The six-year-old girl has been evaluated due to hyperactivity, social interaction impairment, with little social initiative and lack of social or emotional reciprocity, stereotypic and repetitive use of language with echolalia, failure to follow parents/caretakers orders, inconsolable outbursts, and persistent preoccupation with parts of objects. The girl has normal cognitive function. She is the only daughter of a nonconsanguineous young and healthy couple. There are no other similar cases in the family. She was born at term (caesarean delivery), weighing 3800 g and measuring 50.5 cm. She started to walk at 1 year and 4 months, and to speak at 2 years and 6 months. The patient has improved with language and educational therapies. She was examined when she was 6 years and 4 months old, 123 cm (p 50-75) tall, weighing 23,000 g (p 50-75), and had 50.5 cm of head circumference (p 2-50) (Figure 1). The clinical examination showed no physical, neurological, or dysmorphological abnormalities. Hematologic and hormonal test results were normal. The EEG displayed a normal pattern. Bone age was 8 years and 10 months (chronological age was 6 years and 4 months). Brain magnetic resonance imaging was normal. PCR-based assay for sizing CGG repeats at the FRAXA locus showed two normal alleles.
Metaphase chromosome spreads were obtained after lymphocyte temporary cultures, and the slides were subjected to GTG, CBG, and replication bandings.
 FISH was performed only for verification of the X chromosome with total chromosome digoxigenin-labeled probe obtained commercially (Oncorr, Gaithersburg, MD, USA:catalogue no. P5222-DG.5). Hybridization was performed according to manufacturer's instructions, followed by detection with FITC-labeled antidigoxigenin and count stain with propidium iodide (final concentration 0.3 mug/mL in antifade).
A SKY KIT from Applied Spectral Imaging (ASI, Carlsbad, CA, USA) was used for SKY. The slide treatment, posthybridization detection and washes, was performed as per standard protocols and manufacturer's instructions. The slides were mounted with 20 mM Tris-HCl pH 8.0, 90% glycerol containing 2.3% antifade, 1,4,diazabicyclo-(2.2.2)octane (ONCOR Inc., Gaithersburg, MD, USA). Spectral images were acquired and analyzed with a SD 200 spectral bioimaging system (ASI Ltd., MigdalHaemek, Israel) attached to a Zeiss microscope (Axioplan 2) by means of a C-mount consisting of an optical head with a special Fourier transformed spectrophotometer (SAGNAC common path interferometer) to measure the spectrum, and a refrigerated CCD-camera for imaging. Excitation through the custom filter set (Chroma Technology, Brattleboro, VT, USA) allows all dyes to be excited and measured simultaneously, without any image shift. The generation of a spectral image is achieved by acquiring ~100 frames of the same image. Each two frames differ from each other only in the optical path difference (OPD) created by the scanner controller in the interferometer. The images were stored in a computer for further analysis using the SKYVIEW (ASI, Carlsbad, CA, USA) software. Based on the measurement of the spectrum for each chromosome, a spectral classification algorithm was applied. DAPI images were acquired from all metaphases analyzed using a DAPI-specific optical filter.