Novel PITX2 Mutations including a Mutation Causing an Unusual Ophthalmic Phenotype of Axenfeld-Rieger Syndrome
Table 1
Primers for amplifying and sequencing PITX2 and FOXC1.
Gene
Exon
Forward primer (5′-3′)
Reverse primer (5′-3′)
Product size (bp)
Annealing temperature (°C)
PITX2
2
GGCCGCCGCTTCTTACA
CACTGGCGATTTGGTTCTGATTT
254
63
3
CTGCCTCGCCCCTCCCCCTCCTC
TTCAAGCAGCAGGCGCGTCAGGTC
350
64
4
GGGGCAGTAGCCAAGGACT
CAGCTAAGCGGGAATGTCTG
289
64
5
GGCGCGGACACCCACACTTG
GCGCTGCCTTCCACATTCTCT
487
65
6-A
GCGCTGCCTTCCACATTCTCT
GGTGGATAGGGAGGCGGATGTAAG
346
63
6-B
GGTGGATAGGGAGGCGGATGTAAG
CGACGGGCTACTCAGGTTGTTCA
256
64
6-C
TCCCGGGCTCCAGTCTCAACAG
TTTCTTTAGTGCCCACGACCTTCT
355
64
SSCP-1
CGACGACATGTACCCAGGCTATTC
GCGACGGGCTACTCAGGTTGTT
257
68
SSCP-2
GGCGCGGACACCCACACTTG
CTGCCGCCTTTGCCGCTTCTTCTT
269
68
SSCP-3
CTGGCCCTGGTATCTTGGTGTGC
GGTGGATAGGGAGGCGGATGTAAG
346
63
FOXC1
1-A
CCCGGACTCGGACTCGGC
AAGCGGTCCATGATGAACTGG
429
63
1-B
GCGCACGCCGAGCAGTA
CACCGCGTCCTTCTTCTTGA
390
60
1-C
CACCCTGAACGGCATCTACCA
AGGCTGCTGCTGCTGCTGTCG
504
64
1-D
GCCCGTGCGCATCCAGGACATCAA
GCTGCCCGCGCTGGAGGTCTGG
463
64
1-E
AGGGCTTCAGCGTGGACAACATCA
GGTGGGCCGCAGGGTGGTG
482
65
1-F
CAAGCCATGAGCCTGTACG
GGGTTCGATTTAGTTCGGCT
502
60
Primer sequences, sizes of PCR products, and annealing temperatures used for the amplification are listed. Primers 2–6 were used to amplify and sequence the PITX2-coding segments. The sequencing of exon 6 of PITX2 was performed with three overlapping primers A–C. Primers SSCP-1 to SSCP-3 were used to amplify the novel variations detected in PITX2 for heteroduplex-SSCP analysis. The single FOXC1 exon was sequenced with six overlapping primers, A–F.