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Simultaneous Detection of Both Single Nucleotide Variations and Copy Number Alterations by Next-Generation Sequencing in Gorlin Syndrome.

Morita K, Naruto T, Tanimoto K, Yasukawa C, Oikawa Y, Masuda K, Imoto I, Inazawa J, Omura K, Harada H - PLoS ONE (2015)

Bottom Line: In this study, we subjected a cohort of GS-affected individuals from six unrelated families to next-generation sequencing (NGS) analysis for the combined screening of causative alterations in Hedgehog signaling pathway-related genes.Specific single nucleotide variations (SNVs) of PTCH1 causing inferred amino acid changes were identified in four families (seven affected individuals), whereas CNAs within or around PTCH1 were found in two families in whom possible causative SNVs were not detected.Through a targeted resequencing of all coding exons, as well as simultaneous evaluation of copy number status using the alignment map files obtained via NGS, we found that GS phenotypes could be explained by PTCH1 mutations or deletions in all affected patients.

View Article: PubMed Central - PubMed

Affiliation: Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.

ABSTRACT
Gorlin syndrome (GS) is an autosomal dominant disorder that predisposes affected individuals to developmental defects and tumorigenesis, and caused mainly by heterozygous germline PTCH1 mutations. Despite exhaustive analysis, PTCH1 mutations are often unidentifiable in some patients; the failure to detect mutations is presumably because of mutations occurred in other causative genes or outside of analyzed regions of PTCH1, or copy number alterations (CNAs). In this study, we subjected a cohort of GS-affected individuals from six unrelated families to next-generation sequencing (NGS) analysis for the combined screening of causative alterations in Hedgehog signaling pathway-related genes. Specific single nucleotide variations (SNVs) of PTCH1 causing inferred amino acid changes were identified in four families (seven affected individuals), whereas CNAs within or around PTCH1 were found in two families in whom possible causative SNVs were not detected. Through a targeted resequencing of all coding exons, as well as simultaneous evaluation of copy number status using the alignment map files obtained via NGS, we found that GS phenotypes could be explained by PTCH1 mutations or deletions in all affected patients. Because it is advisable to evaluate CNAs of candidate causative genes in point mutation-negative cases, NGS methodology appears to be useful for improving molecular diagnosis through the simultaneous detection of both SNVs and CNAs in the targeted genes/regions.

No MeSH data available.


Related in: MedlinePlus

Flow chart indicating the validation process for variants.After four validation steps, nine genetic variants were selected, as shown in Table 2.
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pone.0140480.g002: Flow chart indicating the validation process for variants.After four validation steps, nine genetic variants were selected, as shown in Table 2.

Mentions: Data were analyzed using Torrent Suite Software v4.2.1 (Thermo Fisher Scientific) and Ion Reporter Software v4.6 (Thermo Fisher Scientific). The flow chart used to select candidate genes is shown in Fig 2. Annotated variants were selected according to the criterion that the causal variants should be segregated in patients with Gorlin syndrome-affected family members. Candidates for pathogenic variants within the PTCH1 were confirmed by Sanger sequencing. The pathogenicity of missense variants was assessed using tools for prediction of possible impact of an amino acid substitution on the structure and function of a human protein, such as Sorting Intolerant From Tolerant (SIFT) v5.1.1 (http://sift.bii.a-star.edu.sg/), PolyPhen-2 v2.2.2r398 (http://genetics.bwh.harvard.edu/pph2/index.shtml), MutationTaster (http://www.mutationtaster.org/index.html). SIFT examines the degree of conservation for amino acid residues across species, and calculated probabilities are used to partition ‘tolerated’ (normalized probability >0.05) from ‘damaging’ (normalized probability ≤0.05) substitutions for prediction of a phenotypic effect. PolyPhen-2 finds change in protein structure and function, and provides both a qualitative prediction (one of 'probably damaging', 'possibly damaging', 'benign' or 'unknown') and a score. MutationTaster checks evolutionary conservation, change in protein structure and function, and additionally effects on splicing or mRNA expression, and predicts the disease potential: ‘disease causing’, which is probably deleterious, ‘disease causing automatic’, which is known to be deleterious, ‘polymorphism’, which is probably harmless, and ‘polymorphism automatic’, which is known to be harmless. Identified SNVs was also evaluated by comparing with known alterations reported in mutation databases, such as the Human Gene Mutation Database (HGMD professional 2015.2, http://www.hgmd.cf.ac.uk/ac/index.php), and ClinVar (http://www.ncbi.nlm.nih.gov/clinvar/).


Simultaneous Detection of Both Single Nucleotide Variations and Copy Number Alterations by Next-Generation Sequencing in Gorlin Syndrome.

Morita K, Naruto T, Tanimoto K, Yasukawa C, Oikawa Y, Masuda K, Imoto I, Inazawa J, Omura K, Harada H - PLoS ONE (2015)

Flow chart indicating the validation process for variants.After four validation steps, nine genetic variants were selected, as shown in Table 2.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4636311&req=5

pone.0140480.g002: Flow chart indicating the validation process for variants.After four validation steps, nine genetic variants were selected, as shown in Table 2.
Mentions: Data were analyzed using Torrent Suite Software v4.2.1 (Thermo Fisher Scientific) and Ion Reporter Software v4.6 (Thermo Fisher Scientific). The flow chart used to select candidate genes is shown in Fig 2. Annotated variants were selected according to the criterion that the causal variants should be segregated in patients with Gorlin syndrome-affected family members. Candidates for pathogenic variants within the PTCH1 were confirmed by Sanger sequencing. The pathogenicity of missense variants was assessed using tools for prediction of possible impact of an amino acid substitution on the structure and function of a human protein, such as Sorting Intolerant From Tolerant (SIFT) v5.1.1 (http://sift.bii.a-star.edu.sg/), PolyPhen-2 v2.2.2r398 (http://genetics.bwh.harvard.edu/pph2/index.shtml), MutationTaster (http://www.mutationtaster.org/index.html). SIFT examines the degree of conservation for amino acid residues across species, and calculated probabilities are used to partition ‘tolerated’ (normalized probability >0.05) from ‘damaging’ (normalized probability ≤0.05) substitutions for prediction of a phenotypic effect. PolyPhen-2 finds change in protein structure and function, and provides both a qualitative prediction (one of 'probably damaging', 'possibly damaging', 'benign' or 'unknown') and a score. MutationTaster checks evolutionary conservation, change in protein structure and function, and additionally effects on splicing or mRNA expression, and predicts the disease potential: ‘disease causing’, which is probably deleterious, ‘disease causing automatic’, which is known to be deleterious, ‘polymorphism’, which is probably harmless, and ‘polymorphism automatic’, which is known to be harmless. Identified SNVs was also evaluated by comparing with known alterations reported in mutation databases, such as the Human Gene Mutation Database (HGMD professional 2015.2, http://www.hgmd.cf.ac.uk/ac/index.php), and ClinVar (http://www.ncbi.nlm.nih.gov/clinvar/).

Bottom Line: In this study, we subjected a cohort of GS-affected individuals from six unrelated families to next-generation sequencing (NGS) analysis for the combined screening of causative alterations in Hedgehog signaling pathway-related genes.Specific single nucleotide variations (SNVs) of PTCH1 causing inferred amino acid changes were identified in four families (seven affected individuals), whereas CNAs within or around PTCH1 were found in two families in whom possible causative SNVs were not detected.Through a targeted resequencing of all coding exons, as well as simultaneous evaluation of copy number status using the alignment map files obtained via NGS, we found that GS phenotypes could be explained by PTCH1 mutations or deletions in all affected patients.

View Article: PubMed Central - PubMed

Affiliation: Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.

ABSTRACT
Gorlin syndrome (GS) is an autosomal dominant disorder that predisposes affected individuals to developmental defects and tumorigenesis, and caused mainly by heterozygous germline PTCH1 mutations. Despite exhaustive analysis, PTCH1 mutations are often unidentifiable in some patients; the failure to detect mutations is presumably because of mutations occurred in other causative genes or outside of analyzed regions of PTCH1, or copy number alterations (CNAs). In this study, we subjected a cohort of GS-affected individuals from six unrelated families to next-generation sequencing (NGS) analysis for the combined screening of causative alterations in Hedgehog signaling pathway-related genes. Specific single nucleotide variations (SNVs) of PTCH1 causing inferred amino acid changes were identified in four families (seven affected individuals), whereas CNAs within or around PTCH1 were found in two families in whom possible causative SNVs were not detected. Through a targeted resequencing of all coding exons, as well as simultaneous evaluation of copy number status using the alignment map files obtained via NGS, we found that GS phenotypes could be explained by PTCH1 mutations or deletions in all affected patients. Because it is advisable to evaluate CNAs of candidate causative genes in point mutation-negative cases, NGS methodology appears to be useful for improving molecular diagnosis through the simultaneous detection of both SNVs and CNAs in the targeted genes/regions.

No MeSH data available.


Related in: MedlinePlus