Screening analysis of candidate gene mutations in a kindred with polycystic liver disease.
OBJECTIVE
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.


CONCLUSIONS
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.
Song Jin, Zi-Qiang Sun, Department of Hepatobiliary and Vascular Surgery, Affiliated Hospital of Jining Medical College, Jining 272029, Shandong Province, China.
Song
S
Jin
Song Jin, Zi-Qiang Sun, Department of Hepatobiliary and Vascular Surgery, Affiliated Hospital of Jining Medical College, Jining 272029, Shandong Province, China.
Kai
K
Cui
Song Jin, Zi-Qiang Sun, Department of Hepatobiliary and Vascular Surgery, Affiliated Hospital of Jining Medical College, Jining 272029, Shandong Province, China.
Zi-Qiang
ZQ
Sun
Song Jin, Zi-Qiang Sun, Department of Hepatobiliary and Vascular Surgery, Affiliated Hospital of Jining Medical College, Jining 272029, Shandong Province, China.
Yang-Yang
YY
Shen
Song Jin, Zi-Qiang Sun, Department of Hepatobiliary and Vascular Surgery, Affiliated Hospital of Jining Medical College, Jining 272029, Shandong Province, China.
Pang
P
Li
Song Jin, Zi-Qiang Sun, Department of Hepatobiliary and Vascular Surgery, Affiliated Hospital of Jining Medical College, Jining 272029, Shandong Province, China.
Zhen-Dan
ZD
Wang
Song Jin, Zi-Qiang Sun, Department of Hepatobiliary and Vascular Surgery, Affiliated Hospital of Jining Medical College, Jining 272029, Shandong Province, China.
Fei-Fei
FF
Li
Song Jin, Zi-Qiang Sun, Department of Hepatobiliary and Vascular Surgery, Affiliated Hospital of Jining Medical College, Jining 272029, Shandong Province, China.
Ke-Nan
KN
Gong
Song Jin, Zi-Qiang Sun, Department of Hepatobiliary and Vascular Surgery, Affiliated Hospital of Jining Medical College, Jining 272029, Shandong Province, China.
Sheng
S
Li
D000328:Adult; D001483:Base Sequence; D003062:Codon; D018389:Codon, Nonsense; D003560:Cysts / Q000175:diagnosis / Q000235:genetics* / Q000601:surgery; D004252:DNA Mutational Analysis; D005091:Exons; D005260:Female; D056726:Genetic Association Studies; D005819:Genetic Markers; D020022:Genetic Predisposition to Disease; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D008107:Liver Diseases / Q000175:diagnosis / Q000235:genetics* / Q000601:surgery; D008297:Male; D008969:Molecular Sequence Data; D010375:Pedigree; D010641:Phenotype; D050396:TRPP Cation Channels / Q000235:genetics*; D014057:Tomography, X-Ray Computed; D055815:Young Adult
D003062:Codon; D018389:Codon, Nonsense; D005819:Genetic Markers; D050396:TRPP Cation Channels
Gene mutation; Kindred; Polycystic liver disease