We identified lymphoblast cell lines (LCLs) of HapMap subjects who were homozygous for either the H1 or H2 haplotypes using the available SNP data of rs1800547 and rs9468, as described previously [Stefansson et al., 2005
]. In brief, rs1800547 is located downstream of MAPT
exon 4 and rs9468 is located in exon 9 of the same gene. H2 is characterized by the alleles G and C of SNPs rs1800547 and rs9468, respectively. Additional homozygous carriers of H1 and H2 were identified in the Human Variation Collection available through the Coriell Institute for Medical Research, New Jersey, USA. Lymphoblast cell lines of these subjects were obtained from the Coriell Cell repository, and the SNP genotyping was performed using TaqMan real-time PCR (Applied Biosystems, Foster City, Calif., USA). Lymphoblast cell lines of probands with the 17q21 microdeletion and their unaffected parents were generated using standard methods at the University Medical Center Nijmegen, the Netherlands. These anonymized frozen samples were shipped to UCLA for FISH analysis and H1/H2 genotyping.
For the FISH analysis, the 23 LCLs were harvested after a 24-h media-starvation to synchronize the cells to being mostly in the G1 phase. Standard FISH methods were employed. Dual-color FISH was done using 3 BAC probes (RP11-403G3, RP11-256F16, RP11-80L9) (fig. ) directly labeled by nick-translation with biotin and digoxigenin and hybridized overnight to interphase cells (probes are less than 1 Mb apart) at 37°C. The cells were stained with DAPI, and a minimum of 50 interphase cells were analyzed under a Nikon fluorescence microscope equipped with appropriate filters. The images were captured with Isis Imaging System (Metasystems Group Inc., Waltham, Mass., USA). The orientation of the dual-colored signals was scored independently by 2 persons. A green-red-green (GRG) signal pattern is the H1 or direct orientation, while a GGR signal pattern on one or 2 chromosomal homologs represents the inverted orientation (figs. , ).
Schematic overview of the 17q21.31 region in direct orientation corresponding with the H1 haplotype, location of segmental duplication regions, and the location of BAC probes used for FISH analysis.
Fig. 2 Representative interphase cells with the FISH probes showing the orientation (green-red-green: non-inverted orientation and red-green-green: inverted orientation) of the 3 BAC probes from one of the nuclear families. A 31919-Father (H1/H1), B 31918-Mother (more ...)
We performed 3-locus interphase FISH experiments and examined the chromosome structure of 17q21.31 in a targeted study sample of subjects homozygous for the H1 and H2 haplotypes, and also of 3 patients with 17q21.31 microdeletion syndrome and their unaffected parents who have been previously described [Koolen et al., 2006
FISH analyses were also performed on 7 cell lines derived from individuals of European descent that were homozygous for the H2 allele (based on the diagnostic SNPs rs1800547 and rs9468). Another 7 cell lines were examined from HapMap subjects, also of European descent and who were either homozygous for H1 (n = 5) or heterozygous H1/H2 (n = 2). In a third series of FISH analyses, we established the inversion status of 3 17q21 deletion patients and their parents.