Underlying Genetic Defects in Diseases of Sexual Development in the Island of Cyprus Support the Founder Effect

Nicos Skordis Paediatric Endocrine Unit, Department of Paediatrics, Makarios III Hospital, 1474 Nicosia, Cyprus.

Department of Molecular Genetics, Function and Therapy, The Cyprus Institute of Neurology & Genetics, P.O. Box 23462, 1683 Nicosia, Cyprus.

Abstract

Hereditary endocrinopathies in Cyprus exhibit evidence of a founder effect and display the influence of past migration patterns. The genetic frequency and mutation pattern of specific Disorder of Sex Development (DSD) which is classified as 46, XX DSD or 46, XY DSD and the Non-Classic form of Congenital Adrenal Hyperplasia (NC-CAH) outline a type of genetic drift.

The high prevalence of the NC-CAH p.V281L mutation but also the rarity of CAH large lesions present genetic diversity similar to that observed in the Middle Eastern countries. In addition, the high frequency of the 5-alpha Reductase Deficiency (5αSRD) IVS1-2A>G mutation and the carrier frequency of 17-beta Hydroxysteroid Dehydrogenase 3 (17β-HSD-3) p.R80Q mutation are both good examples of founder effect. The mutation p.R80Q can be considered as a founder mutation even though it has been identified in patients of Dutch, Brazilian and Portuguese origin. This has led to the speculation that it has a Phoenician origin. Phoenicians as ancient traders migrated around 750 B.C from present day Syria, Lebanon and Israel toward Portugal, Spain and also to nearby Cyprus. While the 5αSRD IVS1-2A>G mutation has already been extensively reported in Turkish patients it is very common in the Eastern Mediterranean region.

This work portrays clearly, through specific endocrine genetic disorders, the past migrational trends in Cyprus that shaped the present day pool of the Greek-Cypriot population.

Address for communication: Dr. Nicos Skordis, Department of Molecular Genetics, Function and Therapy, The Cyprus Institute of Neurology & Genetics, P.O. Box 23462, 1683 Nicosia, Cyprus. E-mail: [email protected] 275 Nicos Skordis

Introduction so far. The genetic defects that cause 5αSRD deficiency in patients of Cypriot origin with their relative frequency Inherited endocrinopathies including congenital adrenal are also presented. The founder effect of 17β-HSD-3 hyperplasia (CAH), 5-alpha deficiency deficiency is finally described, where patients of Greek- (5αSRD) and 17-beta hydroxysteroid dehydrogenase Cypriot origin have mutations that have been identified 3 (17β-HSD-3) deficiency have been found to display in Arabic cohorts. a founder effect in the Greek-Cypriot population. Such conditions can disturb the orderly process of reproductive organ formation leading to what is called Congenital Adrenal Hyperplasia Disorders of Sexual Differentiation (DSD). DSD is defined as a congenital condition in which development Congenital adrenal hyperplasia (CAH) is a recessively of chromosomal, gonadal or anatomical sex is atypical. inherited disorder that is caused by the deficiency of (Houk et al., 2006; Hughes et al., 2006a; Hughes et al., the enzymatic activity required for biosynthesis 2006b; Lee et al., 2006) in the adrenal cortex. In 95% of CAH cases it is due to defects in the steroid 21-hydroxylase (CYP21A2) gene 46, XX DSDs cause virilisation or masculinisation of and display an overall estimated incidence of 1:10,000 to the female foetus. The common pathway of all 46, XX 1:15,000 for the severe classic form and 1:500 to 1:100 DSDs is the final step where excessD ihydrotestosterone live births for the NC-CAH. (Carroll et al., 1985; Urabe (DHT) or potent exogenous effect in the et al., 1990; Morel and Miller, 1991; Pang, 1997; Speiser genital tissue is observed during the critical period of and White, 2003; Forest, 2004; van der Kamp and Wit, sexual differentiation. The extent to which the clinical 2004; Merke and Bornstein, 2005) manifestation occurs depends on both the levels of DHT in circulation and the gestational age when the The CYP21A2 gene is located on the short arm of exposure occurs. The that are accountable 6, within the major histocompatibility for virilisation in 46, XX DSDs are mostly produced by complex (MHC) region, at just 30 kb away from a highly the fetal adrenal glands, while they are only rarely of homologous (>95%) pseudogene CYP21A1P. Mutations maternal origin or from exogenous administration. One observed during 21-OHD deficiency usually result of the most common causes of such condition is due to from the transfer of sequences between the CYP21P 21-hydroxylase deficiency that leads to CAH. pseudogene and the active CYP21A2 gene. The defects that are usually observed in the CYP21A2 gene are 46, XY DSDs consists of a variety of clinical conditions deletions, conversions and single nucleotide mutations, in which the development of fetal gonad is abnormal with more than 100 reported mutations (Human Gene and encompasses both complete and partial forms. Mutation Database: http://www.hgmd.cf.ac.uk/ac/index. (Houk et al., 2006; Hughes et al., 2006a; Hughes et al., php). (Higashi et al., 1986; White et al., 1986; Donohoue 2006b; Lee et al., 2006; George et al., 2010) Included et al., 1989; Tusie-Luna et al., 1990; Urabe et al., 1990; in this category are 5α Steroid Reductase (5αSRD) Wilson et al., 1995a) deficiency that affects the conversion of to its more active metabolite, and Genetic defects in the CYP21A2 gene are classified into 17β-Hydroxysteroid Dehydrogenase-3 (17β-HSD-3) three categories with respect to the residual enzymatic deficiency that disturbs the biosynthesis of sex activity that typically correspond to the three types of causing hormonal imbalance. Both conditions lead to the 21-OHD: salt-wasting (SW), simple virilizing (SV), and formation of undervirilized male genitalia. non classical (NC) CAH. Patients with NC-CAH have no evidence of adrenocorticotropic hormone (ACTH) In this report the genetic defects in endocrinopathies excess and generally present with signs and symptoms that have recently been reported in the island of Cyprus of androgen excess rather than symptoms reflecting are unveiled, which display evidence of a founder effect. glycocorticoid deficiency. (Witchel and Azziz, 2010) The genetic frequency of specificDSD that are classified Children usually present with premature pubarche, tall as 46, XX DSD or 46, XY DSD is underlined. More stature and often accelerated bone maturation whereas specific the major mutations that are found to dominate adolescent females present with hyperandrogenemic the genetic spectrum of Greek Cypriot patients with symptoms such as hirsutism, acne, anovulation, menstrual NC-CAH are described along with a report of the rare dysfunction and decreased fertility. Interestingly, a mutations in the CYP21A2 gene that have been identified significant number of individuals with NC-CAH

Austral - Asian Journal of Cancer ISSN-0972-2556, Vol. 11, No. 4, October 2012 pp 275-288 276 Underlying Genetic Defects in Diseases of Sexual Development ..... identified in family studies are asymptomatic. (Bidet et premature pubarche and were diagnosed in childhood. al., 2009) The remaining fifteen female patients were diagnosed in adolescence and adulthood with clinical signs of The incidence of 21-OHD genetic defects has been hyperandrogenemia. Patients (group B) who carried the extensively studied and ethnic specific distributions in mild severe/mutation genotype as shown in Table 2B the Mediterranean region have been recently reported. were more likely to manifest the disease before puberty (Dracopoulou-Vabouli et al., 2001; Kharrat et al., 2004; as five out of eight females presented with premature Wasniewska et al., 2007; Abid et al., 2008; Sadeghi et pubarche in childhood. Among the fourteen adolescent al., 2008; Wasniewska et al., 2008) The frequency of female patients with the mild/mild genotype PCOS was CAH mutation in the Greek-Cypriot population has been the most common presenting symptom (8/14), followed shown to have p.V281L as the most common with 68.9% by hirsutism (as determined by a Ferrmian Gallway score while the second most frequent mutation is p.P453S at more than 8) in 5/14. 6.75% followed by IVS2-13A/C 4.05%, p.Q318X 4.05%, p.V304M 4.05%, p.M283V 2.7%, p.P482S 2.7%, p.P30L The overall frequency of p.V281L is the highest 2.7%, p.P30L+IVS2-13A/C 1.35%, p.I172N 1.35% while occurring mutation not only for Cyprus but both in large DNA lesions are quite rare in our population and are Greece (Dracopoulou-Vabouli et al., 2001) and other only found at 1.35% (Table 1). (Neocleous et al., 2009; populations in Europe (Wedell et al., 1994; Balsamo Skordis et al., 2011b). et al., 2000; Krone et al., 2000; Ezquieta et al., 2002; Dolzan et al., 2005; Abid et al., 2008; Sadeghi et al., 2008; Wasniewska et al., 2008; Bidet et al., 2009; Finkielstain et Table 1. Mutation frequency of 74 affected alleles al., 2011). The classical form of CAH is considered rare from 37 unrelated NC-CAH patients. in the Cypriot population since only ten cases (6 males, 4 females) have been reported over the past 31 years. Severity of mutations Number of % of alleles (Skordis et al., 2011b) The incidence of classical CAH alleles is 1:30000, which is much less than expected compared to other Mediterranean countries.(Forest, 2004; van der Total Total Kamp and Wit, 2004; Merke and Bornstein, 2005) Mild mutations We have recently reported that IVS2-13A/C>G can be p.V281L 51 68.9 considered as the most frequent molecular defect in the p.P453S 5 6.75 SW form and its high frequency is consistent with most p.P30L 2 2.7 studies reported so far. (Owerbach et al., 1992; Speiser p.V304M 3 4.05 et al., 1992; Ezquieta et al., 1995; Wilson et al., 1995a; p.M283V 2 2.7 Wilson et al., 1995b; Carrera et al., 1996; Dardis et al., p.P482S 2 2.7 1997; Jaaskelainen et al., 1997; Bachega et al., 1998; p.P30L+IVS2- 1 1.35 Fardella et al., 1998; Lako et al., 1999; Krone et al., 2000; 13A/C Dracopoulou-Vabouli et al., 2001; Kotaska et al., 2003; Severe mutations Dolzan et al., 2005; Skordis et al., 2011a) Large lesions IVS2-13A/C 3 4.05 though are quite rare in the Greek-Cypriot population p.Q318X 3 4.05 which is much lower than what is found in the Hellenic p.I172N 1 1.35 population (14.3%) or other ethnic groups (Carrera Large DNA lesion 1 1.35 et al., 1996; Dardis et al., 1997; Fardella et al., 1998; Dracopoulou-Vabouli et al., 2001); this can be attributed to either differences in the genetic background or poor In order to determine the relationship between the number of patients screened.(Skordis et al., 2011a) severity of the mutation and the clinical and biochemical Similarly p.I172N, which is found in 13.5% in Greek data, patients were categorised based on the severity of the patients, has been found at just 1.35% of Greek-Cypriots. mutated alleles (mild/mild and mild/severe genotype) as This genetic defect is observed only 4% in Turkish shown in Table 2. Twenty nine patients (group A), as shown patients, 30% in Morocco and remains unreported from in Table 2A, were found as compound heterozygotes with any patients in Sicily. (Abid et al., 2008; Sadeghi et al., 2 mild mutations (mild/mild genotype). Fourteen patients 2008; Wasniewska et al., 2008) of this group (eleven females, three males) presented with

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Table 2. A) Group A: Mild/Mild genotype (n = 29). Clinical features, basal and adrenocorticotropic hormone (ACTH) stimulated 17-OHP levels in 29 genotyped NC-CAH Cypriot patients with Mild/Mild mutations.

No. Age of Gender Genotype 17- 17-OHP, Clinical Pre- Total DHEA-S Δ4 diagnosis OHP, nmol/l sentation Test ng/ml ng/ml nmol/l, ACTH nmol/l Basal Stimulated 1 6 F p.V281L/p.V281L >60.5 >60.5 PP 0.97 0.18 0.8 2 7 F p.V281L/p.V281L >60.5 >60.5 PP 0.38 5.48 1.8 3 7 F p.V281L/p.V281L 11.58 30.6 PP 1.5 4 0.5 4 7 F p.V281L/p.P453S 17.5 37.8 PP - - - 5 7 F p.V281L/p.P453S >60.5 >60.5 PP, OW 1.39 2.52 2.6 6 7 F p.V281L/p.V281L 17 >60.5 PP 0.69 2.77 0.9 7 7 F p.V281L/p.V281L >60.5 >60.5 PP, OW 2.26 6.9 3.13 8 7 F p.V281L/p.V281L 20.3 >60.5 A, PP, OW 2.7 4.23 3.08 9 8 F p.V281L/p.P453S 33 >60.5 PP - - - 10 8 F p.V281L/p.V281L 40.8 >60.5 PP 0.94 3.15 1.4 11 8 F p.V281L/p.V281L 35.95 >60.5 PP, A 4.74 4.24 2.6 12 8 M p.V281L/p.V281L 13.2 >60.5 PP 1.76 5.39 1.61 13 8 M p.V281L/p.V281L 9.04 >60.5 PP 0.69 3.15 1 14 9 M p.V281L/p.M283V 22.6 57.6 PP 0.69 2.42 - 15 9 F p.V281L/p.V281L >60.5 >60.5 PP 0.86 - 1.1 16 11 F p.V281L/p.P453S 17.8 >60.5 A, H, OW 1.69 3.5 2.5 17 12 F p.P30L/p.V281L 11.5 >60.5 PCOS, A 3.29 5.83 1.6 18 14 F p.V281L/p.V281L >60.5 >60.5 PCOS, A, H 2.63 3.64 4.28 19 14 F p.V281L/p.V281L 24.9 57.6 A, H 2.46 7.68 5.7 20 15 F p.V281L/p.P482S 12.53 57.93 PP, H 2.67 3.94 - 21 15 F p.V281L/p.P482S 11.8 >60.5 PCOS, A, O 2.83 4.64 6.7 22 15 F p.V281L/p.V281L >60.5 >60.5 PCOS 0.87 4.18 7 23 17 F p.V281L/p.V281L >60.5 >60.5 IM, H 3.34 - - 24 17 F p.V281L/p.V281L >60.5 >60.5 PCOS, A 1.6 - 3.2 25 20 F p.V281L/p.V304M >60.5 >60.5 H - - - 26 25 F p.V281L/p.V281L 45 >60.5 PCOS 4.47 6.75 4.75 27 28 F p.V281L/p.P453S 6.2 >60.5 PCOS 2.52 3.75 6.56 28 30 F p.V281L/p.V304M 4.5 48.5 IM, SF - - - 29 36 F p.V281L/p.V304M >60.5 >60.5 PCOS, H 3.31 6.43 6.8 PCOS = Polycystic ovary syndrome; IM = irregular menses; A = acne; H = hirsutism; OW = overweight; PP = premature pubarche; SF = subfertility.

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Table 2. B) Group B: Mild/Severe genotype (n = 8). Clinical features, basal and adrenocorticotropic hormone (ACTH) stimulated 17-OHP levels in 8 genotyped NC-CAH Cypriot patients with Mild/Severe mutations.

No. Age of Gender Genotype 17-OHP, 17-OHP, Clinical Presen- Total DHEA-S Δ4 diagnosis nmol/l nmol/l tation Test ng/ml ng/ Basal ACTH nmol/l ml Stimulated 1 4 F IVS2-13A/p.V281L >60.5 >60.5 PP 4.14 3.59 5.96 2 4 F p. P30L/IVS2-13A/C >60.5 >60.5 PP 9.6 17.2 11 3 7 F p.P30L+IVS2-13A/ >60.5 >60.5 PP - - - C/p.P172N 4 7 F p.V281L/p.Q318stop >60.5 >60.5 PP 2.92 5.89 - 5 8 F IVS2-13A/p.V283M 54.5 >60.5 PP - 2.34 5.6 6 20 F p.V281L/p.Q318stop >60.5 >60.5 PCOS, A, H, 5.06 7.76 5 OW 7 26 F p.V281L/p.Q318stop 9.3 >60.5 H, IM 3.17 6.68 8 32 F p.V281L/Large lesion >60.5 >60.5 PCOS, A, H 3.39 6.45 10

PCOS = Polycystic ovary syndrome; IM = irregular menses; A = acne; H = hirsutism; OW = overweight; PP = premature pubarch

The phenotypic expression of 21-OHD is primarily et al., 2005; Skordis et al., 2011a) Even though p.P30L related to the type of the molecular defect and correlates has been considered as a severe mutation, it sometimes with the severity, although this does not always reflect displays a NC phenotype especially when it is found in the underlying genetic defect.(Dracopoulou-Vabouli et a compound heterozygote form. (Balsamo et al., 2000) al., 2001) Recently we have reported cases with severe NC form that carry in compound heterozygosity the The rare missense mutations p.V304M, p.A391T, as relatively milder p.P30L mutation with the IVS2-13A/C. well as the p.F306insT mutation were detected in these (Skordis et al., 2011a) This milder missense p.P30L five non related females. The rare mutation p.V304M mutation, although known to reduce the enzymatic was detected in three individuals. The first patient, who activity and generally associated with NC form, is often at the age of 28 years presented with irregular menses found in patients with more severe signs of androgen and difficulties in achieving a pregnancy, carried the excess.(Tusie-Luna et al., 1991; White and New, 1992; p.V304M mutation in cis (as determined from the family Wilson et al., 1995a) A study on the Hellenic population studies – data not shown) with 8bpdE3 and in the second identified the mutation p.P30L present in at least 1 of the allele the common p.V281L. In the second patient, aged 2 in 5 out of 6 NC genotypes, associated 14 years with Polycystic Ovary Syndrome (PCOS) the with the SV phenotype (Speiser et al., 1992; Wedell p.V304M mutation was found in cis with 8bpdE3 in et al., 1994; Wilson et al., 1995a; Krone et al., 2000; compound heterozygosity with the common p.V281L. In Dracopoulou-Vabouli et al., 2001; Dolzan et al., 2005). the third girl with NC-CAH who presented at the age of 7 Similar findings were also reported by Krone et al. S( peiser years with premature adrenarche the p.V304M missense et al., 1992; Wedell et al., 1994; Wilson et al., 1995a; mutation was found in the heterozygous state as expected. Krone et al., 2000; Dracopoulou-Vabouli et al., 2001; A screening for the presence of the p.V304M allele in the Dolzan et al., 2005), where the prediction was stronger in Cypriot population to determine its frequency was carried patients in group C when excluding the p.P30L mutation. out. The analysis of 194 healthy unrelated Cypriot adults The presence of p.P30L was found in approximately detected 4 heterozygous for the p.V304M mutation, 25% of Cypriot patients with the SV form either in giving a carrier rate of 2.1 %, or 1 in 49.(Neocleous et homozygosity or in a compound heterozygote state with al., 2009) a second mutation or gene deletion/conversion.(Dolzan

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5a Steroid Reductase deficiency homozygous mutations, whereas some are compound heterozygotes. Deletions and disruptive mutations are 5α Steroid Reductase deficiency (5αSRD) is an rare; mostly missense mutations have been described. autosomal recessive enzymatic deficiency that results from mutations in the 5α Steroid Reductase type 2 gene This A/G splice junction intron1/exon 2 mutation (SRD5A2) that causes male pseudohermaphrodism due is considered to abolish or sometimes partially abolish to decreased dihydrotestosterone (DHT) synthesis. The enzymatic activity, is now regarded as a common mutation development and virilization of the external genitalia in in the Cypriot population.(Skordis et al., 2010) IVS1- the human male depends on the production and action 2A>G mutation has been reported to have a carrier of DHT. DHT is synthesised from testosterone (T) in frequency of 0.98% in the Cypriot population (Skordis a reaction catalysed by the membrane-bound steroid et al., 2010; Skordis et al., 2011c). 5a-Reductase . Impaired DHT synthesis as a result of 5αSRD, leads to incomplete masculinization of The diagnosis of 5αSRD in three out of five patients was the external genitalia of a 46 XY individual (Sultan et suspected based on the biochemical findings, following al., 2001). The clinical spectrum of the 46 XY individual hCG stimulation test, as shown on Table 3. The hCG test with 5αSRD ranges from complete female appearance at was not performed in patient 1, because she had already birth to nearly complete male phenotype. The patients undergone gonadectomy. Therefore, stimulated T and show virilization at puberty without breast development, DHT ratio (T/DHT) was calculated in four out of five which is often accompanied by gender identity change, patients, which showed elevated T/DHT ratio before from female to male (Imperato-McGinley et al., 1974; (20, 16.6 and 13.5) and after stimulation (29, 29.5, 23.7 Wilson et al., 1993; Mendonca et al., 1996; Sinnecker respectively) in three patients. The increased T/DHT et al., 1996). ratio suggested 5αSRD and the diagnosis was confirmed by molecular analysis of the SRD5A2 gene. Elevated T/ Fifty four different mutations of SRD5A2 have currently DHT ratio was not found in patient 5, who was therefore been identified in patients with α5 SRD. (Andersson et al., diagnosed as having Partial Androgen Insensitivity 1991; Thigpen et al., 1992; Russell et al., 1994; Hiort et Syndrome (PAIS). The patient was subsequently given a al., 1996; Hochberg et al., 1996; Mendonca et al., 1996; course of Testosterone treatment (Testosterone depot 25 Canto et al., 1997; Vilchis et al., 1997; Nordenskjold and mg IM monthly x 3), which was not effective in increasing Ivarsson, 1998; Ferraz et al., 1999; Hiort et al., 2002; the penile size (Fig. 1a and 1b). Removal of the testes Ocal et al., 2002; Nicoletti et al., 2005; Adiyaman et was therefore performed and she was raised as a female. al., 2006; Baldinotti et al., 2008; Sahakitrungruang et al., 2008; Vilchis et al., 2008) Approximately two thirds The underlying mutations detected in the patients are of the patients with 5αSRD have been shown to have depicted on Table 3. The same homozygous mutation of

Table 3. Results of the hCG stimulation test and molecular findings in patients with SRD5A2 mutations.

T DHT T/DHT T DHT T/DHT nmol/L nmol/L Ratio nmol/L nmol/L Ratio patient Mutations baseline stimulated 1 ------3 26.5 1.3 20 52.3 1.8 29 Homozygous IVS2-2A>G 4 18.2 1.1 16.6 32.4 1.1 29.5 Heterozygous Heterozygous Pro- 2 19.0 1.4 13.5 23.7 1.0 23.7 IVS2-2A>G 181Leu Heterozygous Heterozygous 5 3.8 0.93 4.1 16.3 3.4 4.8 IVS2-2A>G Arg171Ser

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A B

Figure 1. Phenotypic appearance of patient 5 A. before and B. After Testosterone treatment.

SRD5A2 gene was identified in three out of five patients two mutations which were the same A/G substitution with in genomic DNA analysis. This mutation was A/G at Pro181Leu and Arg171Ser, both in exon 3 respectively splice junction intron1/exon 2. Targeted genetic analysis (Figures 2 and 3). The parents of patients 2 and 5 were also for the IVS1-2A>G identified this mutation to be shared tested for the detection of mutations in the SRD5A2 gene. in heterozygosity in both parents of patients 1, 3 and 4. Mutation IVS1-2A>G was found in the heterozygote Patients 2 and 5 were compound heterozygote carriers of state in both mothers of patients 2 and 5. Mutations p.P181L and p.R171S were respectively identified in the heterozygote state in the fathers of patients 2 and 5.

The exact etiological f a c t o r s t h a t c a u s e undervirilization in males with 46, XY DSD using just clinical characteristic and hormonal findings are not always sufficient without the use of genetic screening. As a result genetic screening for 46, XY DSD in Greek-Cypriot patients has shown that the IVS1-2A>G mutation, found in SRD5A2 gene, is characteristic of the Eastern Mediterranean region and may correspond to a founder effect, which is further supported by its high carrier frequency in our population. (Skordis et Figure 2. A. Partial sequences exon 3 of SRD5A2 gene in patient 2 showing the heterozygous al., 2005). p.P181L. B. Partial sequence of the intron1/exon2 junction showing the A to G mutation at position -2 of the acceptor splice site. The mutation is heterozygous.

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et al., 1999; Rosler, 2006) p.R80Q mutation was also reported in patients of Dutch, Brazilian and Portuguese origin leading to the speculation that the mutation was introduced by the Phoenicians, the ancient traders who migrated from present day Syria and Lebanon and Israel around 750 B.C toward Portugal and Spain. (Geissler et al., 1994; Rosler et al., 1996; Boehmer et al., 1999; Mendonca et al., 1999; Mendonca et al., 2000) Since, p.R80Q is Figure 3. Partial sequence of the intron1/exon2 junction showing the A to G mutation at considered as a founder position -2 of the acceptor splice site. The mutation is homozygous in patients 1, 3 and 4. mutation in Arabic cohorts A normal control is added. it is not surprising that patients with this mutation 17-beta hydroxysteroid have been found within the Greek-Cypriot population dehydrogenase 3 (Neocleous et al. 2012).

17β-HSD-3 deficiency is an inherited autosomal Our patient was the first child from healthy non- recessive disorder that affects the biosynthesis of consanguineous parents. He was born at term by caesarean testosterone. There are at least 14 known isoenzymes of section with birth weight 3640g and he was referred to 17β-HSD that play a role in the final stages of all androgen our clinic because of ambiguous genitalia at birth. He and synthesis. (Labrie et al., 1997; Wu et al., had small penis 1.5cm, perineal blind vaginal pouch and 2007; Kowalik et al., 2009; Moeller and Adamski, 2009) posterior labioscrotal fusion. Gonads were palpable in The17β-hydroxysteroid dehydrogenase (HSD) type 3 the inguinal canal bilaterally and no Müllerian structures isozyme catalyzes the conversion of androstenedione indentified on pelvic ultrasound. Chromosomal analysis (Δ4) to testosterone in the testis and mutations in the showed an apparently normal male 46,XY karyotype. HSD17B3 gene, which is localized on chromosome 9q22 Diagnosis of 17β-HSD-3 deficiency in this newborn was can cause undermasculinization. (Geissler et al., 1994; suspected based on biochemical findings, following hCG Andersson, 1995) The clinical spectrum of a 46, XY stimulation test, which showed low Testosterone levels, individual with 17β-HSD-3 deficiency can vary from high Androstenedione levels and high ratio of serum Δ4/T ambiguous genitalia to almost completely female external in the basal state and after stimulation, thus favoring the genitalia at birth. diagnosis of 17β-HSD-3 deficiency. He was subsequently treated with intramuscular testosterone 25mg monthly for The exact incidence of 17β-HSD-3 deficiency is two months which resulted in an adequate enlargement of unknown, but a study from the Netherlands reported the penis and therefore Androgen Insensitivity Syndrome 1:147,000 newborns with a calculated heterozygous (AIS) was excluded. The diagnosis was confirmed by frequency 1 in 135. (Boehmer et al., 1999) More than molecular analysis of the HSD17B3 gene. Following 20 mutations in the HSD17B3 gene have been reported diagnosis and genetic counseling the decision was to raise with the disease displaying an increased frequency among this newborn as a boy. Hence, the patient underwent the Arab cohort living in Gaza Strip since there is a high repair of cryptorchidism at the age of 15months old and intermarriage rate. (Rosler et al., 1996; Boehmer et al., subsequently repair of hypospadias with urethroplasty at 1999) The most common defect in this Arab population the age of 3years as shown in Figure 4. is p.R80Q mutation that is localized on exon 3 and has an incidence of approximately 1:200-300. (Boehmer Mutation analysis of the HSD17B3 gene by sequencing

Austral - Asian Journal of Cancer ISSN-0972-2556, Vol. 11, No. 4, October 2012 pp 275-288 282 Underlying Genetic Defects in Diseases of Sexual Development ..... analysis and MLPA, revealed the presence of the known p.R80Q missense mutation (Figure 5) and a novel large duplication spanning exons 3-10 . The presence of a possible duplication or deletion was suspected based on the DNA sequencing chromatographs in the patient and his mother. Initial investigation for the identification of possible duplication or deletion was performed by MLPA and RT- PCR analysis (Figure 6). MLPA analysis (using probe mixture P334 which includes 3 probes from gene HSD17B3 on exons 1, 3 and 11) revealed a duplication of exon 3 of gene HSD17B3 in the patient and his mother.

This is a clear indication that Figure 4. Pictures of patient showing his excellent response to testosterone (T) Phoenicians back in the first B.C administration. A: Newborn at clinical presentation. B and C: After treatment with centuries migrated to Cyprus from testosterone. D: After surgical correction of cryptorchidism and hypospadias.

present day Syria, Lebanon and Israel. This is consistent with the high frequency of 1:14 for CFTR gene mutation F508del observed in certain areas of Cyprus (Yiallouros et al., 2007), the frequency of 1:7 to 1:10 for Friedreich Ataxia (Dean et al., 1988) and the recently reported novel COL4A4 gene mutation G871C in a cohort of Greek Cypriot families with thin membrane nephropathy and Focal Segmental Glomerulosclerosis. (Voskarides et al., 2008; Deltas et al., 2011)

Figure 5. Chromatograms showing i. the p.R80Q mutation Figure 6. Results of Real-Time PCR and chromatograms of identified in exon 3 of the HSD17B3 gene in the Cypriot the 11.96 kp novel duplication in the HSD17B3 gene in a patient and ii. control sequence. Cypriot 46, XY individual with 17β-HSD-3 deficiency.

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Discussion present day Syria and Lebanon and Israel around 750 B.C toward Portugal and Spain. (Geissler et al., 1994; CAH has a high carrier frequency in Cyprus (1:10, Rosler et al., 1996; Boehmer et al., 1999; Mendonca et al., unpublished data) similar to what is observed in the 1999; Mendonca et al., 2000) Since, p.R80Q is a founder Eastern Mediterranean countries. But the incidence of mutation in Arabic cohorts migrating to neighboring classical form of CAH is just 1:30000, which is much countries such as Cyprus it is not surprising to find such less than expected compared to other Mediterranean patients in the local population. Similarly, the migration countries. In a similar fashion, large lesions are quite pattern of the Phoenicians back in the firstB .C centuries rare in the Greek-Cypriot population compared to other from present day Syria, Lebanon and Israel to Cyprus is Mediterranean countries suggesting a Middle Eastern very probable. influence.T he high prevalence of the p.V281L mutation (68.9%) in the Greek-Cypriot population is also common The IVS1-2A>G mutation in the SRD5A2 gene is in Ashkenazi Jews (42%). The close proximity of Israel to characteristic in the Cypriot population and displays Cyprus, but also Cyprus being part of the migration path a carrier frequency of 0.98%. This splice site mutation of Ashkenazi Jews to Europe, most likely resulted to a is probably due to a gene founder effect. Such genetic founder effect. (Wilson et al., 2007) abnormalities have been already reported in Turkish patients and it may be characteristic of the Eastern A founder effect is also displayed for the 17β-HSD-3 Mediterranean region (Figure 8). mutation p.R80Q that was found to be common among Arabs in different areas of Israel has been speculated to The phenomenon of founder effect is not unusual in the be from Druze ancestors from Lebanon and Syria (Figure Hellenic population of Cyprus and several recent reports 7). (Rosler et al., 1996; Boehmer et al., 1999; Rosler, have documented several founder mutations in the island. 2006) The same p.R80Q mutation was reported also in The recent evidence of a founder effect unveiled from the patients of Dutch, Brazilian and Portuguese origin leading genetic population profile of certain endocrinopathies to the speculation that the mutation was introduced by describes the past migrational trends in Cyprus (Table 4). the Phoenicians, the ancient traders who migrated from The earliest known human activity on the island dates

Figure 7. Evidence of a founder effect in the Eastern Mediterranean region as observed by the high frequency of 17-beta hydroxysteroid dehydrogenase 3 mutation p.R80Q, the low number of Congenital Adrenal Hyperplasia Large Deletions and the high prevalence of p.V281L.

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Figure 8. Mutations in the SRD5A2 gene as presented in the Mediterranean region.

Table 4. A brief overview of the migration pattern in back to around the 10th millennium BC. The island was Cyprus, from 1400BC until the present time. settled by Mycenaean Greeks in the 2nd millennium BC and the island also experienced long periods of Greek rule ♦ Mycenaean Greeks, 1400-1300 BC. under the Ptolemaic Egyptians and the Byzantines. At a ♦ Achaeans Greeks, 1230-1050 BC. strategic location in the Middle East, Cyprus has been ♦ Phoenicians, 950-850 BC. occupied by several major powers, including the empires ♦ Assyrians, 709-669 BC. of the Assyrians, Egyptians, Persians, Arabs, Lusignans, ♦ Egyptians, 565-546 BC. Venetians, and Ottomans In 333 BC, Alexander the Great ♦ Persians, 546-332 BC. conquered the island from the Persians. ♦ In 333 BC Alexander the Great defeats Persia and Cyprus becomes part of his empire. The Ottoman Empire conquered the island in 1571 ♦ Cyprus under Roman Empire, 58 BC-330 AD. and it remained under Ottoman control for over three ♦ Cyprus under the Byzantine Empire, 330-1191 centuries. It was placed under British administration in AD. 1878 until it was granted independence in 1960, becoming ♦ Cyprus was under constant attack by Arabs and a member of the Commonwealth the following year. The pirates, 647-965 AD. gene pool of the Greek-Cypriot population is constantly ♦ Richard I conquer Cyprus 1191 and establish the changing and it is expected that in the near future it Kingdom of Cyprus, 1191-1192 AD. will display a more eastern European profile. The above ♦ Lusignans, 1192-1474 AD. endocrine genetic profile described may have resulted ♦ Venetians, 1474-1571 AD. from an ancient founder effect, but we cannot rule out ♦ Cyprus under the Ottoman Empire, 1571-1878 that in some cases this might have been created due to a AD. hot spot in the gene, unequal crossing over during meiosis ♦ Cyprus under the British Empire, 1878-1960 AD. or gene conversion of point mutations in the pseudogene ♦ Cyprus gained independence from the British thus the mutation become prevalent in the expanded rule, Sovereign state 1960 – present. population.

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The high prevalence of such endocrinopathies in de novo mutations. J Clin Endocrinol Metab, 84, 4713-4721. 9. brain, C.E., et al., 2010. Holistic management of DSD. Best Pract the local population, 0.018% for DSD and ~7% NC- Res Clin Endocrinol Metab, 24, 335-354. CAH (Sax, 2002; Thyen et al., 2006), emphasizes the 10. canto, P., et al., 1997. Mutations of the 5 alpha-reductase type 2 importance of having a correct system composed of gene in eight Mexican patients from six different pedigrees with 5 clinical endocrinologist, biochemist and geneticists that alpha-reductase-2 deficiency.C lin Endocrinol (Oxf), 46, 155-160. 11. carrera, P., Bordone, L., Azzani, T., Brunelli, V., Garancini, M.P., are essential for the assessment of such difficult and Chiumello, G., Ferrari, M., 1996. Point mutations in Italian complex genetic disorders. The primary aim should be patients with classic, non-classic, and cryptic forms of steroid to achieve a diagnosis; sex assignment and management 21-hydroxylase deficiency. Hum Genet, 98, 662-665. plan as quickly as possible (Brain et al., 2010) but also 12. carroll, M.C., Campbell, R.D., Porter, R.R., 1985. Mapping of steroid 21-hydroxylase adjacent to complement component have a high awareness of the type of DSDs that are found C4 genes in HLA, the major histocompatibility complex in man. in our local population. Proc Natl Acad Sci U S A, 82, 521-525. 13. dardis, A., Bergada, I., Bergada, C., Rivarola, M., Belgorosky, A., 1997. Mutations of the steroid 21-hydroxylase gene in an Argentinian population of 36 patients with classical congenital Author Disclosure Statement adrenal hyperplasia. J Pediatr Endocrinol Metab, 10, 55-61. 14. dean, G., Chamberlain, S., Middleton, L., 1988. Friedreich’s ataxia The author declare that there is no conflict of interest in Kathikas-Arodhes, Cyprus. 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