Limits...
New COL6A6 variant detected by whole-exome sequencing is linked to break points in intron 4 and 3'-UTR, deleting exon 5 of RHO, and causing adRP.

de Sousa Dias M, Hernan I, Delás B, Pascual B, Borràs E, Gamundi MJ, Mañé B, Fernández-San José P, Ayuso C, Carballo M - Mol. Vis. (2015)

Bottom Line: We also performed WES in affected and unaffected members of four families with adRP in whom a disease-causing mutation was previously not found.Moreover, NGS and WES were inefficient for detecting the complete deletion of exon 5 in the RHO gene in one family with adRP.Carriers of this deletion showed variable clinical status, and two of these carriers had not previously been diagnosed with RP.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics Unit, Hospital of Terrassa, Barcelona, Spain.

ABSTRACT

Purpose: This study aimed to test a newly devised cost-effective multiplex PCR assay for the molecular diagnosis of autosomal dominant retinitis pigmentosa (adRP), as well as the use of whole-exome sequencing (WES) to detect disease-causing mutations in adRP.

Methods: Genomic DNA was extracted from peripheral blood lymphocytes of index patients with adRP and their affected and unaffected family members. We used a newly devised multiplex PCR assay capable of amplifying the genetic loci of RHO, PRPH2, RP1, PRPF3, PRPF8, PRPF31, IMPDH1, NRL, CRX, KLHL7, and NR2E3 to molecularly diagnose 18 index patients with adRP. We also performed WES in affected and unaffected members of four families with adRP in whom a disease-causing mutation was previously not found.

Results: We identified five previously reported mutations (p.Arg677X in the RP1 gene, p.Asp133Val and p.Arg195Leu in the PRPH2 gene, and p.Pro171Leu and p.Pro215Leu in the RHO gene) and one novel mutation (p.Val345Gly in the RHO gene) representing 33% detection of causative mutations in our adRP cohort. Comparative WES analysis showed a new variant (p.Gly103Arg in the COL6A6 gene) that segregated with the disease in one family with adRP. As this variant was linked with the RHO locus, we sequenced the complete RHO gene, which revealed a deletion in intron 4 that encompassed all of exon 5 and 28 bp of the 3'-untranslated region (UTR).

Conclusions: The novel multiplex PCR assay with next-generation sequencing (NGS) proved effective for detecting most of the adRP-causing mutations. A WES approach led to identification of a deletion in RHO through detection of a new linked variant in COL6A6. No pathogenic variants were identified in the remaining three families. Moreover, NGS and WES were inefficient for detecting the complete deletion of exon 5 in the RHO gene in one family with adRP. Carriers of this deletion showed variable clinical status, and two of these carriers had not previously been diagnosed with RP.

No MeSH data available.


Related in: MedlinePlus

Pedigree and Sanger sequencing of familly RPT100. A: Pedigree of the adRP family RPT100, which carries the mutation p.Val345Gly in RHO. Capillary Sanger sequencing showed either the presence of the mutation (+) or of the wild-type allele (−). Squares and circles represent men and women, respectively. The open symbols represent unaffected family members, while the completely filled symbols represent patients with retinitis pigmentosa (RP). B: Fluorogram representation of the Sanger sequencing of mutation c.1034T>G in the RHO gene.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4539015&req=5

f1: Pedigree and Sanger sequencing of familly RPT100. A: Pedigree of the adRP family RPT100, which carries the mutation p.Val345Gly in RHO. Capillary Sanger sequencing showed either the presence of the mutation (+) or of the wild-type allele (−). Squares and circles represent men and women, respectively. The open symbols represent unaffected family members, while the completely filled symbols represent patients with retinitis pigmentosa (RP). B: Fluorogram representation of the Sanger sequencing of mutation c.1034T>G in the RHO gene.

Mentions: To investigate the clinical use of our previously described multiplex PCR approach, we used this approach to analyze 18 patients with an uncharacterized adRP-causing mutation. This analysis was performed through three distinct NGS runs, each containing a library constructed using DNA from six patients. Appendix 5 shows the average data from NGS runs. NGS data analysis revealed six different mutations (Table 3), which were confirmed by capillary Sanger sequencing. We detected the novel genetic variant c.1034T>G (Val345Gly) in RHO, which segregated in family RPT100 (Figure 1) and was also tested by capillary Sanger sequencing. In our survey using the multiplex PCR and NGS approach, successful molecular diagnosis was achieved in six (33.3%) of the 18 index patients with adRP.


New COL6A6 variant detected by whole-exome sequencing is linked to break points in intron 4 and 3'-UTR, deleting exon 5 of RHO, and causing adRP.

de Sousa Dias M, Hernan I, Delás B, Pascual B, Borràs E, Gamundi MJ, Mañé B, Fernández-San José P, Ayuso C, Carballo M - Mol. Vis. (2015)

Pedigree and Sanger sequencing of familly RPT100. A: Pedigree of the adRP family RPT100, which carries the mutation p.Val345Gly in RHO. Capillary Sanger sequencing showed either the presence of the mutation (+) or of the wild-type allele (−). Squares and circles represent men and women, respectively. The open symbols represent unaffected family members, while the completely filled symbols represent patients with retinitis pigmentosa (RP). B: Fluorogram representation of the Sanger sequencing of mutation c.1034T>G in the RHO gene.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4539015&req=5

f1: Pedigree and Sanger sequencing of familly RPT100. A: Pedigree of the adRP family RPT100, which carries the mutation p.Val345Gly in RHO. Capillary Sanger sequencing showed either the presence of the mutation (+) or of the wild-type allele (−). Squares and circles represent men and women, respectively. The open symbols represent unaffected family members, while the completely filled symbols represent patients with retinitis pigmentosa (RP). B: Fluorogram representation of the Sanger sequencing of mutation c.1034T>G in the RHO gene.
Mentions: To investigate the clinical use of our previously described multiplex PCR approach, we used this approach to analyze 18 patients with an uncharacterized adRP-causing mutation. This analysis was performed through three distinct NGS runs, each containing a library constructed using DNA from six patients. Appendix 5 shows the average data from NGS runs. NGS data analysis revealed six different mutations (Table 3), which were confirmed by capillary Sanger sequencing. We detected the novel genetic variant c.1034T>G (Val345Gly) in RHO, which segregated in family RPT100 (Figure 1) and was also tested by capillary Sanger sequencing. In our survey using the multiplex PCR and NGS approach, successful molecular diagnosis was achieved in six (33.3%) of the 18 index patients with adRP.

Bottom Line: We also performed WES in affected and unaffected members of four families with adRP in whom a disease-causing mutation was previously not found.Moreover, NGS and WES were inefficient for detecting the complete deletion of exon 5 in the RHO gene in one family with adRP.Carriers of this deletion showed variable clinical status, and two of these carriers had not previously been diagnosed with RP.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics Unit, Hospital of Terrassa, Barcelona, Spain.

ABSTRACT

Purpose: This study aimed to test a newly devised cost-effective multiplex PCR assay for the molecular diagnosis of autosomal dominant retinitis pigmentosa (adRP), as well as the use of whole-exome sequencing (WES) to detect disease-causing mutations in adRP.

Methods: Genomic DNA was extracted from peripheral blood lymphocytes of index patients with adRP and their affected and unaffected family members. We used a newly devised multiplex PCR assay capable of amplifying the genetic loci of RHO, PRPH2, RP1, PRPF3, PRPF8, PRPF31, IMPDH1, NRL, CRX, KLHL7, and NR2E3 to molecularly diagnose 18 index patients with adRP. We also performed WES in affected and unaffected members of four families with adRP in whom a disease-causing mutation was previously not found.

Results: We identified five previously reported mutations (p.Arg677X in the RP1 gene, p.Asp133Val and p.Arg195Leu in the PRPH2 gene, and p.Pro171Leu and p.Pro215Leu in the RHO gene) and one novel mutation (p.Val345Gly in the RHO gene) representing 33% detection of causative mutations in our adRP cohort. Comparative WES analysis showed a new variant (p.Gly103Arg in the COL6A6 gene) that segregated with the disease in one family with adRP. As this variant was linked with the RHO locus, we sequenced the complete RHO gene, which revealed a deletion in intron 4 that encompassed all of exon 5 and 28 bp of the 3'-untranslated region (UTR).

Conclusions: The novel multiplex PCR assay with next-generation sequencing (NGS) proved effective for detecting most of the adRP-causing mutations. A WES approach led to identification of a deletion in RHO through detection of a new linked variant in COL6A6. No pathogenic variants were identified in the remaining three families. Moreover, NGS and WES were inefficient for detecting the complete deletion of exon 5 in the RHO gene in one family with adRP. Carriers of this deletion showed variable clinical status, and two of these carriers had not previously been diagnosed with RP.

No MeSH data available.


Related in: MedlinePlus