Genetic analysis of CSF2RA gene defects. (A) Nucleotide sequence of CSF2RA in genomic DNA from each family member, including nt 580–591 of the coding sequence (numbered relative to the initiation codon; from GenBank/EMBL/DDBJ under accession no. NM_006140.3). The index patient and her sister exhibited only a G→A point mutation at nt 586. The father was heterozygous for this substitution and the mother exhibited only the normal sequence. (B) FISH analysis to detect CSF2RA sequences in genomic DNA from the father and the patient. The probe (CTD-3047L21), which maps to the pseudoautosomal region (Xp22.33 and Yp11.32), hybridized to both X and Y chromosomes in the father (white arrows) and to one (white arrow), but not the other (yellow arrow), X chromosome in the patient. 8–10 metaphase cells and 25 interphase cells were evaluated for each individual. Similar FISH analyses are shown for each family member in the supplemental material . Images were obtained at a total magnification of 1000×. (C) CGH analysis for the patient in the region of Xp22.33. The relative fluorescence of fluorescently labeled DNA from the patient (open circles) compared with a same-sex reference DNA (filled circles) after hybridization to various BAC clones on the SignatureSelect V2 chip representing the Xp22.33 region is shown. Reduced hybridization to several BAC clones (clones B and C) is indicated by the lower fluorescence of the patient's DNA compared with the reference DNA for these BAC clones. The telomeric breakpoint is mapped to between clones A and B and the centromeric breakpoint is mapped to between clones C and D. These data indicate an interstitial deletion of ∼1.264 Mb at Xp22.33 encompassing ∼1,610,183–2,873,864 bp. The dashed line represents a relative fluorescence of zero. (D) High-resolution SNP mapping of the Xp22 region for paternal and maternal X chromosomes. A schematic shows the locations of the point mutation (CSF2RA^G174R) in the paternal X chromosome and the 1.6-Mb deletion at Xp22.33 in the maternal X chromosome and the genes encompassed. The base 2 ratio of normalized hybridization intensities for patient and reference samples (log R ratio) is shown. Similar SNP analyses for each family member are shown in the supplemental material . (E) Map showing a portion of the X chromosome summarizing the genetic analysis used to identify the small maternal X chromosomal deletion at Xp22.33 encompassing CSF2RA. The probe used for FISH analysis (hatched bar) is the same as clone B on the CGH microarray chip. The locations of selected CGH microarray probes in the region of the CSF2RA gene are shown. Those CGH probes showing balanced hybridization to patient and control DNA are shown as clear boxes (A and D), whereas those showing an unbalanced hybridization representing sequences deleted in the patient are shown in black. High-resolution SNP analysis revealed that the deletion (red bar) extended from 1,308,324 to 2,881,011 bp. Sequence analysis (A) and PCR quantification of CSF2RA exon 7 (Fig. S4) demonstrated that the deletion included CSF2RA exon 7. The chromosomal location of the CSF2RA gene (1,347,701–1,388,827 bp) is indicated. (F) Pedigree of the family deduced from sequencing and CSF2RA allelic copy number determination experiments. The index case is indicated (arrow).