Non-USH2A Mutations in USH2 Patients
Thomas Besnard,1–3Christel Vach´ e,1David Baux,1Lise Larrieu,1Caroline Abadie,1Catherine Blanchet,4Sylvie Odent,5
Patricia Blanchet,6Patrick Calvas,7Christian Hamel,4H´ el` ene Dollfus,8Genevi` eve Lina-Granade,9James Lespinasse,10
Albert David,11Bertrand Isidor,11Gilles Morin,12Sue Malcolm,13Sylvie Tuffery-Giraud,2,3Mireille Claustres,1–3and
Anne-Franc ¸oise Roux1,2∗
1CHU Montpellier, Laboratoire de G´ en´ etique Mol´ eculaire, Montpellier, France;2INSERM U827, Montpellier, France;3Univ, Montpellier I,
Montpellier, France;4CHU Montpellier, Centre National de R´ ef´ erence maladies rares “Affections Sensorielles G´ en´ etiques”, Montpellier, France;
5CHU Hˆ opital Sud, Service de G´ en´ etique M´ edicale, Rennes, France;6CHU Montpellier, Service de G´ en´ etique M´ edicale, Montpellier, France;
7Hˆ opital Purpan, Service de G´ en´ etique M´ edicale, Toulouse, France;8Centre de R´ ef´ erence pour les Affections Rares en G´ en´ etique
Ophtalmologique (CARGO), Hˆ opitaux Universitaires de Strasbourg, Strasbourg, France;9CHU Edouard Herriot, Service d’ORL, Lyon, France;10CH
Chamb´ ery, G´ en´ etique M´ edicale, Chambery, France;11CHU de Nantes, Service de G´ en´ etique M´ edicale, Nantes, France;12CHU Amiens, Service de
G´ en´ etique Clinique, Amiens, France;13Clinical and Molecular Genetics, Institute of Child Health, University College London, United Kingdom
Communicated by Andreas Gal
Received 16 September 2011; accepted revised manuscript 28 November 2011.
Published online 2 December 2011 in Wiley Online Library (www.wiley.com/humanmutation).DOI: 10.1002/humu.22004
known minor genes involved in Usher syndrome type 2,
DFNB31 and GPR98, for mutations in a cohort of 31 pa-
tients not linked to USH2A. PDZD7, an Usher syndrome
type 2 (USH2) related gene, was analyzed when indicated.
We found that mutations in GPR98 contribute signifi-
cantly to USH2. We report 17 mutations in 10 individu-
als, doubling the number of GPR98 mutations reported to
date. In contrast to mutations in usherin, the mutational
spectrum of GPR98 predominantly results in a truncated
protein product. This is true even when the mutation af-
fects splicing, and we have incorporated a splicing reporter
minigene assay to show this, where appropriate. Only two
mutations were found which we believe to be genuine mis-
sense changes. Discrepancy in the mutational spectrum
between GPR98 and USH2A is discussed. Only two pa-
tients were found with mutations in DFNB31, showing
that mutations of this gene contribute to only a very small
extent to USH2. Close examination of the clinical details,
where available, for patients in whom no mutation was
found in USH2A, GPR98, or DFNB31, showed that most
of them had atypical features. In effect, these three genes
account for the vast majority of USH2 patients and their
analysis provide a robust pathway for routine molecular
Hum Mutat 33:504–510, 2012.C ?2011 Wiley Periodicals, Inc.
KEY WORDS: Usher syndrome; GPR98; DFNB31; func-
tional analysis; molecular analysis
We have systematically analyzed the two
Additional Supporting Information may be found in the online version of this article.
∗Correspondence to: Anne-Franc ¸oise Roux, Laboratoire de G´ en´ etique Mol´ eculaire,
CHU Montpellier, INSERM U827, IURC, 641 Avenue du Doyen Gaston Giraud, F-34093
Montpellier Cedex 5, France. E-mail: firstname.lastname@example.org
Usher syndrome is the most common form of hereditary syn-
mentosa (RP). The prevalence has long been estimated to be 1 of
17,000 to 1 of 33,000 but recent studies suggest a much higher fre-
the degree of HL and the presence or not of vestibular dysfunction.
To date, nine causative genes have been identified (for review see
Saihan et al., ).
Usher syndrome type 2 (USH2) is the most frequent form and
accounts for more than half of the cases (for review see [Petit,
2001]). Patients present with congenital moderate to severe HL
and normal vestibular function [Abadie et al., 2011]. RP usually
develops from the second decade on. Currently, three genes are
(MIM# 602851), and DFNB31/WHRN (MIM# 607928). Although
USH2A has been extensively studied in terms of mutational screen-
ing over the last 6 years, data on GPR98 and DFNB31 remain scarce
montpellier/). USH2A is by far the most frequently implicated gene
and pathogenic mutations are identified in approximately 75–80%
of cases [Baux et al., 2007]. The prevalence of mutations in each
of the GPR98 and DFNB31 genes is as yet unknown. GPR98 (also
known as VLGR1) was first described as implicated in USH2 in
2004 [Weston et al., 2004] with the identification of four causative
which contains 90 exons remains challenging and cannot be offered
routinely. The DFNB31 gene was first identified as being respon-
sible for nonsyndromic deafness in two separate families [Mburu
et al., 2003]. Its involvement in Usher syndrome was demonstrated
syndrome. Because both identified mutations altered the N termi-
nal portion of the whirlin long isoform, the authors suggest that
two PDZ(Post-synaptic density protien 95, Drosophila disk-large
C ?2011 WILEY PERIODICALS, INC.
tumor supressor, Zona occludens-1 protien) domains, PDZ1 and
PDZ2, involved with binding to other USH2 proteins (usherin and
also for retinal function.
The three USH2 proteins belong to an Usher interactome. PDZ
domains of whirlin interact with the PDZ-binding motif of both
USH2 transmembrane (TM) proteins (G protein-coupled receptor
and play an anchoring role in the connecting cilium with the inner
About 20% of the patients referred to our laboratory are not
linked to USH2A [(Baux et al., 2007] and unpublished results),
which represents a significant proportion of unresolved cases. To
improve our diagnostic service we have tested the involvement of
GPR98 and DFNB31 genes and established the mutational spec-
trum of these two genes. We show that similarly to USH2A, the
whole GPR98 gene needs to be screened. We also confirm that,
integrity of the N-terminal region (PDZ1 and PDZ2 domains) of
the whirlin long isoform is crucial. We performed functional analy-
sis to determine the outcome of the predicted aberrant pre-mRNA
Finally, because PDZD7 has been described as a contributor to
digenism in Usher syndrome and as a modifier of the retinal phe-
notype [Ebermann et al., 2010], its involvement was tested where
Patients and Methods
Patients were recruited from all over France. All patients were
of HL and RP. In most cases, audiograms and electroretinograms
for linkage and segregation analysis.
This study was approved by the local Ethical Committee and
consent to genetic testing was obtained from adult probands or
parents in the case of minors.
Haplotype Analyses of the USH2 Genes
Microsatellites markers were developed at the three different
USH2 loci: USH2A (USH2A gene), USH2C (GPR98 gene), and
USH2D (DFNB31 gene) (see Supp. Fig. S1). Most of the markers
correspond to Genethon markers. However, some additional intra-
genic markers were identified in the laboratory at the USH2A locus
in introns 13, 52, and 70 of the gene, which proved to be more
informative. Their primer sequences are listed in Supp. Figure S1.
PCR Sequencing of the Genes
Primers were designed to amplify all the coding exons and their
flanking intronic sequences of the GPR98 (90), DFNB31 (12), and
PDZD7 (16) genes. They are listed in Supp. Table S1. Most of them
amplified with a uniform PCR condition on a single 96-well plate
(adapted from [Roux et al., 2006]). The same primers were used
for sequencing, except in a few cases, for which new design of in-
ternal primers improved the sequence quality. Sequencing purifica-
tion was carried out with Agencourt CleanSeq on a Biomek 3000
France), following the manufacturer’s recommendations or by us-
ing sephadex (Sephadex G-50 S, GE Healthcare, V´ elizy, France).
Sequences were run on an Applied Biosystems 3130xl Genetic Ana-
lyzer (Applied Biosystems, Courtaboeuf, France) and analyzed with
the ABI Prism SeqScape V2.5 software.
Nomenclature of the variants follows the HGVS (http://www.
hgvs.org/rec.html) recommendations with nucleotide +1 corre-
sponding tothe A of the ATG initiation codon. Genotypes are given
according to HGVS nomenclature.
Large Rearrangement Identification
Detection of large genomic rearrangements was performed on
a custom designed CGH-microarray chip (12 × 135 k), as already
described [Roux et al., 2011].
In Silico Analysis
The pathogenic effect of missense variants was predicted as pre-
viously described [Baux et al., 2007] following a multistep anal-
ysis [Roux et al., 2011] and is available in Usher Syndrome Mis-
sense Analysis (USMA) (https://neuro-2.iurc.montp.inserm.fr/cgi-
bin/USMA/USMA.fcgi). In addition, alignments of orthologs are
accessible in USMA.
Prediction of a splice effect was tested using the Human Splicing
Finder (HSF) tool which gives a position Weight matrices-derived
scores (HSF) for potential splice sites, and also integrates the results
from the MaxEnt program [Desmet et al., 2009; Yeo and Burge,
Splicing Variant Analysis
The potential impact upon splicing of missense and intronic un-
[Le Gu´ edard-Mereuze et al., 2010] and using the pSPL3 exon-
trapping vector [Bottillo et al., 2007]. Briefly, patients’ genomic
DNA was used as a template to generate wild type and mutated in-
serts with the PhusionR ?High fidelity DNA polymerase (Finzymes,
and XhoI restriction sites of pSPL3 vector. Ligation was obtained
with In-FusionR ?Dry-Down PCR cloning kit according to manu-
facturer’s instructions (Clontech, Saint-Germain-en-Laye, France).
Constructs were transiently transfected into HeLa cells and total
RNA was extracted 48 hr after transfection; Reverse Transcription-
PCR(RT-PCR) and vizualization of splicing alterations were per-
formed as already published [Le Gu´ edard-Mereuze et al., 2010].
GenBank reference sequences: GPR98: NM_32119.3; DFNB31:
NM_015404.2; PDZD7: NM_001195263.1.
UNIPROT: G-protein coupled receptor 98: Q8WXG9; Whirlin:
Among the 31 patients analyzed, a likely pathogenic genotype
was identified in 12 of them, 10 in GPR98 and 2 in DFNB31
HUMAN MUTATION, Vol. 33, No. 3, 504–510, 2012
Table 1. Pathogenic GPR98 and DFNB31 Genotypes and Clinical Findings in USH2 Patients
Patient Degree of HL Onset of RP (years) Ophthalmologic findingsGenotype
HL, hearing loss; RP, retinitis pigmentosa; ND, not documented.
Sequencing was performed in the three USH2 genes for all the listed patients with the exception of U734, U831, U797, and U922. For these four patients preliminary haplotypes
analyses were performed for the three USH2 loci.
∗Exclusion of USH2A and USH2D loci.
∗∗Exclusion of USH2A and USH2C loci.
∗∗∗Homozygosity identified for the targeted locus.
GenBank accession numbers:GPR98 : NM_32119.3; DFNB31 : NM_015404.2.
Clinical findings are indicated for each patient. Patients were mainly Caucasians; U831 and U922 were of Turkish and Portuguese origin, respectively.
Table 2. List of Likely Pathogenic Variants Identified in the GPR98 and DFNB31 Genes
Exon/intron Nucleotide exchange Predicted translation effectClassification Prediction of splice effectAllelic frequency
For missense variants, allelic frequency was estimated in control chromosomes.
#Splicing alteration as demonstrated by further ex vivo splicing assays.
##Already described (see USHbases).
In 4 of these 12 families because consanguinity was documented
(U831, U922) or as several affected siblings were available (U734,
U797), haplotype analyses had previously been performed at the
three USH2 loci to prioritize which gene to sequence (Table 1).
Thus, GPR98 sequencing was prioritized in family U734 as USH2A
and USH2D loci had been excluded in the three affected siblings,
while DFNB31 sequencing was performed directly for family U797
after exclusion in the three affected siblings of USH2A and USH2C
analyses at the USH2C and USH2D locus, respectively.
Ten variants were considered “a priori” deleterious in GPR98
as they predict a premature termination codon (PTC) leading to a
deletions, and two small duplications [Tables 1 and 2]). Three sub-
stitutions predicting so-called missense alterations were classified
as likely pathogenic after the multistep analysis already described
of exon 41 (c.9042G>C) was eventually classified as a splicing alter-
ation (see below).
HUMAN MUTATION, Vol. 33, No. 3, 504–510, 2012
electrophoresis of RT-PCR products obtained from the assay (B) for (1–1) c.9042G>C in exon 41; (1–2) c.17204+4_17204+7del in intron 79; (1–3)
c.13232-3C>G in intron 65 and for (1–4) c.3635-2A>G in intron 19. Splicing patterns obtained are specified on the right-hand of each agarose gel.
Underlined nucleotides represent the alteration; the deletion of the four bases is indicated with hyphens (1–2). MaxEnt scores are displayed for
reference sites wild type (Wt), mutated (Mut), and for other used and identified by sequencing of the RT-PCR of the constructs. For 1–3, HSF
score is indicated for the predicted de novo acceptor 3?ss. In Wt constructs 1–1, 1–3, 1–4, traces of exon skipping were detected by sequencing.
(∗)Alternative splice sites used in Wt constructs: (1–2) a cryptic acceptor splice site leads to the retention of 12 intronic nucleotides; (1–4) the use
of an exonic cryptic donor site results in the exclusion of the last 697 bases of exon 20.
Ex vivo analysis of four splicing identified variants in GPR98. Schematic minigene constructs of the region of interest (A) and gel
Three additional putative splicing alterations were found in in-
screening for large genomic rearrangements [Roux et al., 2011] was
performed in cases where only a single deleterious mutation had
been identified after sequencing (U416, U787), and one large du-
plication encompassing several exons was detected (U416).
Because the presence of large rearrangement was excluded in pa-
tient U787, further PDZD7 gene investigation was performed in
order to identify the second alteration, considering the fact that
this gene has been described as a contributor to digenism in Usher
syndrome [Ebermann et al., 2010]. Sequencing of all PDZD7 ex-
ons did not reveal any mutation in combination with the GPR98
Prediction of a splice effect was tested for all the identified
variants (Table 2). For four of them, all within GPR98, a signif-
icant alteration in the score of the wild-type splice sites strength
was highlighted by the two prediction tools (Fig. 1 and Supp.
Two of the variants tested destroy the 5?splice consensus:
c.9042G>C is located at the last position of exon 41 and affects
position–1 of the site, whereas the deletion of the four bases AGTA
5?splice site (ss) of exon 79 by replacing it with +4T and +5C. This
four base deletion arose in an AGTA–AGTA repeat motif. The two
other base changes c.3235-2A>G and c.13232-3C>G are 3?splice
sites affecting variants but with uncertain splicing outcomes: the
results concerning their precise effects are different between both
programs. For c.3235-2A>G, the mutation drastically reduces the
strength of the 3?ss and reinforces the strength of a downstream 3?
ss according to MaxEnt (score of 3.21 > 6.12), while for c.13232-
3C>G, HSF (http://www.umd.be/HSF/) but not MaxEnt indicates
(Supp. Table S2).
HUMAN MUTATION, Vol. 33, No. 3, 504–510, 2012
In order to confirm these splicing predictions, minigene assays
were carried out for the four variants and the results are presented
in Figure 1.
In accordance with predictions, the presence of each variant re-
of the corresponding exon (Fig. 1-1 to 1-4). For the two constructs
affecting 3?ss, the use of either the de novo (c.13232-3C>G) or
the exonic cryptic (c.3235-2A>G) acceptor sites was not detected
(Fig. 1-3; 1-4).
In Silico Studies of the Missense Variants
Two missense variants suspected to be disease causing have been
identified. c.14365C>T (p.(Arg4789Trp)) has been shown to lie
in trans to c.3635-2A>G and has not been found in 176 control
chromosomes. USMA analysis [Roux et al., 2011] shows a global
conservation in 19 of 20 orthologs (95%). The single di-
vergent protein sequence belongs to Anolis carolinensis (EN-
SACAP00000013363), which strongly differs from all others in this
The c.17933A>G (p.(His5978Arg)) variation has been identified
in two unrelated patients, U361 and U919, in trans to c.3945dupA
(p.(Gln1316fs)) and to c.7001T>G (p.(Leu2334∗)), respectively. It
has not been found in 170 control chromosomes. USMA analysis
shows that the residue His is found in 16 of 17 ortholog sequences
(including Homo sapiens). The presence of a divergent residue Glu
is noted in Bos Taurus and includes a totally divergent alignment
in this region. The mutated residue Arg at position 5978 lies in the
first extracellular loop of the 7-TM domain of G-protein coupled
5980. Alignment of 237 sequences of 7-TM proteins shows that His
residue Arg is found only once (0.42%) in this alignment.
Two patients were identified who carried an unambiguous
pathogenic DFNB31 genotype. The two identified alterations
were frameshift mutations predicting a truncated whirlin protein
(Tables 1 and 2).
In addition, among all patients, 87 (68 in GPR98 and 19 in
DFNB31) variants were identified during this study, which could
be neutral. Fifty-eight additional changes were classified as UV1 or
UV2 (unclassified variants likely to be nonpathogenic) in GPR98
(55) and DFNB31 (3). They are all listed in USHbases.
Patients with no Mutation
After exhaustive analysis of USH2A, GPR98, and DFNB31 or
no identified mutation. The coding exons of PDZD7 (considered to
be a potential candidate gene for Usher syndrome) were sequenced
but no pathogenic mutations were identified.
A further assessment of the phenotype was performed (Supp.
Table S3) and several atypical findings were highlighted in most
of these USH2 geno-negative patients, although lack of sufficient
clinical detail was inconclusive for two patients (U767 and U603).
for 12 patients. For two patients, the clinical presentation may in-
dicate the possibility of another syndrome because of additional
clinical findings such as renal impairment (U583) or the presence
of dysmorphic features (U607).
6.4%. On the other hand, mutations in DFNB31 account for a very
small proportion (1.3%). This is to our knowledge the largest study
offering exhaustive molecular analysis of USH2 patients.
Mutations in GPR98 are of all types, including large gene rear-
contains numerous polymorphisms, but in almost all cases lead to
a truncated protein product. As the gene contains 90 exons, this
represents a considerable diagnostic challenge.
Althoughtwopatientswere foundinwhomUSH2was causedby
patient was homozygous for c.737delC and the other patient was a
compound heterozygote for the same allele plus c.680dupG. It is,
spectrum in DFNB31.
Analysis and Outcome of the Likely Pathogenic Mutations
(UV3 and 4) Identified in this Study
more care to analyze the predicted effect. This includes the large
rearrangement, the splicing, and the missense alterations.
The large rearrangement consists of a duplication of exons 79
to 83, identified by means of a CGH array, and predicts an in-
frame duplication of 837 coding nucleotides, resulting in a protein
enlarged by 279 amino acids, from position 5,674 to 5,952. This
receptors (GPCR) Proteolysis Site (GPS), the first TMdomain, and
part of the second. It is very likely that the extracellular structure of
the resulting protein would be strongly affected.
Ex vivo splicing assays have shown that, in fact, the four tested
alterations result in exon skipping. They all predict an out of frame
sequence and a PTC with the exception of c.3635-2A>G, which re-
tween Calx-beta domains 9 and 10. It is difficult to speculate on the
resulting changes, but the local structure is likely to be altered.
p.(Arg4789Trp) and p.(His5978Arg) is not complete due to the lack
of appropriate three-dimensional models. Both missense show per-
fect alignments with ortholog sequences with the exception of one
species in each case (Anolis carolinensis in the first and Bos Taurus
in the second case). Because the surrounding sequences diverge in
both species, the misalignment could be the result of an error in the
translation. In addition, p.(His5978Arg) arises in a well-conserved
position in the 7-TM domain. The fact that both missense lie in
trans to deleterious changes, are absent from a control population,
and are conserved among orthologs is in favor of their involvement
in the disease.
of the two missense
HUMAN MUTATION, Vol. 33, No. 3, 504–510, 2012
protein domains are represented with different pictograms. Pathogenic mutations and UV3–4 are designated at cDNA and protein level: the newly
identified variants are in bold and above the schematic proteins, the known variants are indicated below.
Distribution of the mutations identified in GPR98 (A) and DFNB31 (B). The exon numbering is indicated in blue squares. Identified
Mutation Spectrum of GPR98 and DFNB31 in Usher
A total of 19 likely pathogenic alterations have been identified in
of them are newly described (Fig. 2). They raise the total number to
33 and 4 Usher-linked mutations in GPR98 and DFNB31, respec-
tively. All GPR98 mutations described previously and in this work
are schematically shown, distributed along the G protein-coupled
receptor 98 in Figure 2. Mutations are spread along the whole se-
quence but a cluster emerges in the terminal end. All the mutations
are predicted to lead to a PTC resulting in a truncated protein, with
the exception of four missenses (p.(His3399Pro), p.(Arg4789Trp),
majority of other Usher genes, the spectrum of pathological muta-
deletions and duplications, alterations of splicing of the pre-mRNA
due to exonic or intronic mutations, creation of PTC and missense.
Again, as in the other genes (with maybe the exception of USH2A
c.2299delG), no major recurrent mutations have been identified.
These observations on ∼36 kb of DNA sequence, spread among
the 600 kb of the gene, show again the complexity of the evolution
of the DNA molecule, which modifies itself using various mecha-
Only two mutations, located in the cytoplasmic domain
receptor 98 with a functional extracellular domain and a native TM
domain. Because the PDZ binding motif, located in the COOH ex-
are likely nonfunctional as, if present, are unable to be part of the
The mutational spectrum of GPR98 somewhat differs from that
found in USH2A, as pathogenic missense accounts here for 12%
versus 37%. This discrepancy is surprising considering that the two
proteins share general common properties, such as: (i) a large ex-
tracellular (>5,000 residues) part, (ii) the presence of numerous
repeated domains (35 fibronectin type III domains in usherin, and
35 calx-beta core domains in G protein-coupled receptor 98), (iii)
the localization in the cells, and (iv) of course the phenotype they
can induce, but can be explained by several factors. The first one
is the bias introduced by the number of analyzed patients in each
gene (more than 150 in USH2A, 31 in GPR98), but even consid-
ering this, USH2A clearly shows a tendency to be more prone to
generate pathogenic missense variants. Another clue is the global
organization of the amino acids between the two proteins: 88%
of the residues in usherin belong to identified domains, while this
proportion falls down to 35–60% in G protein-coupled receptor
98 depending on the considered size of the calx-beta repeats. The
40–65% of remaining sequence may tolerate numbers of missense
Based on data supporting the hypothesis that PDZD7 is part
of the Usher interactome, and because Usher patients have been
it was important to screen this gene as a potential Usher candidate
on its own. Implication of the PDZD7 gene has also been excluded
in the 19 patients with no identified mutation.
Among these patients, five remained with typical audiological
and ophthalmologic findings (U277, U496, U585, U654, U391).
These patients will be further screened for the USH1 genes, as some
of them can be associated with variable phenotypes and we have
indeed identified a pathogenic CDH23 genotype in a patient pre-
senting with clinical USH2 (severe HL and Usher type RP) pheno-
HUMAN MUTATION, Vol. 33, No. 3, 504–510, 2012
Usher gene is not to be excluded but is likely to either constitute Download full-text
a small minority or be the result of genotype/phenotype variation.
For example, mutation in the Myo15 gene (whose human ortholog
is MYO15A) is known to be involved in mainly profound deafness,
but has recently been shown to cause an Usher-like phenotype in
rats [Held et al., 2011].
Next-generation sequencing (NGS) of whole genome, exome, or
targeted regions is becoming more cost effective [Choi et al., 2009;
Lim et al., 2011]. More mutations will be found, categorized and
put into the databases and indeed NGS will have a role in solving
these atypical Usher cases efficiently. Sequence capture of Usher
exome is currently being developed and could be implemented in
“routine” diagnostics for any Usher patient in the future. As well,
whole exome sequencing will make a contribution to identify as yet
uncharacterized Usher genes.
Overall, a totalof176 USH2 patients has beeninvestigated inour
laboratory, in either USH2A [Baux et al., 2007] and unpublished
data, or in the other USH2 genes (this study). The USH2 mutation
detection rate has now been raised to 89.2% (157/176) in clinically
diagnosed USH2 patients, which is quite similar to that of USH1
patients [Roux et al., 2011]. Interestingly, most patients remaining
with an uncharacterized pathogenic genotype, present with a ques-
tionable clinical Usher diagnosis. These observations could only be
helpful to clinicians and diagnostic laboratories in their practice.
National 2004, PROM 7802”. The authors are grateful to “UNADEV” and
“SOS R´ etinite” foundations for their support.
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