CLINICS 2012;67(8):981-985 duplicate with a resolution of at least 650 kb and human DNA segments of 100,300 kb distributed through the whole genome. The slides were scanned with InnoScan 710 (Innopsys, Carbonne, France), and MAPIX 4.5 software was used to generate GPR files. Data analysis was conducted with SpectralWareH v2.3.3 aCGH Analysis System software (12) from Perkin Elmer. The computational parameters used were pin linear normalization, a threshold between 0.7 and 1.3, a Loess alpha of 0.1 for normalization and a confidence level of 95%. Median values were used for the interpretation of the results. The Database of Genomics Variants was consulted to determine the size of chromosome regions and to identify CNVs (13). These analyses revealed gains in two neighboring BAC clones on 5q32.2 and losses in 4q22.1, 8q24.3 and 9q34.2-34.3 (Table 1). A spectral image of chromosome 5 from the patient demonstrating the gain in 5q35.2 is presented in Figure 1. The patients revised karyotype was 46,XX.arr 4q22.1(88,282,368-88,336,595)x1, 5q35.2(173,984,926-174,145,340)(174,718,444-174,907,513)x5, 8q24.3(142,215,092-142,412,727)x1, 9q34.2q34.3(136,982,786- 137,166,802)x1[hg18]. DNA samples from the patient and progenitors were used for MSX2 gene copy number assessment. A TaqMan copy number assay (Applied Biosystems, Foster City, CA) consist- ing of an MGB probe labeled with FAM and unlabeled PCR primers for MSX2 (assay Hs01821094_cn) was employed. Unlabeled primers and a VIC dye-labeled TAMRA probe for RNase P gene, which is known to be present in two copies in human diploid genome, were used for normalization. MSX2 COPY number increase and craniosynostosis Pelegrino KO et al. Reactions were performed as recommended by the manufac- turer in triplicate. Duplex real-time polymerase chain reactions were conducted in a 7500 Real-Time PCR System, and the data were analyzed with CopyCallerTM Software v1.0, both from Applied Biosystems. Cycle thresholds (CT) were calculated using the relative quantification method (Applied Biosy- stems). Copy numbers were calculated by determining the difference in the CT between the target and control probes (DCT). Cycle thresholds greater than 32 were excluded from analyses. A quantitative PCR assay to detect MSX2 CNVs revealed two copies for both progenitors and five copies for the patient (Figure 2). Phenotypically normal subjects unre- lated to the studied family also had two copies. The mean delta Ct, calculated as the FAM Ct divided by the VIC Ct, and the standard deviation of the delta Ct were 0.2192¡0.13 for Subject 1, 0.5772¡0.29 for Subject 2, 0.5807¡0.1 for the patient’s mother, -0.8858¡0.12 for the patient, 0.3630¡0.16 for Subject 3 and 0.7518¡0.12 for the patient’s father. DISCUSSION A search for similar abnormalities in DECIPHER (Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources) (14) returned three cases with chromosome 4 alterations related to that observed in the present study (cases 753, 994, and 249573). The phenotypes in these cases included mental retardation/developmental delay, short stature, atrial septum defects, hypotonia and eyelid defects, among other abnormalities. However, it is important to stress that no correlation between a loss in the Figure 2 - Assessment of MSX2 copy number variation. The estimated copy number variations for the patient, the progenitors, and three unrelated subjects are presented. The bars represent the variation in the calculated copy number. The values were ¡0.41 for Subject 1, ¡0.63 for Subject 2, ¡0.23 for the patient’s mother, ¡0.79 for the patient, ¡0.45 for Subject 3 and ¡0.30 for the patient’s father. 983