A 6-year-old boy, the first child of healthy and unrelated parents (mother 35 years old and father 40 years old), and had two healthy siblings,. He was born after 40 weeks of normal gestation. Birth weight was 2,950 g and length 52 cm. There was no family history of intellectual handicap or mental illness. He was referred to the Department of Pediatrics (Çukurova University, Adana, Turkey) because of development and speech delay concerns. He had patent ductus arteriosus in infancy, which had closed. No other sibling had similar features. There were no complications at birth but his psychomotor milestones were always behind those of his peers. He weighted 14 kg (45p for age, ‘p’ for percentile) and was 94 cm (45p for age) long. He was motor retarded, but showed no signs of aggressive behavior. He had mongoloid slant, epicanthal folds, cryptorchidism and umblical hernia, delayed tooth eruption and peg-shaped teeth. His hemogram, serum electrolytes and urine amino acids were normal. Magnetic resonance imaging of the brain showed no myelinization. A GTG banding procedure was used for the analysis of metaphase chromosomes on peripheral blood cell cultures of the family. Twenty metaphases were analyzed microscopically.

Quantitative fluorescent PCR was used for identification of the parental origin of the extra X chromosomes. DNA was extracted from blood samples of the patient and his parents using InstaGene™ Matrix (Bio-Rad Laboratories, Hercules, CA, USA). The QF-PCR amplifications were performed using Aneufast™ (Molgentix SL, Barcelona, Spain) trisomy detection kit which includes fluorescently-labeled primers for 27 predefined STR marker sites on chromosomes 13, 18, 21, X (DXYS218, SBMA, DXS6803, DXS6809, HPRT, DXS8377 and X22) and Y (DXYS218 and X22), and primer pairs for AMXY (at Xp22.1-22.3 and Yp11.2) and SRY (at Yp11.2) regions. The kit also contains dNTPs and HotStart Taq DNA polymerase in an optimized reaction buffer. Two mL of the DNA (5-10 ng) and 3 mL of PCR-grade water were added to 10 mL of each of the master mixes. After initial denaturation at 95°C for 15 min., amplification was achieved by 28 cycles of 95°C for 40 seconds, 58°C for 80 seconds and 72°C for 40 seconds, and final extension was for 30 min. at 60°C. The QF-PCR products (1.5 mL from each mix) were added to 40 mL Hi-Di™ Formamide (Applied Biosystems, Foster City, CA, USA) containing 0.3 mL of GeneScan™ -500 LIZ™ (Applied Biosystems) size standard. After denaturation at 95°C for 3 min., the mixture was cooled to 4°C and then capillary electrophoresis was carried out on an ABI PRISM™ 310 Genetic Analyzer (Applied Biosystems) using POP4 polymer. Analysis of the results was performed using GeneMapper 4.0 software (Applied Biosystems).

By means of the fluorescent primers, the amplified segments could be visualized and quantified as peak areas on automated DNA scanners. As the amount of PCR product was proportional to the initial amount of target DNA, normal heterozygous subjects were expected to show two equal peak areas (ratio 1:1) for each chromosome analyzed. In the case of aneuploidy of a chromosome, the ratio would change according to the change in number of a chromosome [10,11].

The parental origin of the aneuploidy was determined by comparision of the STR alleles of the patient and his parents. Meiotic division errors in meiosis I or meiosis II were inferred on the basis of non reduction/reduction stage of the chromosome by comparision of the proximal (pericentromeric) markers. If parental heterozygosity was retained in the aneuploidic child, we concluded that the error occurred during meiosis I, and if parental heterozygosity was reduced to homozygosity, we concluded that the error occurred during meiosis II or in post-zygotic mitosis. Mitotic errors were distinguished from meiosis II by evaluation of medial and distal markers. If homozygosity was not observed at all informative loci, including at least one each in proximal, medial, and distal portions of the chromosome, a post-zygotic origin was inferred. If homozygosity was observed at one or more loci, the error was assigned to meiosis II [11-15].