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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

The large deletion mutation in SLC25A13 gene of the infant and his father. Figure 2① depicted the positions of the primer Set 3 and the novel large deletion. The primer sequences were 5′-AAGATTGTTGTTTATGGTGAGAC-3′ for IVS4S3 and 5′-ATGGTTTGCCCGACATGAGTAATC-3′ for J5.6KbDelR1, respectively. In Figure 2②, LA-PCR with the primer Set 3 revealed that the patient (P) and his father (F), but not the mother (M), had an unexpected band of 632 bp in size besides the normal product of 6324 bp. Figure 2③ was a segmental sequencing result of the unexpected PCR product. The arrow indicated the breakpoint arising from the large deletion.
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fig2: The large deletion mutation in SLC25A13 gene of the infant and his father. Figure 2① depicted the positions of the primer Set 3 and the novel large deletion. The primer sequences were 5′-AAGATTGTTGTTTATGGTGAGAC-3′ for IVS4S3 and 5′-ATGGTTTGCCCGACATGAGTAATC-3′ for J5.6KbDelR1, respectively. In Figure 2②, LA-PCR with the primer Set 3 revealed that the patient (P) and his father (F), but not the mother (M), had an unexpected band of 632 bp in size besides the normal product of 6324 bp. Figure 2③ was a segmental sequencing result of the unexpected PCR product. The arrow indicated the breakpoint arising from the large deletion.

Mentions: Based on the findings above, a diversity of primers was designed and PCR amplification conducted to analyze a variety of SNPs within the DNA fragment around exon 5 in all 3 family members. Although no clues suggestive of the obscure mutation could be detected by these analyses, semiquantitative PCR had positive findings—when amplified using the primer Set 1 near exon 5, the DNA samples of the patient (P1) and the father (F1) had less PCR products in comparison to that of the mother (M1), but this is not the case when using the primer Set 2 covering the entire exon 2 (Figure 1), indicating that the patient and the father might have a large deletion or insertion involving the positions of the primer Set 1. According to this result, LA-PCR amplification using the primer Set 3 (Figure 2) yielded an unexpected band of 632 bp in size in the patient and father, besides the expected 6324 bp product as in the mother, and subsequent Sanger sequencing of the unexpected product uncovered a large deletion c.329-1687_c.468+3865del5692bp.


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)

The large deletion mutation in SLC25A13 gene of the infant and his father. Figure 2① depicted the positions of the primer Set 3 and the novel large deletion. The primer sequences were 5′-AAGATTGTTGTTTATGGTGAGAC-3′ for IVS4S3 and 5′-ATGGTTTGCCCGACATGAGTAATC-3′ for J5.6KbDelR1, respectively. In Figure 2②, LA-PCR with the primer Set 3 revealed that the patient (P) and his father (F), but not the mother (M), had an unexpected band of 632 bp in size besides the normal product of 6324 bp. Figure 2③ was a segmental sequencing result of the unexpected PCR product. The arrow indicated the breakpoint arising from the large deletion.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: The large deletion mutation in SLC25A13 gene of the infant and his father. Figure 2① depicted the positions of the primer Set 3 and the novel large deletion. The primer sequences were 5′-AAGATTGTTGTTTATGGTGAGAC-3′ for IVS4S3 and 5′-ATGGTTTGCCCGACATGAGTAATC-3′ for J5.6KbDelR1, respectively. In Figure 2②, LA-PCR with the primer Set 3 revealed that the patient (P) and his father (F), but not the mother (M), had an unexpected band of 632 bp in size besides the normal product of 6324 bp. Figure 2③ was a segmental sequencing result of the unexpected PCR product. The arrow indicated the breakpoint arising from the large deletion.
Mentions: Based on the findings above, a diversity of primers was designed and PCR amplification conducted to analyze a variety of SNPs within the DNA fragment around exon 5 in all 3 family members. Although no clues suggestive of the obscure mutation could be detected by these analyses, semiquantitative PCR had positive findings—when amplified using the primer Set 1 near exon 5, the DNA samples of the patient (P1) and the father (F1) had less PCR products in comparison to that of the mother (M1), but this is not the case when using the primer Set 2 covering the entire exon 2 (Figure 1), indicating that the patient and the father might have a large deletion or insertion involving the positions of the primer Set 1. According to this result, LA-PCR amplification using the primer Set 3 (Figure 2) yielded an unexpected band of 632 bp in size in the patient and father, besides the expected 6324 bp product as in the mother, and subsequent Sanger sequencing of the unexpected product uncovered a large deletion c.329-1687_c.468+3865del5692bp.

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