PMC4272946_01
Multiplex ligation-dependent probe amplification analysis of the CHD7 gene. Arrows indicate the reduced ratio of exon 3.
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F1
MLPA P201-C1 CHARGE probemix; MRC-Holland, Amsterdam, Netherlands). MLPA analysis of the CHD7 gene revealed a heterozygous exon 3 deletion (Fig. 1).
Results of the sequence analysis and genomic structure encompassing exons 2-4 of the CHD7 gene. (A) Electrophoresis of a PCR product using the forward and reverse primers designated in (C) showed an apparent band in the patient. (B) The proximal and distal parts had no homologous sequences near the breakpoints. (C) Schematic diagram showing the range of the 38,304-bp deletion from c.1665+10039 to c.2097-3547. Alu sequences (blue triangles) were not adjacent to the breakpoints. Orange, green, and violet arrows indicate stepwise long-range PCR primers. (D) Sequence analysis revealed a 4-bp insertion (TAAC) between the breakpoints.. Abbreviations: Pt., patient; NC, normal control.
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F2
A total of nine Alu sequences were located in intron 2 and three were located in intron 3 (Fig. 2C). Finally, a 1kb-sized PCR fragment was obtained by using the following primers: GGTGGGCTGTGAAGTGTTCTGGC (forward primer; located in intron 2) and ACCCACAGTGCACTCCTCCCC (reverse primer; located in intron 3) (Fig. 2A). Sequence analysis revealed the exact breakpoints (Fig. 2B). The deleted region totaled 38,304 bp from c.1665+10039 in intron 2 to c.2097-3547 in intron 3, and it was accompanied by a TAAC insertion (Fig. 2D).