Figure 1.
A and B: Short and medially deviated index finger (above) and second toe (below). C and D: Radiograph of the triangular-shaped middle phalanx of the index finger (above) and the second toe (below).
Table 1.
Two-Point LOD Score Obtained from the Linkage Analysis between the BDA2 Locus and Chromosome 20p12.3 in the Pedigree.
Figure 2.
Pedigree structure and haplotypes of the family.
The marker order was determined from the Marshfield map and the UCSC Human Genome database (Mar 2006). The open symbols indicate the unaffected individuals, the blackened symbols indicate the affected individuals, the squares indicate the males, and the circles indicate the females. The blackened bars indicate the chromosome region shared by the affected members of the pedigree.
Figure 3.
A. Genomic profile of the microduplication as detected on the NimbleGen Human CGH 385K Chromosome 20 Tiling Array. The detected breakpoints are indicated by arrows. The duplication comprises ∼4.6 kb. The x axis shows the genomic positions on chromosome 20, and the y axis shows the log2 ratio. B. Microduplication confirmed by quantitative real-time PCR (Q-PCR) utilizing a ViiATM 7 Real-Time PCR system. The mean values of the relative quantification were exported from the ViiATM 7 software 1.0. The mean values and standard deviations (error bars) for each target amplicon relative to albumin, which was used as a two-copy reference gene, were calculated for eight affected (blue bars) and eight unaffected (green bars) individuals. The primers P1–P5 (P2–P4 are within the duplicate region, and P1 and P5 flank the region) were designed by Su et al. (2011). One duplicated allele plus one normal allele resulted in three copies of the amplicons of primers P2–P4 in the affected individuals and in a ratio of 1.5 relative to the two copies of the healthy control. The localization of the Q-PCR amplicons is illustrated in Figures 4A and 4B.
Figure 4.
Results of long PCR, breakpoint identification, and positions of primers used.
A. Structure of duplicated region in the unaffected members. F7 and R7: primers used in the breakpoint identification. P1–P5 are the primer positions used in the Q-PCR. The red and green bars indicate the breakpoint region. The numbers above the BMP2 denote the genomic position. Tel: telomere; Cen: centromere. B. Structure of duplicated region in patients. The duplicated region is between Chr20: 6,809,218 and 6,813,888. PCR using primers F7 and R7 amplified the 655 bp sequences spanning the breakpoint. C. Sequencing results of the breakpoint PCR product. The nucleotides of microhomology A (5 bp) are marked by a green box, and the nucleotides of upstream microhomology B (52 bp), including the rs6085706 SNP, are underlined. In addition, the microhomologous nucleotides GTGACC (7 bp) [22] and TTGCAGTGAGC (11 bp) [23] reported previously were also observed downstream of microhomology A (data not shown).
Figure 5.
Agarose gel electrophoresis of PCR product with the breakpoint primers F7 and R7.
There is a 655-bp band in the patient lanes (1 and 2) but no product in the lanes of the unaffected family member (3 and 4). M: marker.
Figure 6.
The chromosomal positions of the three previously reported duplications (Chr20: 6,808,129–6,814,024; Chr20: 6,808,477–6,814,024; and Chr20: 6,809,382–6,814,044) are indicated by the horizontal arrows.
Comparison with the reported duplications shows that our duplication (Chr20: 6,809,218–6,813,888) overlaps with them but has different ends. Furthermore, the causal sequences of BDA2 could be narrowed down to 4,507 bp (Chr20: 6,809,382–6,813,888).