**4.4 Molecular diagnosis**

*HSD17B3* gene alterations have been identified in patients showing clinical and biochemical characteristics of 17β-HSD3 deficiency. The disease is genetically heterogeneous and genotype-phenotype correlations have not been found.

To date, 27 mutations in the *HSD17B3* gene have been reported. These include intronic splice junction abnormalities, exonic deletions and missense mutations (Table 2) (Mains et al., 2008). The majority are missense mutations inherited as homozygous or compound heterozygous mutations, occurring most frequent in exons 3,9,10 of the gene; 4 are splice junction abnormalities (Andersson et al., 1996; Boehmer et al., 1999), 1 is a small deletion (777-783), and 1 is a thymidine deletion resulting in a frame shift mutation which alters the amino acid sequence from codon position 187 onward with a premature termination in codon 226 (Boehmer et al., 1999; Twesten et al., 2000).

Fig. 2. A diagnostic algorithm to elucidate the various etiologies of 46,XY DSD. The diagram shows the importance of hCG stimulation in the diagnosis of 46,XY DSD. Upon hCG stimulation, if the T/4-A ratio is >0.8, the diagnosis of 17- HSD3 can be suspected; if the T/DHT ratio is >20, a diagnosis of 5-reductase deficiency can be suspected. If the response

*HSD17B3* gene alterations have been identified in patients showing clinical and biochemical characteristics of 17β-HSD3 deficiency. The disease is genetically heterogeneous and

To date, 27 mutations in the *HSD17B3* gene have been reported. These include intronic splice junction abnormalities, exonic deletions and missense mutations (Table 2) (Mains et al., 2008). The majority are missense mutations inherited as homozygous or compound heterozygous mutations, occurring most frequent in exons 3,9,10 of the gene; 4 are splice junction abnormalities (Andersson et al., 1996; Boehmer et al., 1999), 1 is a small deletion (777-783), and 1 is a thymidine deletion resulting in a frame shift mutation which alters the amino acid sequence from codon position 187 onward with a premature termination in

of T is >100 ng/dl, androgen insensivity syndrome (AIS) is possible. However, if the response is <100 ng/dl, causes of gonadal dysgenesis should be sought. Once a diagnosis is

suspected, molecular genetic studies can be used for definitive diagnosis.

genotype-phenotype correlations have not been found.

codon 226 (Boehmer et al., 1999; Twesten et al., 2000).

**4.4 Molecular diagnosis** 


17β-Hydroxysteroid Dehydrogenase Type 3 Deficiency:

ambiguous genitalia, clitoromegaly (1.5 cm) and posterior fusion and scrotalization of the labia majora which contained palpable masses

46,XY DSD; inguinal mass, mild clitoromegaly

46,XY DSD;

gender role

46,XY DSD;

minora, severe hypospadias with undermasculinization– raised as males and

females

masses

46,XY DSD; prominent clitoris,

palpable inguinal gonads

17 years 46,XY DSD;

15 years 46,XY DSD;

12, 14 years 46,XY DSD;

6 months 46,XY DSD;

female or ambiguous genitalia at birth, male behaviors in childhood, pubertal virilization, absence of menses, male

 clitoromegaly, primary amenorrhea, absent labia

clitoromegaly, primary amenorrhea, inguinal

testes in herniorrhaphy sac, failure to menstruate

clitoromegaly, failure of breast development and deepening of voice

normal female prepubertal genitalia, bilateral inguinal hernia at sonography

Newborn 46,XY DSD;

2 years, 3 months

14, 15, 21 years

2 months, 2,

6, 17 years

5, 18 months, 2–4 years

Diagnosis, Phenotypic Variability and Molecular Findings 131

White Brazilian, English

African-American, South Asian

African-American, Italian

Unknown 46,XY DSD French p.His271Arg missense/

White American, Dutch

Italian, West Indian

Table 2. Mutations reported to date in patients with 17-HSD3 deficiency phenotype

Polish c.777-

Pakistani p.Cys268YT yr

783delGAT AACC

German p.Phe208Ile missense/

Italian p.Leu212Gln missense/

p.Glu215Asp missense/

p.Ser232Leu missense/

p.Met235Val missense/

inactivates enzyme

inactivates enzyme

inactivates enzyme

inactivates enzyme

inactivates enzyme

deletion/ frame shift truncates protein

missense/ inactivates enzyme

inactivates enzyme

inactivates enzyme

p.Pro282Leu missense/

p.Gly289Ser polymorphism / unknown

Andersson et al.,

Geissler et al., 1994 Bertelloni et al., 2006

Mendonca et al.,

Lee et al., 2007 Andersson et al.,1996

Geissler et al.,1994 Lee et al., 2007 Moghrabi et al., 1998

Geissler et al.,1994 Bertelloni et al., 2006 Moghrabi et al., 1998

Andersson et al.,1996

Lee et al., 2007 Lindqvist et al., 2001

Bachelot et al., 2006

Boehmer et al., 1999 Andersson et al.,1996

Boehmer et al., 1999 Bertelloni et al., 2009

1996

2000


American (Italian, German, Irish)

Syrian, Turkish, Dutch, Greek-American

African-Brazilian, Italian

Southern Italian

White American, English

junction

premature stop codon

inactivates enzyme

alters secondary protein structure

junction/ disrupts splice acceptance site

inactivates enzyme

p.Ala203Glu missense Mendonca et al.,

inactivates enzyme

German c.608delT downstream

Turkish p.Ala188Val missense/

Afghan p.Met197Lys missense/

c.655–1,G-A splice

p.Ala203Val missense/

p.Val205Glu missense/

p.Gln176Pro missense Andersson et al.,

Mueller § Coovadia,

Moghrabi et al., 1998

Twesten et al., 2000

Boehmer et al., 1999

Lee et al., 2007

Geissler et al., 1994 Boehmer et al., 1999 Andersson et al.,

Moghrabi et al., 1998 Ademola Akesode et al., 1977

Mendonca et al.,

Geissler et al., 1994 Mendonca et al., 1999 Moghrabi et al.,

Bertelloni et al., 2009

Lee et al., 2007 Andersson et al.,

1996

2000

1998

2000

1996

2009

1996

Unknown 46,XY DSD unknown c.538–1,G-A splice

13 years 46,XY DSD;

12 years 46,XY DSD;

10 years 46,XY DSD;

12 years 46,XY DSD;

10,16,17 years

13, 18, 21, 26 years

Newborn, 20 years

masses

canal

mass

46,XY DSD; prepubertal female external genitalia, pubertal virilization, male gender

46,XY DSD;

and female

46,XY DSD; prepubertal female externalgenitalia to perineoscrotal hypospadias, primary amenorrhea, mild clitoromegaly

Unknown 46,XY DSD;

absence of menses, failure of breast development, facial and chest hair and clitoral enlargement, male

gender identity in siblings

pubertal virilization

rol

clitoromegaly and coarsening of voice, scrotalization of labia majora and inguinal

female prepubertal development, clitoral enlargement at 12 years of age, testes in inguinal

prepubertal female external genitalia, inguinal

pubertal virilization, facial hair, 4–8 cm phallus and labioscrotal folds


Table 2. Mutations reported to date in patients with 17-HSD3 deficiency phenotype

17β-Hydroxysteroid Dehydrogenase Type 3 Deficiency:

Patients Epididimus Testes

2 Yes 1.0 –0.5 Present

4 Yes 9.0 1.3 Absent/

a mean of the two gonads; SDS: SD score.

**4.8 Gender behavior** 

adulthood (Tab. 3) (Bertelloni et al., 2009a; Rosler et al., 1996).

mla SDS

Table 3. Gonadal findings in 4 subjects with 17β-HSD3 deficiency

Diagnosis, Phenotypic Variability and Molecular Findings 133

gonadal tissue may show normal testicular structures, which can help to exclude any structural abnormalities (testicular dysgenesis) as the cause for the 46,XY DSD. Despite an early orchidopexy, an absent spermatogenesis has been seen in patients affected with 17- HSD3 deficiency raised as males (Dumic et al., 1985). So far, no patient with 17-HSD3 deficiency was fertile although raised as male, thus infertility appears to be the rule in

Spematogonia

1 Yes 1.4 –1.0 Scarce Normal Normal No

(sub-normal)

3 Yes 2.0 2.0 Present Normal Normal No

very scarce

Normal values from Cassorla et al., 1981 for patients 1-3 and from Taranger et al., 1976 for patient 4.

In the absence of a correct diagnosis before puberty, most patients with 17-HSD deficiency are raised as females and undergo virilization during adolescence due to extratesticular conversion of 4-A to T, secondary to some residual function of the enzyme and increased substrate availability in 4-A at puberty (Andersson et al., 1996). In cases with partial virilization, early post-natal diagnosis and consequence successful androgen treatment may result in a male sex assignment and in a nearly normal male phenotype in adulthood. Gonadectomy is recommended before puberty for those individuals who have been raised as females and wish to remain so. In these subjects, female sex characteristics should be induced or maintained with appropriate hormone replacement therapy (Hiort et al., 2003). Vaginal dilation using the modified Frank's procedure or vaginal reconstruction surgery may be necessary to create a vaginal cavity with adequate capacity for sexual relations (Castro-Magana et al., 1993). The patient and family will need appropriate psychological counseling to accept the diagnosis and the infertility that accompanies it (Gooren, 2002). In patients with a male attitude, it is possible to achieve adequate male development without medical intervention, when corrective surgery has been judged to be warranted (Boehmer et al., 1999; Farkas § Rosler, 1993; Rosler et al., 1996). Exogenous T treatment does not seem to yield additional benefits in adulthood (Mendonca et al., 2000; Farkas § Rosler, 1993), while pre-operative T administration may result in a better cosmetic appearance of the external genitalia (Farkas § Rosler., 1993). Gender role changes have been reported in 39-60% of cases of 17-HSD3 deficiency who have been raised as girls (Wilson, 1999). Genetic and endocrine evidence indicates that androgens play an important role in male gender behavior and identity. However the fact that many individuals with mutations of the 5-reductase and 17-HSD3 encoding genes do not change their gender role behavior implies that other

Sertoli cells

Leydig Micro-

Normal Normal Yes

Normal Hypertrophic No

calcifications

cells

Two missense mutations, the 239 G to A resulting in an Arg to Gln (R80Q) substitution, which is the most frequent alteration described in the Arab population living in the Gaza Strip (Boehmer et al.,1999; Mains et al., 2008; Rosler et al., 1996), and the 238 C to T resulting in an Arg to Trp (R80W) substitution (Bilbao et al, 1998; Faienza et al., 2007) involve the same arginine residue in exon 3 at position 80. This site has been extensively studied by systematic replacement of the wild-type arginine at position 80 and has been shown to be extremely important for both forming the salt bridge with the terminal phosphate moiety of the NADPH, as well as providing for a hydrophobic pocket for the purine ring of the adenosine portion of the NADPH (McKeever et al., 2002). Thus, this arginin is critical for cofactor binding and the substitution by different amino acids results in alteration of cofactor preference, switching from NADPH to NADH (Payne § Hales, 2004).

One polymorphic substitution (G289S) has been described in a heterozygous form in apparently normal individuals. This polymorphism does not impair the kinetic properties of the normal enzyme (Moghrabi et al., 1998). A possible role of the G289S variation has been demonstrate in prostate cancer (Margiotti et al., 2002).

Most gene alterations severely compromise the enzyme activity, but the R80Q mutation results in a 17-HSD3 residual enzyme activity (20%), showing a significantly lower reaction velocity as compared to the normal enzyme (Geissler et al., 1994).

### **4.5 Worldwide distribution of ancient and** *de novo* **mutations**

Haplotype analysis of genetic markers flanking the *HSD17B3* gene has been performed to establish the ancient or *de novo* occurrence of mutations described in European, North American, Latin American, Australian and Arab populations (Boehmer et al., 1999). Dutch, German, white Australian and white American patients carrying the 325+4,A –T mutation share the same genetic markers and seem to have a common European ancestor. A founder effect was also demonstrated for the R80Q mutation that is common in Dutch, Arab (in Gaza), white Brazilian, and white Portuguese patients. As this mutation is associated with a specific haplotype, a common ancestor introduced during the Phoenician migration has been hypothesized (Rosler et al., 2006). An additional founder effect has been suggested for 655–1,G-T mutation found in Greeks, Turks and Syrians patients that may have spread to the Mediterranean area during Ottoman Empire (Boehmer et al., 1999). On the contrary, patients harboring the 326-1,G-C and the c.Pro282Leu mutations have a different marker genotype suggesting that these are the novo mutations (Boehmer et al., 1999).

## **4.6 Genotype-phenotype correlation**

No phenotype-genotype correlation has been noted in 17-HSD3 deficiency, as exemplified by members of the same family who have different phenotypes despite the same genotype (Lee et al., 2007). A variable T/4-A ratio after human chorionic gonadotropin (hCG) stimulation was also seen despite the same homozygous mutation in different subjects of the same pedigree. This can be attributed to the extratesticular ability of some subjects to convert 4-A to T by other enzymes such as 17-HSD5 (Qiu et al., 2004).

### **4.7 Imaging studies**

Imaging studies that reveal the absence of mullerian structures and persistent wolffian structures also point to the diagnosis of 17-HSD3 deficiency, but this is not pathognomonic as 5-reductase type 2 deficiency will also have similar findings. Histological evidence from

gonadal tissue may show normal testicular structures, which can help to exclude any structural abnormalities (testicular dysgenesis) as the cause for the 46,XY DSD. Despite an early orchidopexy, an absent spermatogenesis has been seen in patients affected with 17- HSD3 deficiency raised as males (Dumic et al., 1985). So far, no patient with 17-HSD3 deficiency was fertile although raised as male, thus infertility appears to be the rule in adulthood (Tab. 3) (Bertelloni et al., 2009a; Rosler et al., 1996).


a mean of the two gonads; SDS: SD score.

Normal values from Cassorla et al., 1981 for patients 1-3 and from Taranger et al., 1976 for patient 4.

Table 3. Gonadal findings in 4 subjects with 17β-HSD3 deficiency
