Balkan Journal of Medical Genetics

MOLECULAR ANALYSIS OF RING Y CHROMOSOME IN A 10-YEAR-OLD BOY WITH MIXED GONADAL DYSGENESIS AND GROWTH HORMONE DEFICIENCY
Milenkovic T1,*, Guc-Scekic M2, Zdravkovic D1,3, Topic V4, Liehr T5, Joksic G6, Radivojevic D2, Lakic N2
*Corresponding Author: Tatjana Milenkovic, Department of Endocrinology, Institute for Mother and Child Healthcare of Serbia “Dr. Vukan Cupic,” Radoja Dakica 6-8, 11 070 Belgrade, Serbia; Tel.: +381-11-3018-109; Fax: +381-11-3108-257; E-mail: tanjamil@eunet.rs
page: 71

MATERIALS AND METHODS

Clinical Report. A 10-year-old boy of healthy and unrelated parents was referred for evaluation of perineal hypospadias and unilateral cryptorchidism, anomalies that were incompletely surgically corrected in early childhood in a regional hospital. He was the second child in the family, born at term after a normal pregnancy and delivery: birth weight was 2850 g and length 49 cm. His motor and mental development was normal. He was obese from early childhood. On admission to our hospital, his height was 129.3 cm (3rd- 10th centile), but inconsistent for the target height of 173.5 cm (25th-50th centile). His weight was 53.6 kg, body mass index (BMI) was 32.99 kg/m2 (>97th centile) and he had significant acanthosis nigricans on the neck, in the axillae and the cubital area. Bilateral pseudogynecomastia was also noticed. His external genitalia appeared predominantly male, his penis was 4.5 cm, and showed scars from previous operations. The large scrotal sac contained a 4 ml testis on the left, and an irregular mass of 0.5 ml on the right. Basal gonadotrophin levels were: luteinizing hormone (LH) 0.1 IU/L (0.03-0.43 IU/L), follicle-stimulating hormone (FSH) 1.7 IU/L (0.22-1.92 IU/L) and testosterone 0.1 nmol/L (0.06-0.8 nmol/L). Testosterone response to a short human chorionic gonadotrophine (HCG) test showed 1.2 and 2.6 nmol/L on days 3 and 4, respectively. Peak value of GH in the clonidine test was 1.2 mIU/Ll (>20 mIU/L), while values during overnight profile of GH were as follows: for peak value 1.1 mIU/L (>13.1 mIU/L) and for mean value 0.23 mIU/L (>3.2 mIU/L). The level of insulin-like growth factor-1 (IGF- 1) was 164 ng/mL (88-452 ng/mL) and IGF binding protein-3 (IGFBP-3) was 4.9 μg/mL (2.1-7.7 μg/mL). Serum free thyroxine and thyroid-stimulating hormone were normal. An oral glucose tolerance test showed no glucose intolerance but the insulin levels peaked at 656.8 mIU/L (100 mIUL/L). The homeostasis model assessment of insulin resistance (HOMA-IR) was 6.6 (3.16). Levels of high density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterorl (LDL-c) and triglycerides were normal. Bone age was estimated at 12 years. Magnetic resonance imaging (MRI) of the brain did not reveal any structural abnormality in the hypothalamic-pituitary region. Histopathology of surgical biopsy of the left gonad confirmed the presence of normal testicular tissue with early spermatogenesis. The irregular mass from the right side of the scrotal sac was histopathologically described as a streak gonad with the presence of a Fallopian tube and rudimentary uterus. Growth hormone therapy, 1.0 mg/m2/day, was started. During the first year of treatment his height increased by 11.0 cm and reached the 25th centile. He remained extremely obese. Cytogenetic and Molecular Cytogenetic Studies. Cytogenetic analysis was performed on G-banded metaphase chromosomes obtained from cultures of PHA-stimulated blood lymphocytes from the proband and his parents according to standard procedures [14,15]. Fluorescence in situ hybridization (FISH) was performed on peripheral blood lymphocytes using a Y chromosome cen satellite probe (Q-Biogene, Carlsbad, CA, USA) to detect and enumerate the Y chromosomespecific DYZ3 locus, according to the manufacturer’s recommendations, using an Axioskop microscope and MC 80 camera (Carl Zeiss, Jena, Germany). Subcentromere- specific multicolor FISH (subcenM-FISH) was performed using RP11-115H13, RP11.71M14 and a centromeric probe for the Y chromosome, as reported previously [16]. Fluorescent in situ hybridization with a subtelomeric TELVysion Xp/Yp probe, spectrum green (Abbott Laboratories, Abbott Park, IL, USA) and CEPY (DYZ1) probe for Yq12 (Abbott Laboratories) were used for precise characterization of the ring Y chromosome. The FISH images were captured on a Zeiss Axioplan microscope (Zeiss) with the IKAROS and ISIS digital FISH imaging system (MetaSystems, Altlussheim, Germany). Telomeric FISH (Telo-FISH) was performed as described in [17], using the Cy-3 labeled telomeric PNA probe (CCCTAA) 3 and staining with DAPI-mounting medium (Vector Laboratories). Chromosomal analysis was performed using a Zeiss- Axioimager A1 microscope equipped with a CCD camera and Axiocam image acquisition software (Imaging Associate) and software, Image J. Fifteen metaphases were analyzed for the presence of telomeric signals. Molecular Genetic Studies. Genomic DNA was extracted from a peripheral blood sample by standard procedures [18]. The heterochromatic region of the Y chromosome was analyzed using polymerase chain reaction (PCR) with primers flanking part of a Y chromosome- specific region (the repetitive 3.4 kb sequence from the DYZ1 family) [19]. Diagnostic testing of Y chromosome-specific microdeletions was performed using multiplex PCR amplification directed against three distinct azoospermia (AZF) regions [AZFa (s84, s86), AZFb (s127, s134) and AZFc (s254, s255)] on the long arm of the Y chromosome, the SRY gene on the short arm of the Y chromosome, and the ZFX/ZFY gene as an internal control [20]. Positive and negative controls were provided by DNA samples from a man with normal spermatogenesis and from a healthy woman, respectively.

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