocorticoid that causes hypertension and hypokalemia. Typical features include suppressed plasma renin activity (or renin) and aldosterone. The elevation of corticosterone (compound B) compensates for the decrease in cortisol. In the gonads, the lack of 17,20-lyase activity prevents gonadal sex steroid production and leads to undervirilization in 46,XY individuals, as well as failure of pubertal development. Frequently, affected subjects are raised as social females. 46,XX individuals usually present primary amenorrhea and lack of development of secondary sex characteristics.

The Biochemical diagnosis of 17OHD, as for other steroidogenic enzyme deficiencies, is established by measuring precursor-to-product ratios in the course of an ACTH stimulation test (13, 14). A 5- to 10-fold increase in the 17-deoxysteroids, B, DOC, and progesterone (P) after ACTH administration leads to diagnosis of 17OHD. In addition, 17OHD is characterized by elevated production of 18-hydroxycorticosterone and 18-hydroxy-DOC, in contrast to 11-hydroxylase and 21-hydroxylase deficiencies. P is elevated in 17OHD and available in most hospital laboratories, but P is also elevated in other forms of CAH. Basal P measurement seems a useful and practical screen for diagnosis of 17OHD, particularly if the clinical presentation excludes other forms of CAH, as reported by Berenice Mendonca et al. (15). The contributions of the brazilian investigators to the study of this mutation have been remarkable.

LHR

FETAL LEYDIG CELL

Mitoch.

StAR

Chol.

P5 17OHP5 DHEA

P450scc

P450c17 P450c17 P5

7-dehydro chol.

7-dehydro- sterol-Δ7-red.

Chol.

CG/LH

LHR

FETAL LEYDIG CELL

Mitoch.

StAR

Chol.

P5 17OHP5 DHEA

P450scc

P450c17 P450c17 P5

POR

7-dehydro chol.

7-dehydro- sterol-Δ7-red.

Chol.

CG/LH

Copyright ©2004 The Endocrine Society

Costa-Santos, M. et al. J Clin Endocrinol Metab 2004;89:49-60

FIGURA 9. Schematic representation of the CYPc17 gene, indicating the approximate localization and size of exons (numbered box), primer oligonucleotides used and mutations found (asteriscs) (13).

FIGURE 10

Coming back to our biosynthetic pathway in fetal Leydig cell, Figure 10 shows that cP450c17 requires the POR enzyme for its appropriate function. The gene for cP450 oxidoreductase is located on chromosome 7q11.2. It encodes eight exons over 2.5 kb DNA. The protein (POR) has 677 amino acids. POR is an endoplasmic reticulum membrane oxidoreductase with an FAD-binding domain and a flavodoxin-like domain.

POR is required for the activity of all 50 human Type II P450 enzymes, and ablation of the POR gene in mice causes embryonic lethality (16). Nevertheless, mutation of the human POR gene is compatible with life, causing multiple steroidogenic defects and a skeletal dysplasia called Antley-Bixler syndrome.

P450 oxidoreductase deficiency typically presents a steroid profile suggesting combined deficiencies of steroid 21-hydroxylase and 17alpha-hydroxylase/17,20-lyase activities. These and other enzymes require electron donation from P450 oxidoreductase.

The clinical spectrum of P450 oxidoreductase deficiency ranges from severely affected, 46,XX children with ambiguous genitalia, adrenal insufficiency and the Antley-Bixler skeletal malformation syndrome to normal or mildly affected 46,XY individuals, or 46,XX girls with polycystic ovary syndrome.

P450 oxidoreductase deficiency, with or without Antley-Bixler syndrome, is autosomal recessive, whereas Antley-Bixler syndrome without disordered steroidogenesis is caused by autosomal dominant fibroblast growth factor receptor 2 mutations. The causal connection between P450 oxidoreductase deficiency and disordered bone formation remains unclear.

Clinical Diagnosis (17). Signs of maternal virilization during pregnancy. Cortisol deficiency: variable from mild to severe. Manifestations are often subtle. 46,XY DSD: normal genitalia or micropenis, undescended testes. Poor virilization at puberty.

46,XX DSD: hypoplastic labia majora, fused labia minora, large clitoris and/or vaginal atresia. Primary amenorrhea. Enlarged cystic ovaries.

Antley-Bixler syndrome (ABS): craniosynostosis, brachycephaly, severe midface hypoplasia, radiohumeral synostosis, multiple joint contractures. Cognitive functioning ranges from moderate mental retardation to normal intelligence.

Biochemical diagnosis. Serum concentrations of cholesterol are grossly normal, Lanosterol and

dihydrolanosterol accumulate, but they are not usually measured. Pregnenolone, progesterone, 17-OH pregnenolone, and 17-OH progesterone

serum concentration are often elevated at baseline and/or after ACTH stimulation. Dehyroepiandrosterone and androstenedione serum concentrations are normal or decreased before and/or after ACTH stimulation (18). Steroid Anomalies and Pregnancy. The partial blockages, which occur at each step catalyzed by cytochrome p450 (CYP) dependent enzymes, explain the finding of low maternal serum uE3 during pregnancy. A proposed alternative (or "backdoor") pathway of androgen synthesis, may explain why some affected females develop ambiguous genitalia, and why some mothers develop symptoms of androgen excess during pregnancy. The following step in testosterone biosynthesis is the conversion of dehydroepiandrosterone in androstendione by type II 3� -hydroxysteroid dehydrogenase, as depicted in Figure 11. The structure of each of the HSD3B2 and HSD3B1 genes consists of four exons included on a 7.8 kb fragment of chromosome 1p13.1 (19). Five related pseudogenes have also been cloned.

FIGURE 11

Copyright ©2005 The Endocrine Society

Simard, J. et al. Endocr Rev 2005;26:525-582

FIGURE 12. Chromosomal localization of the two genes, HSD3B1 and HSD3B2, as well as the five pseudo genes {Psi}1-5.

LHR

FETAL LEYDIG CELL

Mitoch.

StAR

Chol.

P5 17OHP5 DHEA

P450scc

P450c17 P450c17 P5

Adione

3β-HSD II

POR

7-dehydro chol.

7-dehydro- sterol-Δ7-red.

Chol.

CG/LH

The type II gene (HSD3B2), which encodes a protein of 371 amino acids, shares 93.5% identity with the type I gene, and is almost exclusively expressed in the adrenals, the ovary, and testis. The type I gene (HSD3B1) encodes an enzyme of 372 amino acids predominantly expressed in the placenta and peripheral tissues, such as the skin, mammary gland, prostate, and several other normal and tumor tissues. 3� -HSD subcellular localization patterns are unique in that they show various degrees of endoplasmic reticulun and mitochondrial distribution. In Figure 12, reproduced from Simard et al. (19) short and long arms of chromosome 1 is shown. Several genomic markers can be seen after amplification of region p13.1. In an additional amplification, the two HSD3B genes with the 4 exons (roman numerals), the mRNA and the type I and type II proteins are depicted. Clinical presentation (20). 46,XY subjects. Combined adrenal and testicular deficits. Salt wasting with both aldosterone and cortisol deficiency. Perineal hypospadias or perineoscrotal hypospadias. Or mild salt wasting and premature pubarche. 46,XX subjects. Salt wasting as in 46,XX subjects. Absent or minimal virilization. Mild salt wasting and premature pubarche. Pubertal hirsutism (infrequent). Biochemical diagnosis. The basal plasma levels of � 5-3ß-hydroxy steroids such as PREG, 17OH-PREG, and DHEA are elevated in affected individuals. ACTH-stimulated (1 h post iv bolus of 250 microg Cortrosyn) serum 17-hydroxypregnenolone (� 5-17P) levels and basal and ACTH-stimulated ratios of � 5-17P to cortisol are the most consistent finding in the genotypic proven patients (21). Hormonal criteria for the diagnosis of 3� -HSD deficiency have been controversial because they were not based on genetic evidence. Mermelo et al. (22) established that basal and post-ACTH serum 17-hydroxypregnenolone and the 17-hydroxypregnenolone/cortisol ratio are useful biochemical parameters for diagnosis, although molecular studies for diagnostic confirmation are advisable. We come back to our fetal Leydig cell for the final biosynthetic step, the conversion of androstendione to testosterone, activated by type III 17� -hydroxysteroid dehydrogenase (Figure 13). As for other enzymes, congenital defects in the gene for this enzyme have been reported (23-25). The HSD17B3 gene contains 11 exons and is located on chromosome 9q22. The protein has 310 aa. At least 14 isozymes have been described. They can be predominantly reductive (types 1, 3, 5, 7) or oxidative (types 2, 4, 8) . Many are involved in the estrogen balance in peripheral tissues. Type 1 is expressed in the ovary. Type 3 catalyzes the reduction of androstenedione to testosterone, and it is almost exclusively expressed in the testis.

FIGURA 13

Clinical presentation 46,XY DSD. Female external genitalia with slight masculinization. Gonads in inguinal region, no uterus. Absence of sexual development at puberty. 46,XX subjects. Normal female external and internal genitalia. Normal sexual development at puberty (normal phenotype). Biochemical diagnosis. Basal and/or post hCG serum androstenodione/testosterone ratio is the more important biochemical parameter to suspect 17� -HSD deficiency. However, this relationship might turn out to give a false positive diagnosis. CASE PRESENTATION A 2-month-old baby was referred to the Garrahan Pediatric Hospital because of ambiguous genitalia, in October 2004. A curved phallus, 1.8 cm long with a single perineal orifice, was surrounded by labio-scrotal folds. Symmetrical inguinal gonads were palpable. Karyotype was 46,XY. Serum LH was 2.66 and serum FSH 11.2 U/L. Serum 17 OH-Progesterone was 13.6, androstenedione 0.18 and testosterone 0.63 ng/ml. Under hCG stimulation serum androstenedione increased to 5.8 and testosterone 1.61 ng/ml, giving and abnormally elevated androstenedione/testosterone ratio of 3.6. Sex of rearing was feminine. Tentative diagnosis was 17� -HSD deficiency on the basis of clinical and laboratory findings. Therefore, analysis of the HSD17B3 gene was carried out. However, after sequencing all exons and intron/exon flanking regions of this gene, no mutation was found.

LHR

FETAL LEYDIG CELL

Mitoch.

StAR

Chol.

P5 17OHP5 DHEA

P450scc

P450c17 P450c17 P5

Adione

3β-HSD II

POR

7-dehydro chol.

7-dehydro- sterol-Δ7-red.

Chol.

CG/LH

T

17β-HSD III

T

The etiology of this 46,XY DSD remains undetermined. This is an example of the difficulties and frustrations arising from the study of these patients. In summary, 46,XY DSD secondary to defects in testosterone biosynthesis by the fetal testis show a variable phenotype, strongly depending in the particularly mutated gene. Table 1 summarizes the main characteristics of these gene defects. The predominant phenotype is female or poorly virilized external genitalia and absence of uterus and fallopian tubes. Two defects associate severe adrenal insufficiency and a third one partial adrenal failure. In one case arterial hypertension can be observed. The last column shows laboratory results. In three gene defects absence of all steroids are observed: LHR, StAR, and cP450scc. In another five defects, elevation of precursors are of diagnostic value: SLOS, cP450c17, POR, 3� -HSD and 17� -HSD. However, hormone determinations often give just diagnostic orientation and confirmation requires the demonstration of inactivating mutations in involved genes.

NONDYSGENETIC ANDROGEN SYNTHESIS DEFECTS RATIONAL DIAGNOSTIC APPROACH IN 46,XY NEWBORNS (CONFIRMATION: GENE ANALYSIS)

GENE PREDOMINANT PHENOTYPE

ADRENAL FUNCTION

LABORATORY

LHR FEMALE EXTERN GENITALIA PALPABLE GONADS

NORMAL hCG: no testosterone

∆7dehydr Reduct.

SMITH-LEMNI-OPITZ SYNDROME

USUALLY NORMAL LOW CHOLESTER. HIGH 7-DEHYDROC.

StAR or P450scc

FEMALE EXTERN GENITALIA PALPABLE GONADS

ADRENAL CRISIS ABSENCE OF ALL STEROIDS

P450c17 FEMALE EXTERN GENITALIA PALPABLE GONADS

LOW-RENIN HYPER- TENSION

HIGH P4, B, DOC LOW F, RENIN

POR MILD GENITAL AMBIGUITY ANTLEY-BIXLER SYNDROME

NORMAL HIGH P5,17OHP5, P4, 17OHP4

Type II 3� -HSD AMBIGUOUS EXT. GENITALIA PALPABLE GONADS

ADRENAL CRISIS HIGH 17OHP5, DHEA

Type III 17� -HSD

FEMALE EXTERN GENITALIA PALPABLE GONADS

NORMAL HIGH ∆4ANDROS- TENDIONE

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