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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 gel electrophoresis analysis of family RPT65. A: Pedigree of the adRP family RPT65, which carries the genetic variant c.307G> (p.Gly103Arg) causing the COL6A6/827-bp deletion (g.9281_10108del) in RHO. The genetic varian in COL6A6 was detected by capillary Sanger sequencing and fluorescence resonance energy transfer (FRET) assay, with (+) indicating the presence of genetic variants and (−) indicating wild-type alleles. Squares and circles represent men and women, respectively. The open symbols represent unaffected family members. Completely filled symbols represent patients with retinitis pigmentosa who underwent ophthalmic examination before genetic variant analysis was performed. Semifilled symbols represent carriers of the genetic variants who were not clinically diagnosed with RP before the molecular analysis. Ophthalmic examination of III:8 and III:10 showed a RP phenotype. B: Gel electrophoresis of the PCR products obtained by amplification of genomic DNA from the family members showing the deletion (g.9281_10108del) in RHO.
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f2: Pedigree and gel electrophoresis analysis of family RPT65. A: Pedigree of the adRP family RPT65, which carries the genetic variant c.307G> (p.Gly103Arg) causing the COL6A6/827-bp deletion (g.9281_10108del) in RHO. The genetic varian in COL6A6 was detected by capillary Sanger sequencing and fluorescence resonance energy transfer (FRET) assay, with (+) indicating the presence of genetic variants and (−) indicating wild-type alleles. Squares and circles represent men and women, respectively. The open symbols represent unaffected family members. Completely filled symbols represent patients with retinitis pigmentosa who underwent ophthalmic examination before genetic variant analysis was performed. Semifilled symbols represent carriers of the genetic variants who were not clinically diagnosed with RP before the molecular analysis. Ophthalmic examination of III:8 and III:10 showed a RP phenotype. B: Gel electrophoresis of the PCR products obtained by amplification of genomic DNA from the family members showing the deletion (g.9281_10108del) in RHO.

Mentions: Appendix 6 shows the candidate genetic variants found by WES in family RPT65, which were validated using direct capillary Sanger sequencing and segregated in the family. The new genetic variant c.1034T>G (p.Gly103Arg) in COL6A6 was the only variant in the family that cosegregated with the phenotype (Figure 2). The presence of the genetic variant c.1034T>G in the RPT65 family was validated by FRET assay (Appendix 7). We also used FRET to screen for this genetic variant in 120 index patients with adRP (in which the adRP-causing mutation is still unknown) and in 200 controls, none of whom exhibited the p.Gly103Arg genetic variant in COL6A6. PCR amplification of a fragment containing the juxtaposing sequences of exons 2 and 3 from a retinal cDNA sample revealed retinal expression of COL6A6.


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 gel electrophoresis analysis of family RPT65. A: Pedigree of the adRP family RPT65, which carries the genetic variant c.307G> (p.Gly103Arg) causing the COL6A6/827-bp deletion (g.9281_10108del) in RHO. The genetic varian in COL6A6 was detected by capillary Sanger sequencing and fluorescence resonance energy transfer (FRET) assay, with (+) indicating the presence of genetic variants and (−) indicating wild-type alleles. Squares and circles represent men and women, respectively. The open symbols represent unaffected family members. Completely filled symbols represent patients with retinitis pigmentosa who underwent ophthalmic examination before genetic variant analysis was performed. Semifilled symbols represent carriers of the genetic variants who were not clinically diagnosed with RP before the molecular analysis. Ophthalmic examination of III:8 and III:10 showed a RP phenotype. B: Gel electrophoresis of the PCR products obtained by amplification of genomic DNA from the family members showing the deletion (g.9281_10108del) in RHO.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Pedigree and gel electrophoresis analysis of family RPT65. A: Pedigree of the adRP family RPT65, which carries the genetic variant c.307G> (p.Gly103Arg) causing the COL6A6/827-bp deletion (g.9281_10108del) in RHO. The genetic varian in COL6A6 was detected by capillary Sanger sequencing and fluorescence resonance energy transfer (FRET) assay, with (+) indicating the presence of genetic variants and (−) indicating wild-type alleles. Squares and circles represent men and women, respectively. The open symbols represent unaffected family members. Completely filled symbols represent patients with retinitis pigmentosa who underwent ophthalmic examination before genetic variant analysis was performed. Semifilled symbols represent carriers of the genetic variants who were not clinically diagnosed with RP before the molecular analysis. Ophthalmic examination of III:8 and III:10 showed a RP phenotype. B: Gel electrophoresis of the PCR products obtained by amplification of genomic DNA from the family members showing the deletion (g.9281_10108del) in RHO.
Mentions: Appendix 6 shows the candidate genetic variants found by WES in family RPT65, which were validated using direct capillary Sanger sequencing and segregated in the family. The new genetic variant c.1034T>G (p.Gly103Arg) in COL6A6 was the only variant in the family that cosegregated with the phenotype (Figure 2). The presence of the genetic variant c.1034T>G in the RPT65 family was validated by FRET assay (Appendix 7). We also used FRET to screen for this genetic variant in 120 index patients with adRP (in which the adRP-causing mutation is still unknown) and in 200 controls, none of whom exhibited the p.Gly103Arg genetic variant in COL6A6. PCR amplification of a fragment containing the juxtaposing sequences of exons 2 and 3 from a retinal cDNA sample revealed retinal expression of COL6A6.

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