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Identification of a Large SLC25A13 Deletion via Sophisticated Molecular Analyses Using Peripheral Blood Lymphocytes in an Infant with Neonatal Intrahepatic Cholestasis Caused by Citrin Deficiency (NICCD): A Clinical and Molecular Study.

Zheng QQ, Zhang ZH, Zeng HS, Lin WX, Yang HW, Yin ZN, Song YZ - Biomed Res Int (2016)

Bottom Line: Ultimately, a large deletion c.329-1687_c.468+3865del5692bp, which has never been described in any other references, was identified via intensive study on the genomic DNA around exon 5 of SLC25A13 gene.Conclusions.The large deletion enriched the SLC25A13 mutation spectrum, and its identification supported the concept that cDNA cloning analysis, along with other molecular tools such as semiquantitative PCR, could provide valuable clues, facilitating the identification of obscure SLC25A13 deletions.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, The First Affiliated Hospital, Jinan University, Guangzhou 510630, China.

ABSTRACT
Background. Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) is a Mendelian disorder arising from biallelic SLC25A13 mutations, and SLC25A13 genetic analysis was indispensable for its definite diagnosis. However, conventional SLC25A13 analysis could not detect all mutations, especially obscure large insertions/deletions. This paper aimed to explore the obscure SLC25A13 mutation in an NICCD infant. Methods. Genomic DNA was extracted to screen for 4 high-frequency SLC25A13 mutations, and then all 18 exons and their flanking sequences were analyzed by Sanger sequencing. Subsequently, cDNA cloning, SNP analyses, and semiquantitative PCR were performed to identify the obscure mutation. Results. A maternally inherited mutation IVS16ins3kb was screened out, and then cDNA cloning unveiled paternally inherited alternative splicing variants (ASVs) featuring exon 5 skipping. Ultimately, a large deletion c.329-1687_c.468+3865del5692bp, which has never been described in any other references, was identified via intensive study on the genomic DNA around exon 5 of SLC25A13 gene. Conclusions. An NICCD patient was definitely diagnosed as a compound heterozygote of IVS16ins3kb and c.329-1687_c.468+3865del5692bp. The large deletion enriched the SLC25A13 mutation spectrum, and its identification supported the concept that cDNA cloning analysis, along with other molecular tools such as semiquantitative PCR, could provide valuable clues, facilitating the identification of obscure SLC25A13 deletions.

No MeSH data available.


Related in: MedlinePlus

Semiquantitative PCR in positioning analysis of the novel large deletion. Figure 1① was a representative electrophoresis of the semiquantitative PCR products. Compared with the mother (M1), the patient (P1) and the father (F1) had less signal intensity of the PCR products when using primer Set 1, while this is not the case when using primer Set 2; note that the signal intensity of the PCR products in the patient (P2) and the father (F2) was similar to that in the mother (M2). Figure 1② depicted the positions of the primer Sets 1 and 2. The primer sequences in Set 1 were 5′-GAGCTTCTTAGAAACCACCATGTGG-3′ (IVS5S5) and 5′-TCCAATGAGG AAGAAGACTACAGGAAG-3′ (IVS5A6), while in Set 2, 5′-TTTATGCACTGGGGCAACATG-3′ (IVS 1NF) and 5′-TGCCGGGCTGACACTTTGG-3′ (IVS 2NB), respectively. The results suggested that the patient (P) and the father (F) might harbor an obscure large deletion around the primer Set 1 but not Set 2.
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fig1: Semiquantitative PCR in positioning analysis of the novel large deletion. Figure 1① was a representative electrophoresis of the semiquantitative PCR products. Compared with the mother (M1), the patient (P1) and the father (F1) had less signal intensity of the PCR products when using primer Set 1, while this is not the case when using primer Set 2; note that the signal intensity of the PCR products in the patient (P2) and the father (F2) was similar to that in the mother (M2). Figure 1② depicted the positions of the primer Sets 1 and 2. The primer sequences in Set 1 were 5′-GAGCTTCTTAGAAACCACCATGTGG-3′ (IVS5S5) and 5′-TCCAATGAGG AAGAAGACTACAGGAAG-3′ (IVS5A6), while in Set 2, 5′-TTTATGCACTGGGGCAACATG-3′ (IVS 1NF) and 5′-TGCCGGGCTGACACTTTGG-3′ (IVS 2NB), respectively. The results suggested that the patient (P) and the father (F) might harbor an obscure large deletion around the primer Set 1 but not Set 2.

Mentions: According to the ASV structures, to further locate the DNA span around exon 5 that might contain the obscure mutation, semiquantitative PCR was performed in a total volume of 50 μL, containing 5 μL of 10x Buffer (Mg2+ plus) (TaKaRa), 4 μL of dNTP (10 mM), 37.75 μL of sterilized distilled water, 0.25 μL of Taq (TaKaRa), 2 μL of the forward and reverse primers together, and 1 μL of DNA template. Then, all tubes were placed into a thermal cycler and the parameters were set in 94°C for 5 minutes followed by 28 to 30 cycles of 94°C for 30 seconds, 60°C for 40 seconds, 72°C for 40 seconds, and a final extension step at 72°C for 10 minutes. The two primer pairs were IVS5S5 and IVS5A6 in Set 1 and IVS 1NF and IVS 2NB in Set 2, whose sequences and locations were displayed in Figure 1, respectively.


Identification of a Large SLC25A13 Deletion via Sophisticated Molecular Analyses Using Peripheral Blood Lymphocytes in an Infant with Neonatal Intrahepatic Cholestasis Caused by Citrin Deficiency (NICCD): A Clinical and Molecular Study.

Zheng QQ, Zhang ZH, Zeng HS, Lin WX, Yang HW, Yin ZN, Song YZ - Biomed Res Int (2016)

Semiquantitative PCR in positioning analysis of the novel large deletion. Figure 1① was a representative electrophoresis of the semiquantitative PCR products. Compared with the mother (M1), the patient (P1) and the father (F1) had less signal intensity of the PCR products when using primer Set 1, while this is not the case when using primer Set 2; note that the signal intensity of the PCR products in the patient (P2) and the father (F2) was similar to that in the mother (M2). Figure 1② depicted the positions of the primer Sets 1 and 2. The primer sequences in Set 1 were 5′-GAGCTTCTTAGAAACCACCATGTGG-3′ (IVS5S5) and 5′-TCCAATGAGG AAGAAGACTACAGGAAG-3′ (IVS5A6), while in Set 2, 5′-TTTATGCACTGGGGCAACATG-3′ (IVS 1NF) and 5′-TGCCGGGCTGACACTTTGG-3′ (IVS 2NB), respectively. The results suggested that the patient (P) and the father (F) might harbor an obscure large deletion around the primer Set 1 but not Set 2.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835617&req=5

fig1: Semiquantitative PCR in positioning analysis of the novel large deletion. Figure 1① was a representative electrophoresis of the semiquantitative PCR products. Compared with the mother (M1), the patient (P1) and the father (F1) had less signal intensity of the PCR products when using primer Set 1, while this is not the case when using primer Set 2; note that the signal intensity of the PCR products in the patient (P2) and the father (F2) was similar to that in the mother (M2). Figure 1② depicted the positions of the primer Sets 1 and 2. The primer sequences in Set 1 were 5′-GAGCTTCTTAGAAACCACCATGTGG-3′ (IVS5S5) and 5′-TCCAATGAGG AAGAAGACTACAGGAAG-3′ (IVS5A6), while in Set 2, 5′-TTTATGCACTGGGGCAACATG-3′ (IVS 1NF) and 5′-TGCCGGGCTGACACTTTGG-3′ (IVS 2NB), respectively. The results suggested that the patient (P) and the father (F) might harbor an obscure large deletion around the primer Set 1 but not Set 2.
Mentions: According to the ASV structures, to further locate the DNA span around exon 5 that might contain the obscure mutation, semiquantitative PCR was performed in a total volume of 50 μL, containing 5 μL of 10x Buffer (Mg2+ plus) (TaKaRa), 4 μL of dNTP (10 mM), 37.75 μL of sterilized distilled water, 0.25 μL of Taq (TaKaRa), 2 μL of the forward and reverse primers together, and 1 μL of DNA template. Then, all tubes were placed into a thermal cycler and the parameters were set in 94°C for 5 minutes followed by 28 to 30 cycles of 94°C for 30 seconds, 60°C for 40 seconds, 72°C for 40 seconds, and a final extension step at 72°C for 10 minutes. The two primer pairs were IVS5S5 and IVS5A6 in Set 1 and IVS 1NF and IVS 2NB in Set 2, whose sequences and locations were displayed in Figure 1, respectively.

Bottom Line: Ultimately, a large deletion c.329-1687_c.468+3865del5692bp, which has never been described in any other references, was identified via intensive study on the genomic DNA around exon 5 of SLC25A13 gene.Conclusions.The large deletion enriched the SLC25A13 mutation spectrum, and its identification supported the concept that cDNA cloning analysis, along with other molecular tools such as semiquantitative PCR, could provide valuable clues, facilitating the identification of obscure SLC25A13 deletions.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, The First Affiliated Hospital, Jinan University, Guangzhou 510630, China.

ABSTRACT
Background. Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) is a Mendelian disorder arising from biallelic SLC25A13 mutations, and SLC25A13 genetic analysis was indispensable for its definite diagnosis. However, conventional SLC25A13 analysis could not detect all mutations, especially obscure large insertions/deletions. This paper aimed to explore the obscure SLC25A13 mutation in an NICCD infant. Methods. Genomic DNA was extracted to screen for 4 high-frequency SLC25A13 mutations, and then all 18 exons and their flanking sequences were analyzed by Sanger sequencing. Subsequently, cDNA cloning, SNP analyses, and semiquantitative PCR were performed to identify the obscure mutation. Results. A maternally inherited mutation IVS16ins3kb was screened out, and then cDNA cloning unveiled paternally inherited alternative splicing variants (ASVs) featuring exon 5 skipping. Ultimately, a large deletion c.329-1687_c.468+3865del5692bp, which has never been described in any other references, was identified via intensive study on the genomic DNA around exon 5 of SLC25A13 gene. Conclusions. An NICCD patient was definitely diagnosed as a compound heterozygote of IVS16ins3kb and c.329-1687_c.468+3865del5692bp. The large deletion enriched the SLC25A13 mutation spectrum, and its identification supported the concept that cDNA cloning analysis, along with other molecular tools such as semiquantitative PCR, could provide valuable clues, facilitating the identification of obscure SLC25A13 deletions.

No MeSH data available.


Related in: MedlinePlus