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Molecular Characterization of 6 Unrelated Italian Patients With 5-Reductase Type 2 Deficiency

S. MAJORE

G. MARROCCO

S. TUMINI

B. BOSCHERINI

S. SCOMMEGNA

R. RINALDI

P. GRAMMATICO

From the ^* Cytogenetic and Molecular Genetic Unit, S. Chiara Hospital, Pisa, Italy; the Centre for the Disorders of Sexual Development, Medical Genetics, University "La Sapienza," S. Camillo-Forlanini Hospital, Rome, Italy; the Department of Pediatrics, University of Chieti, Ospedale Policlinico, Chieti, Italy; the Pediatric Unit, Policlinico Tor Vergata, Rome, Italy; and the ^|| Medical School of Endocrinology, Garibaldi Hospital, Catania, Italy.

Correspondence to: Dr Fulvia Baldinotti, Unitû Operativa Cito-genetica e Genetica Molecolare, Dipartimento di Ginecologia e Ostetricia, Ospedale S.Chiara: via Roma, 67, 56100 Pisa, Italy (e-mail: f.baldinotti{at}ao-pisa.toscana.it).

Received for publication April 12, 2007; accepted for publication June 27, 2007.

	Abstract

Top Abstract Patients and Methods Results Discussion References

 
Steroid 5-reductase (5R) deficiency (OMIM number #264600) is a rare 46,XY disorder of sex differentiation caused by mutations in the 5R type 2 gene (SRD5A2) resulting in dihydrotestosterone deficiency during fetal development. We report on the analysis of the SRD5A2 gene in 6 unrelated 46,XY Italian patients with external genitalia morphology ranging from predominantly female to nearly completely male. Three subjects were seen and assessed at birth, 1 patient was referred to us before puberty, and 2 at postpubertal age. Six different causative mutations (5 missense and 1 nonsense) and a rare polymorphism were identified. Four patients presented homozygous single-base substitutions. These SRD5A2 mutations were located in exon 2 (variant Cys133Gly), exon 4 (Gly196Ser and Ala207Asp) and exon 5 (Tyr235Phe). A fifth subject was a compound heterozygote who carried a nonsense mutation in exon 1 (Trp53X) and a second SRD5A2 alteration in exon 5 (Tyr235Phe). The final patient presented a mutation in only 1 allele (Gly34Trp) together with the Ala49Thr variant. The molecular characterization of these patients made it possible to identify novel mutations and to confirm, before gender assignment or any surgical approach, the suspected 5R deficiency in 2 newborns, 1 of whom had inconclusive hormonal data. 5R deficiency in subjects without parental consanguinity and the presence of compound heterozygotic patients suggest that SRD5A2 mutations carrier frequency may be higher than previously thought.

     Key words: 5-reductase deficiency, SRD5A2 gene, hypospadias

Affected patients show a deficiency of the 5R type 2 enzyme, which becomes partially or totally unable to convert testosterone (T) into dihydrotestosterone (DHT), the latter being responsible for the development of external genitalia, prostate, and urethra in the male fetus (Wilson et al, 1993; Imperato-McGinley and Zhu, 2002). The abnormal 5R enzyme is the result of mutations in the 5R type 2 gene (SRD5A2), which is located on chromosome 2p23 (Wilson et al, 1993; Griffin et al, 2001; Imperato-McGinley and Zhu, 2002). The coding region of the SRD5A2 gene consists of 5 exons, and it is translated into a protein of 254 amino acids that presents an androgen-binding domain at its N-terminal end (Wilson et al, 1993; Griffin et al, 2001). So far more than 45 different mutations scattered throughout the gene have been reported (Thigpen et al, 1992; Wilson et al, 1993; Griffin et al, 2001; Imperato-McGinley and Zhu, 2002; Hackel et al, 2005; Human Gene Mutation Database), the majority being missense mutations; however, premature stop codons and small deletions have also been described (Griffin et al, 2001; Human Gene Mutation Database). In 2 patients, the entire SRD5A2 coding sequence was found to be deleted (Andersson et al, 1991). In some individuals, with clinical findings suggestive of 5R deficiency, mutations in only 1 allele have been found (Thigpen et al, 1992; Griffin et al, 2001; Hackel et al, 2005). Several mutations are recurrent and have been reported in different populations (Wilson et al, 1993), whereas other mutations have been described in specific ethnic groups (Thigpen et al, 1992; Wilson et al, 1993; Hochberg et al, 1996; Mazen et al, 2003) and their recurrence is probably the result of a founder gene effect in people geographically isolated with a large coefficient of inbreeding (Griffin et al, 2001; Imperato-McGinley and Zhu, 2002). Interestingly, consanguinity is present in approximately one third of the reported affected patients, and the family history is positive in approximately 40% of families (Wilson et al, 1993).

Differential diagnosis of steroid 5R deficiency from other forms of 46,XY DSD can be difficult (Griffin et al, 2001). Abnormally high levels of baseline and/or human chorionic gonadotropin (hCG)–stimulated T:DHT ratio are the endocrine hallmarks of the disorder (Wilson et al, 1993; Imperato-McGinley and Zhu, 2002). However, the biochemical parameters allow identification of subjects with the classic disorder, in which DHT synthesis is severely impaired. Patients with partial enzyme deficiencies, especially prepubertal subjects after short hCG stimulation tests, may remain undiagnosed because of T:DHT ratios within the normal range (Wilson et al, 1993; Hochberg et al, 1996; Hiort et al, 1996b). Thus, molecular analysis of the entire coding region of the SRD5A2 gene may be a useful tool to confirm diagnosis in patients with endocrine features of 5R deficiency or to investigate individuals with inconclusive hormonal data but clinical findings suggestive of the disorder.

In this article, we report on molecular analysis of the SRD5A2 gene in 6 unrelated Italian patients with 46,XY DSD, showing clinical and/or endocrine features of steroid 5R deficiency. Our data confirm the previously observed allelic heterogeneity, moreover suggesting that the carrier frequency of SRD5A2 mutations in the Italian population may be higher than previously thought.

	Patients and Methods

Top Abstract Patients and Methods Results Discussion References

 
Patients

Six patients from unrelated families and their available parents were studied. All patients had normal male karyotypes and were affected by 46,XY DSD. The clinical data suggesting possible 5R deficiencies are summarized in Table 1. Genital ambiguity was staged according to the classification proposed by Sinnecker et al (1996) and is described in Table 2. The study was approved by the Institutional Review Board. Informed consent was provided by each minor patient's parents and by patient 6.

View this table: [in this window] [in a new window]   Table 2. Classification of phenotypes in 46,XY DSD due to steroid 5-Reductase deficiency (Sinnecker et al, 1996)

     Patient 1— At the infant's birth, micropenis (length = 1.3 cm) was detected and was the only genital abnormality seen (Figure 1a). He was analyzed according to the multidisciplinary protocol for newborns with ambiguous genitalia (Corsello et al, 2003), and karyotype and hormonal studies were performed during the first days of life. Serum T concentration was normal, but the DHT value was markedly low and the basal T:DHT ratio was 42 (Table 1). T and DHT evaluations, repeated at age 20 days, resulted in a T:DHT ratio of 23. At 2 months, the penile length was 1.6 cm. Transdermal 25% DHT gel was administered for 3 months, resulting in an increase in penile length to 3.9 cm. At 4 years, weight and stature were normal, and the extended phallus length was 4 cm.

View larger version (122K): [in this window] [in a new window]   Figure 1. (a) Patient 1: An isolated micropenis is the only detectable genital abnormality. (b) Patient 2: Ambiguous external genitalia with perineoscrotal hypospadias and reduced phallic size. (c) Patient 3: Aspect of external genitalia after 2 doses of intramuscular testosterone enanthate and 2 months of percutaneous DHT; perineoscrotal hypospadias, a single perineal opening, partially transposed scrotum, and bilaterally descended testes. (d) Patient 6: The patient before sigmoidovaginoplasty; the testes had been removed 6 months earlier. There is an enlarged clitoris with labia minora and single perineal opening.

     Patient 3— A 15-day-old patient was observed, having already been assigned to the female gender. On physical examination, the newborn showed perineoscrotal hypospadias with a single perineal opening, a clitoral-like phallus, and a bifid scrotum/labia majora in which gonads were palpable (Figure 1c). At ultrasonography, no müllerian remnants were detectable. Serum luteinizing hormone and follicle-stimulating hormone concentrations were normal. T and DHT basal levels are reported in Table 1. Twenty-five milligrams of testosterone enanthate was initiated intramuscularly (once per month for 3 months), together with local DHT twice a day. The penile length increased from 1.2 cm to 3 cm (150% increase) and reached a circumference of 1.5 cm. In agreement with the parents, reassignment to the male sex was made.

     Patient 4— This 8-year-old boy was referred to us because of micropenis, severe perineoscrotal hypospadias, and bilateral cryptorchidism that had already been surgically treated. An hCG test, performed at 1 year, was reported to have shown a normal level of plasma T. At 3 years, surgical repair of hypospadias was performed. Subsequently treatment with T and transdermal DHT was begun. At our first observation, he showed 3-ml testicular volume bilaterally, microphallus (length 3.0 cm), and persistent perineoscrotal hypospadias related to postsurgical fistulization.

     Patient 5— At the infant's birth, clitoromegaly and mobile masses in the labia majora were detected. Upon ultrasound, the presence of a uterus was erroneously reported, and the female gender was assigned. Her parents are first cousins. She had been raised as a girl until 15 years. She was then referred to us because neither menstruation nor female pubertal development had been observed, but signs of progressive virilization were evident. Later the patient developed a deepening voice, labia majora enlargement with hyperpigmentation, and phallus growth. She also showed, at that point, a male habitus with muscular development. Genitography showed a urogenital sinus of 2 cm in length and no evidence of vagina or müllerian remnants. Hormonal studies (Table 1) revealed normal male T levels and an increased T:DHT ratio, suggesting steroid 5R deficiency. After a careful psychologic assessment of gender identity, the patient decided to continue in the female sex, and the appropriate medical and surgical approaches were applied (testes removal, estrogen substitution therapy, and laparoscopic sigmoidovaginoplasty) (Figure 1d). The follow-up has been uneventful to date, and psychologic evaluation over a period of time has revealed that the patient shows satisfaction with the surgical outcome and effects of estrogen replacement.

     Patient 6— At birth, she showed ambiguous external genitalia, with clitoromegaly, rudimental blind-ending vagina, and palpable gonads in the inguinal region. At that time, she was assigned the female gender. Her parents were first-degree cousins. The patient was reared as a girl until puberty, when virilization occurred (muscle mass increase, deepening of the voice, acne, and facial hair). When the patient was 15 years, physical examination identified a phallus 5 cm in length, perineal hypospadias, gonads in the inguinal region, blind-ending vagina, beard, sideburns, and lack of breast development. Chromosome analysis revealed a normal male karyotype. Hormone values obtained at that age are shown in Table 1. Despite clinical features and in opposition to the patient's feelings about gender identity, she was submitted to bilateral gonadectomy, vaginoplasty, and clitoral reduction. The patient was referred to us at 29 years because she presented with gender dysphoria and male attitude and wanted external male genitalia reconstruction.

Hormonal Studies

Total T measurements were performed in different laboratories using commercial radioimmunoassay (RIA) kits, and DHT was assayed with a high-performance liquid chromatography–RIA method, except for patient 3, for whom DHT was assayed with an RIA ¹²⁵I test.

Molecular Analysis

Blood samples were collected in EDTA-containing tubes, and DNA was extracted by standard procedures from white blood cells. Exons 1–5 of the SRD5A2 gene (GenBank accession number L03843) were amplified by polymerase chain reaction (PCR) using the oligonucleotides listed in Table 3. Briefly, PCR amplification was performed in 50-µL total volume containing 200 ng of genomic DNA, 25 pmol each oligonucleotide primer, 200 µM each of 4 deoxyribonucleotide triphosphates, 1.5 to 2 mM MgCl₂, 1X buffer, and 2 U of Taq DNA polymerase (Invitrogen, Milan, Italy). Amplification conditions consisted of 35 cycles of 1 minute at 94°C and 1 minute at 68°C, followed by a final cycle of 1 minute at 94°C and 3 minutes at 68°C. PCR products were verified for correct length on an agarose gel and purified using the QIAQuick PCR Purification kit (QIAGEN, Milan, Italy). Purified PCR products were bidirectionally sequenced using the ABIPrism BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Monza, Italy), and the sequencing reaction products were separated on an ABIPrism 3100 Genetic Analyzer (Applied Biosystems) after removal of unincorporated dye terminators and analyzed by DNA sequencing analysis and SeqScape software (Applied Biosystems). Exons 1 and 2, in which the novel mutations were identified, were sequenced for 40 normal male controls.

	Results

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In the present study, 6 different SRD5A2 point mutations and the Ala49Thr variant were identified (Table 4). Four of 6 patients presented with homozygous single-base substitutions, a fifth subject was a compound heterozygote, and 1 patient presented with a single mutation together with the Ala49Thr variant.

Patient 1 was a heterozygote who presented with 2 different substitutions located in exon 1 (Gly34Trp and Ala49Thr variant). The previously unreported Gly34Trp mutation was caused by a g.843G>T transversion (codon TGG instead of GGG), and the Ala49Thr substitution was caused by a g.888G>A transition (codon ACC instead of GCC) (Figure 2a). Investigation of maternal DNA demonstrated that the mother was a heterozygous carrier of the Gly34Trp change. The father, of white American origin, refused gene analysis.

View larger version (43K): [in this window] [in a new window]   Figure 2. Partial sequences of the SRD5A2 gene showing the molecular alterations detected in the present study. (a) Patient 1. (b) Patient 3. (c) Patient 2. (d) Patient 4. (e) Patient 5. (f) Patient 6. Arrows indicate mutations. w.t. indicates wild type.

In patient 3, 2 different mutations, located in exons 1 and 5, were identified. In exon 1, an unreported g.901G>A transition at codon 53 replaced the Trp residue with a stop codon (codon TAG instead of TGG); in exon 5, a g.2246A>T transversion at codon 235 was responsible for the Tyr235Phe substitution (codon TTC instead of TAC) (Figure 2b). Parental DNA studies revealed the Trp53X mutation in the mother and the Tyr235Phe change in the father.

Patient 4 showed a homozygous mutation in exon 5 (g.2246A>T), replacing a Tyr residue with Phe at codon 235 (Figure 2d). Because parental DNA was not available for patient 4, a deletion cannot be excluded in this patient. Parental consanguinity was not verified, but the parents come from the same small village in Sicily.

In subject 5, a g.1339T>G homozygous transversion was detected at codon 133 in exon 2 (Figure 2e). This previously undescribed point mutation was responsible for a Cys to Gly change (codon GGT instead of TGT). The parents, coming from Campania and first-degree cousins, were both heterozygous for this mutation.

Patient 6 presented a g.1962C>A transversion in exon 4. This homozygous mutation replaced an Ala residue at position 207 with an Asp (codon GAC instead of GCC) (Figure 2f). Both parents were from Calabria, but parental consanguinity was not ascertained and blood samples were not available for DNA analysis.

The 2 previously undescribed mutations (Gly34Trp and Cys133Gly) were not present in the DNA from 40 normal male controls.

	Discussion

Top Abstract Patients and Methods Results Discussion References

 
The present report describes the clinical and molecular characteristics of 6 unrelated subjects of Italian origin with 5R deficiencies. Endocrine data (T:DHT ratio) or in vivo sensitivity tests for T and/or DHT were used to select this series of patients with 46,XY DSD for SRD5A2 gene analysis. Our patients reflect the phenotypic and genetic heterogeneity of steroid 5R deficiency.

Subject 1, with micropenis as the only genital abnormality detected at birth, carried the unreported Gly34Trp mutation together with the Ala49Thr variant. The mutation from Gly to Trp is an exchange of a polar for a nonpolar molecule caused by the G>T transversion in a cytosine-guanosine dinucleotide in exon 1 of the gene, which is at a site that experiences a high frequency of mutations; indeed, 2 different substitutions (GGG>AGG and GGG>CGG) result in the already reported Gly34Arg amino acid change, found in patients from Sicily, Mexico, Egypt, and Vietnam (Hochberg et al, 1996). The Ala49Thr molecular change has not been reported to date in association with 5R deficiency, but its presence has been correlated with masculinization defects, and it may represent a genetic risk factor for the occurrence of hypospadias (Silver and Russel, 1999; Wang et al, 2004). This allelic variant has been studied extensively as a possible prognostic factor in prostate cancer, and it has been demonstrated that this variant increases 5R in vitro activity, enhancing the conversion of T to DHT sixfold (Makridakis et al, 1999; Makridakis et al, 2000; Makridakis et al, 2004). On the other hand, it has been further demonstrated that the Ala49Thr allele, both in the homozygous or heterozygous condition, leads to lower serum androgen levels, suggesting that this variant may be less efficient than initially thought (Allen et al, 2003). It is difficult to evaluate the contribution of the Ala49Thr substitution to the development of our patient's phenotype, resulting at birth in only the presence of micropenis together with a persistently high T:DHT ratio.

In the other 2 patients (patients 2 and 3) with ambiguous genitalia at birth (3b grade), early diagnoses was made in the first weeks of life, allowing reassignments to the male sex. In both cases, short-term treatments with parenteral T and local DHT led to significant increases in penile length and reinforced the decisions of male sex assignment. The Gly196Ser mutation, found in patient 2, has been reported in both the homozygous and compound heterozygous forms in patients from Italy (Nicoletti et al, 2005), the Mediterranean area (Carpenter et al, 1990; Thigpen et al, 1992; Sinnecker et al, 1996), and Sweden (Nordenskjold and Ivarsson, 1998). It has also been reported in Brazilian patients of European origin (Hackel et al, 2005), suggesting that this mutation was probably spread in South America by European conquerors or migrants. Biochemical analysis and transfection studies have widely demonstrated that the Gly196Ser mutation reduces enzyme activity to approximately 8% of normal by decreasing the 5R type 2 affinity for NADPH, and its affinity for T is not altered (Thigpen et al, 1992). Because the great majority of reported patients show sufficiently masculinized phenotypes at birth to be raised as boys, it is conceivable that the residual 5R activity may be sufficient to induce partial masculinization of external genitalia (Thigpen et al, 1992) and to promote spermatogenesis and paternity (Nordenskjold and Ivarsson, 1998).

Patient 3 was a compound heterozygote for Tyr235Phe and Trp53X, the latter being a nonsense mutation previously reported in a single patient (Griffin et al, 2001). Nonsense mutations are known to cause severe forms of the disease; nevertheless, compound heterozygosity with a missense mutation could give rise to partially functioning enzymes, which might explain milder phenotypes. The Tyr235Phe mutation, also identified in patient 4 in the homozygous state, has been described in a white patient (Wigley et al, 1994) and in an Egyptian newborn (Mazen et al, 2003), both with predominantly female phenotypes. In a recent report, the Tyr235Phe mutation was described in homozygosity in 2 Italian prepubertal patients, 1 of whom showed a predominantly female phenotype and the other, reared as male, presented only with perineoscrotal hypospadias and descended testes (Nicoletti et al, 2005). The same mutation was also found in a Jewish Israeli patient with micropenis and hypospadias (Mazen, unpublished data, 2003), who, like patient 4 in this report, was reared as a boy. The reasons for the observed variability are unclear, but factors other than the impairment of the 5R enzyme may contribute to the clinical expression of the disorder (Manson and Carr, 2003, Ellis et al, 2005; Thiele et al, 2005).

The last 2 patients, 5 and 6, had the classic "historical" form with predominantly female external genitalia at birth and partial virilization at puberty. Patient 5 harbored the Cys133Gly novel mutation, which is to date a uniquely reported change in the SRD5A2 gene. Even though no in vitro studies were performed to investigate the functional consequence of the Cys133Gly mutation on enzymatic activity, it is interesting to note that this cysteine residue is conserved in both human type 1 and type 2, as well as in rat, monkey, and mouse 5-reductase enzymes (Normington and Russel, 1992; Levi et al, 1995), suggesting a possible relevant role for the activity of the enzyme. Patient 6 presented with the homozygous mutation Ala207Asp that was previously identified in a compound heterozygous patient from Austria (Thigpen et al, 1992), in 2 Mexican siblings with the homozygous condition (Méndez et al, 1995; Canto et al, 1997), and recently in a Brazilian male of African-European descent in the heterozygous state (Hackel et al, 2005). The study of 5R activity in cultured skin fibroblasts from the Austrian patient showed a qualitatively abnormal enzyme with altered affinity for T, thermolability, and a shift in optimum pH, supporting the pathogenicity of the mutation (Johnson et al, 1986; Jenkins et al, 1992; Thigpen et al, 1992). The identification of this mutation in different ethnic groups may be due to the existence of mutational hot spots in the SRD5A2 gene or to an ancient European or North African ancestor who spread the mutation. The clinical histories of patients 5 and 6 emphasize the necessity of early diagnosis before gender is assigned. In fact, although the female gender was assigned at birth to these patients and bilateral gonadectomies and feminizing surgical repairs of the genitalia were performed at puberty, 1 of the 2 patients retained predominantly male psychosexual orientation and identity. These observations should lead physicians to consider male gender assignment in individuals affected by 5R deficiency, even if the external genitalia are predominantly female at birth.

In conclusion, in this study, we identified 2 novel and 4 previously described SRD5A2 gene mutations in 6 Italian patients suspected to be affected by 5R deficiency by clinical and/or endocrine findings. Each of the detected novel mutations affects a residue that is conserved among 5R enzymes and was not detected in 80 chromosomes from normal controls, suggesting that these molecular changes are in fact the cause of the disease rather than the result of DNA polymorphisms. Our data confirm the heterogeneity of clinical and molecular spectra of 5R deficiency (Wilson et al, 1993; Griffin et al, 2001; Imperato-McGinley and Zhu, 2002; Hackel et al, 2005) and underline the importance of SRD5A2 gene analysis in selected subjects with 46,XY DSD to confirm diagnosis and plan correct management for each patient before gender assignment or surgical interventions are done (Hughes et al, 2006). In addition, our results indicated that 5R deficiency should be considered in patients with female or overtly ambiguous genitalia, with mild signs of undermasculinization, and with inconclusive (patient 3) or unavailable (patient 4) T:DHT ratios.

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