Published online Feb 28, 2015. doi: 10.3748/wjg.v21.i8.2343
Peer-review started: July 14, 2014
First decision: August 15, 2014
Revised: September 3, 2014
Accepted: November 19, 2014
Article in press: November 19, 2014
Published online: February 28, 2015
AIM: To find potential mutable sites by detecting mutations of the candidate gene in a kindred with polycystic liver disease (PCLD).
METHODS: First, we chose a kindred with PCLD and obtained five venous blood samples of this kindred after the family members signed the informed consent form. In the kindred two cases were diagnosed with PCLD, and the left three cases were normal individuals. All the blood samples were preserved at -85 °C. Second, we extracted the genomic DNA from the venous blood samples of the kindred using a QIAamp DNA Mini Kit and then performed long-range polymerase chain reaction (PCR) with different primers. The exons of PKD1 were all sequenced with the forward and reverse primers to ensure the accuracy of the results. Next, we purified the PCR products and directly sequenced them using Big Dye Terminator Chemistry version 3.1. The sequencing reaction was conducted with BiomekFX (Beckman). Finally, we analyzed the results.
RESULTS: A total of 42 normal exons were identified in detecting mutations of the PKD1 gene. A synonymous mutation occurred in exon 5. The mutation was a homozygous T in the proband and was C in the reference sequence. This mutation was located in the third codon and did not change the amino acid encoded by the codon. Missense mutations occurred in exons 11 and 35. These mutations were located in the second codon; they changed the amino acid sequence and existed in the dbSNP library. A nonsense mutation occurred in exon 15. The mutation was a heterozygous CT in the proband and was C in the reference sequence. This mutation was located in the first codon and resulted in a termination codon. This mutation had an obvious influence on the encoded protein and changed the length of the protein from 4303 to 2246 amino acids. This was a new mutation that was not present in the dbSNP library.
CONCLUSION: The nonsense mutation of exon 15 existed in the proband and in the third individual. Additionally, the proband was heterozygous for this mutation, so the mutable site was a pathogenic mutation.
Core tip: This study explored the mutable sites in the PKD1 gene and found the potential mutable sites by detecting mutations of the candidate gene in a kindred with polycystic liver disease (PCLD). We analysed five blood samples obtained from the kindred by using long-range polymerase chain reaction and direct sequencing with different primers. We found synonymous mutations in exons 5, 11, 15, and 35 of the PKD1 gene. Only the mutation in exon 15 was not in the dbSNP library, and it was a heterozygous CT in the proband and was C in the reference sequence. This mutation was located in the first codon and resulted in a termination codon. This nonsense mutation existed also in the third individual, so the mutable site was a pathogenic mutation.