1. health and fitness
2. disease

CLINICAL REPORT
Re-Evaluation Casts Doubt on the Pathogenicity of
Homozygous USH2A p.C759F
Marı´a Gonza´lez-del Pozo,1,2 Nereida Bravo-Gil,1,2 Cristina Me´ndez-Vidal,1,2
Ignacio Montero-de-Espinosa,3 Jose´ M Milla´n,2,4,5 Joaquı´n Dopazo,2,6,7 Salud Borrego,1,2
and Guillermo Antin˜olo1,2,6*
1
Department of Genetics, Reproduction and Fetal Medicine, Institute of Biomedicine of Seville, University Hospital Virgen del Rocı´o/CSIC/
University of Seville, Seville, Spain
2
Centro de Investigacio´n Biome´dica en Red de Enfermedades Raras (CIBERER), Spain
3
Department of Ophthalmology, University Hospital Virgen Macarena, Seville, Spain
Grupo de Investigacio´n en Enfermedades Neurosensoriales, IIS-La Fe, Valencia, Spain
5
Unidad de Gene´tica y Diagno´stico Prenatal, Hospital Universitario La Fe´, Valencia, Spain
4
6
Genomics and Bioinformatics Platform of Andalusia (GBPA), Seville, Spain
Computational Genomics Department, Centro de Investigacio´n Prı´ncipe Felipe (CIPF), Valencia, Spain
7
Manuscript Received: 14 July 2014; Manuscript Accepted: 19 January 2015
Mutations in USH2A are a common cause of Retinitis Pigmentosa (RP). Among the most frequently reported USH2A variants,
c.2276G>T (p.C759F) has been found in both affected and
healthy individuals. The pathogenicity of this variant remains
controversial since it was detected in homozygosity in two
healthy siblings of a Spanish family (S23), eleven years ago.
The fact that these individuals remain asymptomatic today,
prompted us to study the presence of other pathogenic variants
in this family using targeted resequencing of 26 retinal genes in
one of the affected individuals. This approach allowed us to
identify one novel pathogenic homozygous mutation in exon 13
of PDE6B (c.1678C>T; p.R560C). This variant cosegregated with
the disease and was absent in 200 control individuals. Remarkably, the identified variant in PDE6B corresponds to the mutation responsible of the retinal degeneration in the naturally
occurring rd10 mutant mice. To our knowledge, this is the first
report of the identification of the rd10 mice mutation in a RP
family. These findings, together with a review of the literature,
support the hypothesis that homozygous p.C759F mutations are
not pathogenic and led us to exclude the implication of p.C759F
in the RP of family S23. Our results indicate the need of reevaluating all families genetically diagnosed with this mutation.
Ó 2015 Wiley Periodicals, Inc.
Key words: C759F; inherited retinal dystrophies; retinitis
pigmentosa; Usher syndrome; gene panel sequencing; next
generation sequencing; rd10
INTRODUCTION
Inherited retinal dystrophies (IRDs) are a heterogeneous group of
disorders characterized by primary dysfunction or loss of photoreceptors leading to progressive visual impairment and blindness.
Ó 2015 Wiley Periodicals, Inc.
How to Cite this Article:
Gonza´lez-del Pozo M, Bravo-Gil N, Me´ndezVidal C, Montero-de-Espinosa I, Milla´n JM,
Dopazo J, Borrego S, Antin˜olo G. 2015. Reevaluation casts doubt on the pathogenicity
of homozygous USH2A p.C759F.
Am J Med Genet Part A. 9999:1–4.
Retinitis Pigmentosa (RP; OMIM #268000) represents the most
prevalent clinical subtype of IRDs, affecting 1 in 4,000 individuals.
To date, more than 60 genes have been associated with RP. The
USH2A gene (OMIM# 608400), along with EYS (OMIM# 612424),
is the most commonly mutated gene in autosomal recessive RP
(ARRP) [Bernal et al., 2003; Glockle et al., 2014]. Although most
causative mutations associated with IRDs are rare (MAF<<0.01),
Marı´a Gonza´lez-del Pozo and Nereida Bravo-Gil have contributed
equally to this work.
Conflict of interest: none.
Grant sponsor: Instituto de Salud Carlos III (ISCIII); Grant sponsor:
Spanish Ministry of Economy and Competitiveness, Spain;
Grant numbers: PI1102923, CIBERER ACCI, CDTI FEDER
EXP00052887/ITC-20111037; Grant sponsor: The Foundation Ramon
Areces; Grant number: CIVP16A1856.
Correspondence to:
Guillermo Antin˜olo, M.D., Ph.D., Department of Genetics, Reproduction and Fetal Medicine, University Hospital Virgen del Rocı´o, Avenida
Manuel Siurot s/n, 41013 Seville, Spain.
E-mail: [email protected]
Article first published online in Wiley Online Library
(wileyonlinelibrary.com): 00 Month 2015
DOI 10.1002/ajmg.a.37003
1
2
some exceptions have been described. A frequent change is p.C759F
(USH2A; rs80338902) which is responsible of 4.5% of patients
with ARRP [Rivolta et al., 2000; Seyedahmadi et al., 2004], and has
been found in a heterozygotic state in 20 control individuals out of
6,503 exomes in the Exome Variant Server (EVS). This USH2A
mutation is present in a large number of families with IRD in both a
homozygous and heterozygous state, but the pathogenicity of the p.
C759F variant has been questioned because of non-segregation in a
Spanish family (S23) with two asymptomatic homozygous siblings
[Bernal et al., 2003].
According to the relatively high prevalence of the p.C759F
variant and the discrepancies between clinical data and genetic
findings in family S23, we set out to evaluate the pathogenicity of
this variant using a NGS panel of 26 retinal genes [Mendez-Vidal
et al., 2014].
MATERIALS AND METHODS
Patients and Clinical Evaluation
This study involved a consanguineous Spanish family (S23) comprising two affected siblings diagnosed with ARRP and seven
asymptomatic members (Supplemental online Fig. S1). All affected
individuals were referred from the Ophthalmology Department to
the Genetic, Reproduction, and Fetal Medicine Department. Additionally, a group of 200 ethnically matched control individuals
was studied. This research project was reviewed and approved by
our research ethics committee and followed the tenets of the World
Medical Association Declaration of Helsinki. Written informed
consent was obtained from all participants. Clinical diagnosis of
ARRP was ascertained as described [Mendez-Vidal et al., 2014].
Individual II:1 underwent ophthalmogic examination including
fundoscopy and electroretinography. Peripheral blood of all subjects was collected for genomic DNA isolation from leukocytes
using standard procedures. A DNA sample of the affected individual II:4 was processed for gene panel sequencing.
AMERICAN JOURNAL OF MEDICAL GENETICS PART A
gating variants were subsequently screened in 200 healthy matched
control individuals by Sanger sequencing. The pathogenicity of
missense substitutions was estimated by Polyphen-2 and SIFT
scores, whereas the impact on disulfide bonds was predicted using
the DiANNA web server.
Additional Genetic Analyses of USH2A
USH2A regions with low coverage and the most common pathogenic intronic mutation of this gene (c.7595–2144A>G) were
further analyzed by Sanger sequencing. To test if a recombination
event or a large deletion had occurred in USH2A, linkage analysis in
all family members was conducted using the microsatellite markers
D1S2629, D1S2827, D1S229, and D1S490.
RESULTS
Clinical Data
The index patient (II:3) (Supplemental online Fig. S1) had a clinical
diagnosis of ARRP characterized by night blindness as the initial
symptom at age four, followed by progressive visual field constriction and decreased visual acuity by the age of 28 (OD:1/3 and OS:1/
2). Fundus examination showed typical signs of RP with pale optic
nerve disc, narrowed blood vessels, and bone spicule pigmentation
in the periphery. Her ERG responses were not detectable, consistent
with severe, generalized rod cone dysfunction. Her sister (II:4)
showed an age of onset and clinical features similar to the proband.
Individuals II:1 and II:2, harboring the homozygous USH2A mutation p.C759F, were clinically re-evaluated but no signs of RP were
observed at the ages of 57 and 55, respectively. The ERG and fundus
photography of individual II:1 (Supplemental online Fig. S2) confirmed normal visual function in this family member.
Prevalence of the p.C759F Mutation
Library Preparation and Gene Panel Sequencing
Direct sequencing of exon 13 of USH2A in 200 matched controls
showed the presence of the heterozygous p.C759F mutation in three
individuals. Also, four out of 252 Spanish healthy exomes [GarciaAlonso et al., 2014] were heterozygous for this mutation. A review of
the literature showed that p.C759F is present in a total of 41 families,
including 15 families with homozygous individuals (Supplemental
online Table SI) and 26 with heterozygous carriers. Regarding the
families with homozygous individuals, segregation analyses were
performed in only five families (S23, M286, RP0332, RP0930, and
RP0849) [Avila-Fernandez et al., 2010; Bernal et al., 2003]. Moreover, not all patients were screened for mutations in the entire gene
and, in the majority of families, only exon 13 of USH2A was
analyzed by Sanger sequencing.
Library preparation, targeted sequencing on a 454 Roche GS Junior
sequencer and data analysis were performed as described [MendezVidal et al., 2014].
NGS Data and Identification of Variants.
Analysis of p.C759F Prevalence
The p.C759F mutation was screened in 200 control individuals of
our cohort by Sanger sequencing and in 252 Spanish healthy
exomes [Garcia-Alonso et al., 2014]. Also, we have performed an
extensive literature review of families with RP harboring p.C759F,
using the following search terms in PubMed: (Retinitis Pigmentosa
OR USH2A) AND (C759F). We also explored other informative
sources of data such as LOVD-USHbases, HGMD and OMIM.
Assessment of the Pathogenicity of Candidate
Variants
Sanger sequencing was used to verify each predicted disease-causing variant and to perform cosegregation analyses. Novel cosegre-
To identify the causative variant underlying RP in Family S23, we
performed NGS for individual II:4 using our panel of 26 IRD genes.
This analysis generated a data output of 68 Mb and 155,475 reads
with a mean length of 438 bp. This sample showed a percentage of
reads on target of 83% and a mean coverage of 129. The
percentage of covered bases was 99.8% and, although this is a fairly
POZO ET AL.
high value, exon 59 of USH2A remained uncovered. Application of
variant calling, filtering, and annotation of the sequencing data
from individual II:4 was performed as described above. Output data
for each step of the analysis pipeline are shown in Supplemental
online Table SII. A total of 669 SNVs and 2,479 indels were found
but only four SNVs remained once the procedure was completed.
Three of these four changes were the known USH2A variants p.
C759F, p.H752H, and p.L555V, all previously detected and assessed
in this family [Bernal et al., 2003; Aller et al., 2004], but we
concluded that none were pathogenic. The remaining variant
was a rare homozygous mutation not previously associated with
human RP located in exon 13 of PDE6B at genomic position 4:
655986 (c.1678C>T; p.R560C) (Supplemental online Fig. S3A,B).
The specific coverage for this position was 29.
Pathogenicity Evaluation of the PDE6B
Mutation.
Sanger sequencing was performed using primers that amplified
exon 13 of PDE6B. The c.1678C>T (p.R560C) mutation cosegregated with the disease in Family S23 (Supplemental online
Fig. S1) and was absent in 200 control individuals. This variant
was not present in EVS, but one heterozygous allele out of 428
chromosomes was found as a result of another large scale genome
sequencing project [Biesecker et al., 2009]. Although this variant
has not been previously associated with RP in humans, it has
been reported as the causative mutation of the retinal degeneration in the naturally occurring rd10 mutant mouse [Chang et al.,
2007]. The mutated residue lies in an evolutionarily conserved
domain that corresponds to the catalytic domain of the protein
(Supplemental online Fig. S3C). In silico pathogenicity prediction tools showed that p.R560C is probably damaging (Polyphen2 score ¼ 0.997, SIFT score ¼ 0). Additionally, the DiANNA web
server predicted that the newly introduced cysteine alters the
disulfide bonding by establishing a new disulfide bridge with the
cysteine at position 717 (Supplemental online Fig. S3D). We
conclude that this PDE6B mutation is the likely disease-causing
variant in Family S23.
Given that exon 59 of USH2A was poorly covered during targeted
sequencing, we performed Sanger sequencing to check this region,
but no additional variants were detected. Likewise, direct full
sequencing of the USH2A intronic mutation c.7595–2144A>G
excluded the presence of this variant in the family. Finally, to exclude
recombination events or large deletions in this gene, we carried out a
linkage analysis in the family. The haplotypes showed a recombination event in individual II:5, but no other complex events were
detected (Supplemental online Fig. S4). This, together with the fact
that both affected and two unaffected individual (II:6 and II:7)
shared the same haplotype, led us to exclude USH2A as the RPcausing gene.
DISCUSSION
In this report, we have applied NGS panel sequencing to identify
causal gene mutations in a family with RP, in which the homozygous p.C759F variant was previously ruled out as the cause of the
disease, questioning the pathogenic role of this mutation.
3
According to the literature, 15 families have been described
harboring the homozygous p.C759F variant. In all reviewed cases,
the segregation data were not shown or were inconclusive. Of those,
Family S23 was one of the most interesting since p.C759F was
homozygous in two healthy, currently asymptomatic, siblings
[Bernal et al., 2003]. The other family reported by the same authors
(M286) showed segregation of p.C759F with the disease. However,
the offspring of that consanguineous family was comprised of only
two affected individuals, and the probability of having obtained this
result by chance alone is not negligible. Furthermore, Rivolta et al.
[2000] described another two families with homozygous p.C759F
but the segregation analysis was carried out only in two affected
individuals of one of these families (#E685). The authors also
identified the homozygous p.C759F mutation inherited by uniparental paternal disomy in one patient, whereas her affected second
cousin was heterozygous for the same variant [Rivolta et al., 2002].
Although the authors concluded that the p.C759F variants in both
affected individuals were likely of independent origin, the identification of a second mutant allele would help to clarify the molecular
diagnosis of this complex family. Finally, another patient homozygous for p.C759F has been reported [Glockle et al., 2014], but the
symptoms of his homozygous sibling were unclear. Of note, in most
families, only the affected members were analyzed, which may be
the reason for the absence of other occurrences of unaffected
individuals homozygous for this mutation.
Furthermore, in the majority of families, only exon 13 of USH2A
was analyzed by Sanger sequencing. Therefore, screening of additional USH2A regions including exons encoding for the large
isoform, duplications, deletions, and the common pathogenic
deep intronic variant (c.7595–2144A>G), should had been carried
out to detect variants acting in cis with p.C759F. Likewise, the
involvement of other genes could not be excluded. In this regard,
clinical data such as the onset age of symptoms and the average rates
of visual acuity and visual field loss, can be helpful to identify
candidate genes. Family S23 was diagnosed of early onset RP and
Patient II:4 reached legal blindness by the age of 30, which is not
consistent with the typical disease progression associated with
USH2A mutations [Sandberg et al., 2008]. Another factor against
the p.C759F pathogenicity is its relative high allele frequency
compared to other IRD-associated mutations.
Collectively, these results indicated that p.C759F is not implicated in the RP of Family S23. The application of targeted
sequencing led to the identification of a predicted pathogenic
homozygous PDE6B mutation (c.1678C>T; p.R560C). Notably,
this variant is equivalent to the mutation that causes the IRD in
the rd10 mutant mice with a homozygous p.R560C mutation in
exon 13 of Pde6b (Pde6bR560C) [Chang et al., 2007]. This is the
first report of a family affected with ARRP caused by the same
homozygous mutation present in the rd10 mice. The location of
the variant in a domain responsible for the catalytic function of
the protein and the conservation between mouse and human
PDE6B may explain that the same mutation cause similar phenotypes in both species. Clinical features in affected individuals
of the S23 family were consistent with those of RP caused by
PDE6B mutations. Indeed, RP associated with PDE6B mutations
is considered one of the earliest onset and most aggressive forms
of IRD [McLaughlin et al., 1993].
4
Our work shows for the first time that p.C759F might not be
pathogenic. Therefore, genetic re-evaluation of other reported
families with homozygous individuals should be considered, paying
special attention to the screening of regions that were not initially
analyzed, such as additional exons encoding the long isoform, deep
intronic regions and copy number variations. Moreover, the involvement of additional RP genes should not be dismissed, especially in those families whose clinical manifestations may be
indicative of mutations in other loci.
ACKNOWLEDGMENTS
The authors are grateful to the families described in this study. This
work was supported by Instituto de Salud Carlos III (ISCIII),
Spanish Ministry of Economy and Competitiveness, Spain
(PI1102923, CIBERER ACCI and CDTI FEDER EXP00052887/
ITC-20111037), and the Foundation Ramon Areces
(CIVP16A1856). The CIBERER is an initiative of the ISCIII,
Spanish Ministry of Economy and Competitiveness. NB-G is
supported by fellowship FI12/00545 from ISCIII.
INTERNET RESOURCES
RetNet: https://sph.uth.edu/retnet/home.htm
Exome Variant Server: http://evs.gs.washington.edu/EVS/
LOVD-USHbases: https://grenada.lumc.nl/LOVD2/Usher_montpellier/USHbases.html
HGMD: http://www.hgmd.cf.ac.uk/ac/index.php
OMIM: http://www.ncbi.nlm.nih.gov/omim
Polyphen-2: http://genetics.bwh.harvard.edu/pph2/
SIFT: http://sift.bii.a-star.edu.sg
DiANNA web server: http://clavius.bc.edu/~clotelab/DiANNA/
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