Limits...
A detailed clinical and molecular survey of subjects with nonsyndromic USH2A retinopathy reveals an allelic hierarchy of disease-causing variants.

Lenassi E, Vincent A, Li Z, Saihan Z, Coffey AJ, Steele-Stallard HB, Moore AT, Steel KP, Luxon LM, Héon E, Bitner-Glindzicz M, Webster AR - Eur. J. Hum. Genet. (2015)

Bottom Line: Overall, 23 of 186 probands (discovery cohort) were found to harbour two likely disease-causing variants in USH2A.Some of these variants were predominantly associated with nonsyndromic retinal degeneration ('retinal disease-specific'); these included the common c.2276 G>T, p.(Cys759Phe) mutation and five additional variants: c.2802 T>G, p.(Cys934Trp); c.10073 G>A, p.(Cys3358Tyr); c.11156 G>A, p.(Arg3719His); c.12295-3 T>A; and c.12575 G>A, p.(Arg4192His).The following model is proposed: the presence of at least one 'retinal disease-specific' USH2A allele in a patient with USH2A-related disease results in the preservation of normal hearing.

View Article: PubMed Central - PubMed

Affiliation: UCL Institute of Ophthalmology and Moorfields Eye Hospital, University College of London, London, UK.

ABSTRACT
Defects in USH2A cause both isolated retinal disease and Usher syndrome (ie, retinal disease and deafness). To gain insights into isolated/nonsyndromic USH2A retinopathy, we screened USH2A in 186 probands with recessive retinal disease and no hearing complaint in childhood (discovery cohort) and in 84 probands with recessive retinal disease (replication cohort). Detailed phenotyping, including retinal imaging and audiological assessment, was performed in individuals with two likely disease-causing USH2A variants. Further genetic testing, including screening for a deep-intronic disease-causing variant and large deletions/duplications, was performed in those with one likely disease-causing change. Overall, 23 of 186 probands (discovery cohort) were found to harbour two likely disease-causing variants in USH2A. Some of these variants were predominantly associated with nonsyndromic retinal degeneration ('retinal disease-specific'); these included the common c.2276 G>T, p.(Cys759Phe) mutation and five additional variants: c.2802 T>G, p.(Cys934Trp); c.10073 G>A, p.(Cys3358Tyr); c.11156 G>A, p.(Arg3719His); c.12295-3 T>A; and c.12575 G>A, p.(Arg4192His). An allelic hierarchy was observed in the discovery cohort and confirmed in the replication cohort. In nonsyndromic USH2A disease, retinopathy was consistent with retinitis pigmentosa and the audiological phenotype was variable. USH2A retinopathy is a common cause of nonsyndromic recessive retinal degeneration and has a different mutational spectrum to that observed in Usher syndrome. The following model is proposed: the presence of at least one 'retinal disease-specific' USH2A allele in a patient with USH2A-related disease results in the preservation of normal hearing. Careful genotype-phenotype studies such as this will become increasingly important, especially now that high-throughput sequencing is widely used in the clinical setting.

No MeSH data available.


Related in: MedlinePlus

(a) RT-PCR analysis of the c.12295-3T>A mutation. RT-PCR was performed on RNA extracted from nasal epithelial cells of subject D13 and an unrelated control individual, using primers located in exons 58 and 64 of USH2A. In subject D13, RT-PCR produced a shorter product of 1033 bp corresponding to skipping of USH2A exon 63 (partial sequence chromatogram of this transcript is shown in the box; the dashed line indicates the splice junction between exons 62 and 64). The other allele of subject D13 harbouring a c.12093C>A, p.(Tyr4031*) mutation did not amplify. Amplification on control template produced a band of 2550 bp, corresponding to wild-type sequence. (b) Partial sequence chromatogram of genomic DNA from subject D13 showing a heterozygous c.12093C>A variant in exon 62. Sequence analysis of the corresponding RT-PCR product (1033 bp, see above) revealed a normal sequence at c.12093; this implies that the c.12295-3T>A and c.12093C>A variants reside on different alleles. NTC stands for no template control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4592079&req=5

fig2: (a) RT-PCR analysis of the c.12295-3T>A mutation. RT-PCR was performed on RNA extracted from nasal epithelial cells of subject D13 and an unrelated control individual, using primers located in exons 58 and 64 of USH2A. In subject D13, RT-PCR produced a shorter product of 1033 bp corresponding to skipping of USH2A exon 63 (partial sequence chromatogram of this transcript is shown in the box; the dashed line indicates the splice junction between exons 62 and 64). The other allele of subject D13 harbouring a c.12093C>A, p.(Tyr4031*) mutation did not amplify. Amplification on control template produced a band of 2550 bp, corresponding to wild-type sequence. (b) Partial sequence chromatogram of genomic DNA from subject D13 showing a heterozygous c.12093C>A variant in exon 62. Sequence analysis of the corresponding RT-PCR product (1033 bp, see above) revealed a normal sequence at c.12093; this implies that the c.12295-3T>A and c.12093C>A variants reside on different alleles. NTC stands for no template control.

Mentions: The effect of c.12295-3 T>A, one of the probably ‘retinal disease-specific' changes, on splicing was investigated in subject D13. Primers were used to amplify a 2550 bp fragment between exons 58 and 64. PCR products of the expected size were amplified for the control sample, whereas a much smaller band was observed in the patient sample. Further cDNA analysis (Figure 2a) revealed the presence of an abnormal transcript (1033 bp) associated with an out-of-frame skipping of exon 63; this would result in a premature termination codon. Amplification of a 2371 bp fragment between exons 62 and 66 combined with direct sequencing of the RT-PCR product confirmed the above findings. Notably, direct sequencing of the RT-PCR product demonstrated a normal sequence at position c.12093, where the sequencing of the genomic DNA identified a c.12093C>A, p.(Tyr4031*) change in heterozygous state. This suggests that the two likely disease-causing variants identified in this patient (c.12295-3 T>A and c.12093C>A) reside on two different alleles (Figure 2b). The non-amplification of the allele with c.12093C>A could be due to nonsense-mediated decay or preferential amplification of the smaller, exon-skipped PCR product from the other allele.


A detailed clinical and molecular survey of subjects with nonsyndromic USH2A retinopathy reveals an allelic hierarchy of disease-causing variants.

Lenassi E, Vincent A, Li Z, Saihan Z, Coffey AJ, Steele-Stallard HB, Moore AT, Steel KP, Luxon LM, Héon E, Bitner-Glindzicz M, Webster AR - Eur. J. Hum. Genet. (2015)

(a) RT-PCR analysis of the c.12295-3T>A mutation. RT-PCR was performed on RNA extracted from nasal epithelial cells of subject D13 and an unrelated control individual, using primers located in exons 58 and 64 of USH2A. In subject D13, RT-PCR produced a shorter product of 1033 bp corresponding to skipping of USH2A exon 63 (partial sequence chromatogram of this transcript is shown in the box; the dashed line indicates the splice junction between exons 62 and 64). The other allele of subject D13 harbouring a c.12093C>A, p.(Tyr4031*) mutation did not amplify. Amplification on control template produced a band of 2550 bp, corresponding to wild-type sequence. (b) Partial sequence chromatogram of genomic DNA from subject D13 showing a heterozygous c.12093C>A variant in exon 62. Sequence analysis of the corresponding RT-PCR product (1033 bp, see above) revealed a normal sequence at c.12093; this implies that the c.12295-3T>A and c.12093C>A variants reside on different alleles. NTC stands for no template control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4592079&req=5

fig2: (a) RT-PCR analysis of the c.12295-3T>A mutation. RT-PCR was performed on RNA extracted from nasal epithelial cells of subject D13 and an unrelated control individual, using primers located in exons 58 and 64 of USH2A. In subject D13, RT-PCR produced a shorter product of 1033 bp corresponding to skipping of USH2A exon 63 (partial sequence chromatogram of this transcript is shown in the box; the dashed line indicates the splice junction between exons 62 and 64). The other allele of subject D13 harbouring a c.12093C>A, p.(Tyr4031*) mutation did not amplify. Amplification on control template produced a band of 2550 bp, corresponding to wild-type sequence. (b) Partial sequence chromatogram of genomic DNA from subject D13 showing a heterozygous c.12093C>A variant in exon 62. Sequence analysis of the corresponding RT-PCR product (1033 bp, see above) revealed a normal sequence at c.12093; this implies that the c.12295-3T>A and c.12093C>A variants reside on different alleles. NTC stands for no template control.
Mentions: The effect of c.12295-3 T>A, one of the probably ‘retinal disease-specific' changes, on splicing was investigated in subject D13. Primers were used to amplify a 2550 bp fragment between exons 58 and 64. PCR products of the expected size were amplified for the control sample, whereas a much smaller band was observed in the patient sample. Further cDNA analysis (Figure 2a) revealed the presence of an abnormal transcript (1033 bp) associated with an out-of-frame skipping of exon 63; this would result in a premature termination codon. Amplification of a 2371 bp fragment between exons 62 and 66 combined with direct sequencing of the RT-PCR product confirmed the above findings. Notably, direct sequencing of the RT-PCR product demonstrated a normal sequence at position c.12093, where the sequencing of the genomic DNA identified a c.12093C>A, p.(Tyr4031*) change in heterozygous state. This suggests that the two likely disease-causing variants identified in this patient (c.12295-3 T>A and c.12093C>A) reside on two different alleles (Figure 2b). The non-amplification of the allele with c.12093C>A could be due to nonsense-mediated decay or preferential amplification of the smaller, exon-skipped PCR product from the other allele.

Bottom Line: Overall, 23 of 186 probands (discovery cohort) were found to harbour two likely disease-causing variants in USH2A.Some of these variants were predominantly associated with nonsyndromic retinal degeneration ('retinal disease-specific'); these included the common c.2276 G>T, p.(Cys759Phe) mutation and five additional variants: c.2802 T>G, p.(Cys934Trp); c.10073 G>A, p.(Cys3358Tyr); c.11156 G>A, p.(Arg3719His); c.12295-3 T>A; and c.12575 G>A, p.(Arg4192His).The following model is proposed: the presence of at least one 'retinal disease-specific' USH2A allele in a patient with USH2A-related disease results in the preservation of normal hearing.

View Article: PubMed Central - PubMed

Affiliation: UCL Institute of Ophthalmology and Moorfields Eye Hospital, University College of London, London, UK.

ABSTRACT
Defects in USH2A cause both isolated retinal disease and Usher syndrome (ie, retinal disease and deafness). To gain insights into isolated/nonsyndromic USH2A retinopathy, we screened USH2A in 186 probands with recessive retinal disease and no hearing complaint in childhood (discovery cohort) and in 84 probands with recessive retinal disease (replication cohort). Detailed phenotyping, including retinal imaging and audiological assessment, was performed in individuals with two likely disease-causing USH2A variants. Further genetic testing, including screening for a deep-intronic disease-causing variant and large deletions/duplications, was performed in those with one likely disease-causing change. Overall, 23 of 186 probands (discovery cohort) were found to harbour two likely disease-causing variants in USH2A. Some of these variants were predominantly associated with nonsyndromic retinal degeneration ('retinal disease-specific'); these included the common c.2276 G>T, p.(Cys759Phe) mutation and five additional variants: c.2802 T>G, p.(Cys934Trp); c.10073 G>A, p.(Cys3358Tyr); c.11156 G>A, p.(Arg3719His); c.12295-3 T>A; and c.12575 G>A, p.(Arg4192His). An allelic hierarchy was observed in the discovery cohort and confirmed in the replication cohort. In nonsyndromic USH2A disease, retinopathy was consistent with retinitis pigmentosa and the audiological phenotype was variable. USH2A retinopathy is a common cause of nonsyndromic recessive retinal degeneration and has a different mutational spectrum to that observed in Usher syndrome. The following model is proposed: the presence of at least one 'retinal disease-specific' USH2A allele in a patient with USH2A-related disease results in the preservation of normal hearing. Careful genotype-phenotype studies such as this will become increasingly important, especially now that high-throughput sequencing is widely used in the clinical setting.

No MeSH data available.


Related in: MedlinePlus