Non-USH2A mutations in USH2 patients

Article (PDF Available)inHuman Mutation 33(3):504-10 · March 2012with116 Reads
DOI: 10.1002/humu.22004 · Source: PubMed
We have systematically analyzed the two known minor genes involved in Usher syndrome type 2, DFNB31 and GPR98, for mutations in a cohort of 31 patients not linked to USH2A. PDZD7, an Usher syndrome type 2 (USH2) related gene, was analyzed when indicated. We found that mutations in GPR98 contribute significantly to USH2. We report 17 mutations in 10 individuals, 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 affects 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 missense changes. Discrepancy in the mutational spectrum between GPR98 and USH2A is discussed. Only two patients 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 diagnosis.
Mutations in USH2 Patients
Thomas Besnard,
Christel Vach
David Baux,
Lise Larrieu,
Caroline Abadie,
Catherine Blanchet,
Sylvie Odent,
Patricia Blanchet,
Patrick Calvas,
Christian Hamel,
ene Dollfus,
eve Lina-Granade,
James Lespinasse,
Albert David,
Bertrand Isidor,
Gilles Morin,
Sue Malcolm,
Sylvie Tuffery-Giraud,
Mireille Claustres,
Anne-Franc¸oise Roux
CHU Montpellier, Laboratoire de G
etique Mol
eculaire, Montpellier, France;
INSERM U827, Montpellier, France;
Univ, Montpellier I,
Montpellier, France;
CHU Montpellier, Centre National de R
erence maladies rares “Affections Sensorielles G
etiques”, Montpellier, France;
opital Sud, Service de G
etique M
edicale, Rennes, France;
CHU Montpellier, Service de G
etique M
edicale, Montpellier, France;
opital Purpan, Service de G
etique M
edicale, Toulouse, France;
Centre de R
erence pour les Affections Rares en G
Ophtalmologique (CARGO), H
opitaux Universitaires de Strasbourg, Strasbourg, France;
CHU Edouard Herriot, Service d’ORL, Lyon, France;
ery, G
etique M
edicale, Chambery, France;
CHU de Nantes, Service de G
etique M
edicale, Nantes, France;
CHU Amiens, Service de
etique Clinique, Amiens, France;
Clinical 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 ( 10.1002/humu.22004
ABSTRACT: We have systematically analyzed the two
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 t he 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,orDFNB31, 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.
2011 Wiley Periodicals, Inc.
KEY WORDS: Usher syndrome; GPR98; DFNB31; func-
tional analysis; molecular analysis
Additional Supporting Information may be found in the online version of this article.
Correspondence to: Anne-Franc¸oise Roux, Laboratoire de G
etique Mol
CHU Montpellier, INSERM U827, IURC, 641 Avenue du Doyen Gaston Giraud, F-34093
Montpellier Cedex 5, France. E-mail:
Usher syndrome is the most common form of hereditary syn-
drome associating sensorineural hearing loss (HL) and retinitis pig-
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-
quency of 1 of 6,000 [Kimberling et al., 2010]. This group of recessive
disorders is divided into three clinical subtypes defined according to
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., [2009]).
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
known to be responsible for USH2: USH2A (MIM# 608400), GPR98
(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
(see LOVD-USHbases:
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 genesisasyetunknown.GPR98 (also
known as VLGR1) was first described as implicated in USH2 in
2004 [Weston et al., 2004] with the identification of four causative
mutations in five unrelated female patients. However, only a limited
number of USH2-related deleterious mutations (n
= 17) is known to
date (see USHbases), possibly because molecular testing of this gene,
which contains 90 exons remains challeng ing and cannot be offered
routinely. The DFNB31 gene was first identified as being respon-
sible for nonsyndromic deafness in two s eparate families [Mburu
et al., 2003]. Its involvement in Usher syndrome was demonstrated
by Ebermann et al. [2007], in a family presenting with typical USH2
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
tumor supressor, Zona occludens-1 protien) domains, PDZ1 and
PDZ2, involved with binding to other USH2 proteins (usherin and
G protein-coupled receptor 98) are not only cr itical for auditory but
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
98 and usherin). These proteins are major components of the ankle-
link of the hair bundle of the hair cells cochlea [Michalski et al., 2007]
and play an anchoring role in the connecting cilium with the inner
segment of the photoreceptors [Maerker et al., 2008; van Wijk et al.,
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
splicing alterations.
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
referred with a clinical diagnosis of Usher type 2 based on the degree
of HL and RP. In most cases, audiograms and electroretinograms
were provided.
When possible, blood samples of relatives were collected and used
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
Laboratory Automation Workstation (Beckman Coulter, Villepinte,
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. recommendations with nucleotide
sponding to the 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) (
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-
known variants was functionally established as previously described
[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 ty pe and mutated in-
serts with the Phusion
High fidelity DNA polymerase (Finzymes,
Espoo, Finlande). Amplified products were cloned between the NheI
and XhoI restriction sites of pSPL3 vector. Ligation was obtained
with In-Fusion
Dry-Dow n 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 Numbers
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
(Table 1).
HUMAN MUTATION, Vol. 33, No. 3, 504–510, 2012 505
Table 1. Pathogenic
Genotypes and Clinical Findings in USH2 Patients
Patient Degree of HL Onset of RP (years) Ophthalmologic findings Genotyp e
U229 Moderate 17 Typical RP c.[14365C
U347 Severe 20 ND c.[7770delC(;)17204
U361 Severe 47 Rod-cone dystrophy c.[3945dupA];[17933A
U380 Moderate 35 Typical RP c.[10935_10938del(;)9042G
U416 Moderate 13 Typical RP c.[17668_17669del(;)17020-?_17856
U457 Severe 30 ND c.[13320dupC(;)16940delT]
Severe 40 Typical RP c.[5671A
U787 Moderate 35 Typical RP c.[17062C
Moderate 20 Typical RP c.[2984_2988del(;)2984_2988del]
U919 Moderate 35 ND c.[7001T
Moderate 30 Typical RP c.[680dupG];[737delC]
Moderate 30 Typical RP c.[737delC];[737delC]
HL, hear ing 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
Exon/intron Nucleotide exchange Predicted translation effect Classification Prediction of splice effect Allelic frequency
16 c.2984_2988del p.(Leu995fs) Pathogenic No
Intron 19 c.3635-2A
20 c.3945dupA p.(Gln1316fs) Pathogenic No
28 c.5671A
T p.(Arg1891
) Pathogenic No
32 c.7001T
G p.(Leu2334
) Pathogenic No
33 c.7770delC p.(Glu2591fs) Pathogenic No
41 c.9042G
C p.(Met3014Ile)
UV4 Yes
52 c.10935_10938del p.(Ser3646fs) Pathogenic No
Intron 65 c.13232-3C
66 c.13320dupC p.(Ser4441fs) Pathogenic No
70 c.14365C
T p.(Arg4789Trp) UV3 No 0/176
78 c.16940delT p.(Val5647fs) Pathogenic No
79 c.17062C
T p.(Arg5688
) Pathogenic No
Intron 79 c.17204
7del p.(?) UV4 Yes
82 c.17668_17669del p.(Met5890fs)
Pathogenic No
84 c.17933A
G p.(His5978Arg) UV3 No 0/170
79–83 c.17020-?_17856
?dup p.(Ile5674_Gln5952dup) UV4 ?
2 c.680dupG p.(Tyr228fs) Pathogenic No
2 c.737delC p.(Pro246fs)
Pathogenic No
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
loci. In the two consanguineous families U831 and U922, GPR98 and
DFNB31 weresequenced due to homozygosity revealedby haplotype
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
truncated G protein-coupled receptor 98 (three nonsenses, fivesmall
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
[Roux et al., 2011]; however, one of them lying at the last nucleotide
of exon 41 (c.9042G
C) was eventually classified as a splicing alter-
ation (see below).
506 HUMAN MUTATION, Vol. 33, No. 3, 504–510, 2012
Figure 1. Ex vivo analysis of four splicing identified variants in
. Schematic minigene constructs of the region of interest (A) and gel
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.
Three additional putative splicing alterations were found in in-
trons and likely to alter the strength of consensus splice sites. Finally,
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 i dentify 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
Splicing Alterations
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.
Table S2).
Two of the variants tested destroy the 5
splice consensus:
C is located at the last position of exon 41 and affects
1 of the site, whereas the deletion of the four bases AGTA
7del) abolishes the
4A and
5G positions of the
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
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 d rastically 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-
G, HSF ( but not MaxEnt indicates
the potential use of the de novo 3
ss (77.36) created by the mutation
(Supp. Table S2).
HUMAN MUTATION, Vol. 33, No. 3, 504–510, 2012 507
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-
sulted in an aberrant splicing pattern, and in all cases to the skipping
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
particular region.
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
receptor 98, the third TM region being predicted to begin at position
5980. Alignment of 237 sequences of 7-TM proteins shows that His
is the major residue at this position (97 sequences, 40.93%). Mutant
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,andDFNB31 or
exclusion of their involvement by linkage, 19 patients remained with
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 se veral atypical findings were highlighted in most
of these USH2 geno-negative patients, althoug h lack of sufficient
clinical detail was inconclusive for two patients (U767 and U603).
Atypical audiological and/or ophthalmologic impairment was noted
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).
We have confirmed in this study that mutations in GPR98 account
for a small but significant proportion of mutations in USH2, that is,
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-
rangements, and are spread throughout the gene, which additionally
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.
Although two patients were found in whom USH2 was caused by
mutations in DFNB31, only two mutant alleles were identified as one
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
Many of the predicted mutations in GPR98 could be deduced with
some confidence as they predicted premature chain termination due
to small frameshifts or single changes, but several alterations needed
more care to analyze the predicted effect. This includes the large
rearrangement, the s plicing, 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
protein would contain a duplication of the entire G protein-coupled
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-
sults in an in-frame deletion, predicting a protein lacking 248 amino
acids (positions 1213 to 1460). This deletion removed the region be-
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.
Unfortunately, in silico analysis of the two missense
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.
508 HUMAN MUTATION, Vol. 33, No. 3, 504–510, 2012
Figure 2. Distribution of the mutations identified in
(A) and
(B). The exon numbering is indicated in blue squares. Identified
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.
Mutation Spectrum of GPR98 and DFNB31 in Usher
A total of 19 likely pathogenic alterations have been identified in
the cohort described here, 17 in GPR98 and 2 in DFNB31. Seventeen
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),
p.(His5978Arg), and p.(Thr6044Cys)). It can be noted that as in the
majority of other Usher genes, the spectrum of pathological muta-
tions in GPR98 encompasses all possible type of DNA/RNA/protein
alterations, including at the DNA level: substitutions, small and large
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-
nisms occurring by chance which can sometimes lead to devastating
Only two mutations, located in the cytoplasmic domain
p.(Ala6216fs)and p.(Thr6244fs), would predict a G protein-coupled
receptor 98 with a functional extracellular domain and a native TM
domain. Because the PDZ binding motif, located in the COOH ex-
tremity, is then lacking in both cases, the resulting mutated proteins
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 the y
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,31inGPR98), 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 hy pothesis that PDZD7 is part
of the Usher interactome, and because Usher patients have been
found carrying one PDZD7 mutation in association with one GPR98
mutation or a pathogenic USH2A genotype [Ebermann et al., 2010],
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-
type (unpublished results). Implication of an as yet uncharacterized
HUMAN MUTATION, Vol. 33, No. 3, 504–510, 2012 509
Usher gene is not to be excluded but is likely to either constitute
a small minority or be the result of genotype/phenoty pe 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 reg ions 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 s equencing will make a contribution to identify as yet
uncharacterized Usher genes.
Overall, a total of 176 USH2 patients has been investigated in our
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
done a posteriori and in these terms, we believe that our work is very
helpful to clinicians and diagnostic laboratories in their practice.
This work has been supported in par t by le Minist
ere de la Recherche “PHRC
National 2004, PROM 7802”. T he authors are grateful to “UNADEV” and
etinite” foundations for their support.
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510 HUMAN MUTATION, Vol. 33, No. 3, 504–510, 2012
    • "These findings are consistent with mapped pathogenic variants in human patients and their respective phenotypes: DFNB31 patients , who suffer from profound prelingual sensorineural hearing loss (Mustapha et al., 2002), have mutations within exons 10 and 11 of DFNB31 (Mburu et al., 2003; Mustapha et al., 2002; Tlili et al., 2005), which affect the PDZ3 domain of both WHRN-L and WHRN-S, likely resulting in truncated, non-functional proteins. Mutations in patients with USH2D, who have milder and more variable hearing abnormalities, have been localized to exons 1 and 2 and intron 2 of DFNB31 (Audo et al., 2011; Besnard et al., 2012; Ebermann et al., 2007), which affect PDZ1 and PDZ2 of WHRN-L, but likely have no impact on the expression of WHRN-S. Our findings are also consistent with the report of a different mutation targeting exon 1 of Whrn in the mouse, where IHCs appeared normal and OHCs showed similar abnormalities as we report here (Yang et al., 2010). "
    [Show abstract] [Hide abstract] ABSTRACT: WHRN (DFNB31) mutations cause diverse hearing disorders: profound deafness (DFNB31) or variable hearing loss in Usher syndrome type II. The known role of WHRN in stereocilia elongation does not explain these different pathophysiologies. Using spontaneous and targeted Whrn mutants, we show that the major long (WHRN-L) and short (WHRN-S) isoforms of WHRN have distinct localizations within stereocilia and also across hair cell types. Lack of both isoforms causes abnormally short stereocilia and profound deafness and vestibular dysfunction. WHRN-S expression, however, is sufficient to maintain stereocilia bundle morphology and function in a subset of hair cells, resulting in some auditory response and no overt vestibular dysfunction. WHRN-S interacts with EPS8, and both are required at stereocilia tips for normal length regulation. WHRN-L localizes midway along the shorter stereocilia, at the level of inter-stereociliary links. We propose that differential isoform expression underlies the variable auditory and vestibular phenotypes associated with WHRN mutations.
    Full-text · Article · Apr 2016
    • "Patients reach legal blindness at a median age of 58 years [Sandberg et al., 2008]. Mutations in one of three known genes (USH2A, GPR98 [ADGRV1] and DFNB31) can be responsible for USH2, but USH2A (MIM #608400) is by far the most frequently involved and accounts for up to 82% of cases [Dreyer et al., 2008; Garcia-Garcia et al., 2011; Besnard et al., 2012; Le Quesne Stabej et al., 2012]. The USH2A gene, located at chromosome 1q41, spans 800 kb, comprises 72 exons with introns varying from 127 bp to 78 kb in length [van Wijk et al., 2004]. "
    [Show abstract] [Hide abstract] ABSTRACT: Deep intronic mutations leading to pseudoexon (PE) insertions are underestimated and most of these splicing alterations have been identified by transcript analysis, for instance, the first deep intronic mutation in USH2A, the gene most frequently involved in Usher syndrome type II (USH2). Unfortunately, analyzing USH2A transcripts is challenging and for 1.8%-19% of USH2 individuals carrying a single USH2A recessive mutation, a second mutation is yet to be identified. We have developed and validated a DNA next-generation sequencing approach to identify deep intronic variants in USH2A and evaluated their consequences on splicing. Three distinct novel deep intronic mutations have been identified. All were predicted to affect splicing and resulted in the insertion of PEs, as shown by minigene assays. We present a new and attractive strategy to identify deep intronic mutations, when RNA analyses are not possible. Moreover, the bioinformatics pipeline developed is independent of the gene size, implying the possible application of this approach to any disease-linked gene. Finally, an antisense morpholino oligonucleotide tested in vitro for its ability to restore splicing caused by the c.9959-4159A>G mutation provided high inhibition rates, which are indicative of its potential for molecular therapy.
    Full-text · Article · Oct 2015
    • "Three of the six rearrangements found in this study had been previously described. The USH2A deletions of exon 14 and exon 44 were described by Glockle et al. [35] and the GPR98 duplication was previously described by Besnard et al. [52]. The deletion of exon 14 in USH2A has also been detected by our group, in one Spanish USH patient in a homozygous state (unpublished results). "
    [Show abstract] [Hide abstract] ABSTRACT: Background Usher syndrome is an autosomal recessive disease that associates sensorineural hearing loss, retinitis pigmentosa and, in some cases, vestibular dysfunction. It is clinically and genetically heterogeneous. To date, 10 genes have been associated with the disease, making its molecular diagnosis based on Sanger sequencing, expensive and time-consuming. Consequently, the aim of the present study was to develop a molecular diagnostics method for Usher syndrome, based on targeted next generation sequencing.MethodsA custom HaloPlex panel for Illumina platforms was designed to capture all exons of the 10 known causative Usher syndrome genes (MYO7A, USH1C, CDH23, PCDH15, USH1G, CIB2, USH2A, GPR98, DFNB31 and CLRN1), the two Usher syndrome-related genes (HARS and PDZD7) and the two candidate genes VEZT and MYO15A. A cohort of 44 patients suffering from Usher syndrome was selected for this study. This cohort was divided into two groups: a test group of 11 patients with known mutations and another group of 33 patients with unknown mutations.ResultsForty USH patients were successfully sequenced, 8 USH patients from the test group and 32 patients from the group composed of USH patients without genetic diagnosis. We were able to detect biallelic mutations in one USH gene in 22 out of 32 USH patients (68.75%) and to identify 79.7% of the expected mutated alleles. Fifty-three different mutations were detected. These mutations included 21 missense, 8 nonsense, 9 frameshifts, 9 intronic mutations and 6 large rearrangements.Conclusions Targeted next generation sequencing allowed us to detect both point mutations and large rearrangements in a single experiment, minimizing the economic cost of the study, increasing the detection ratio of the genetic cause of the disease and improving the genetic diagnosis of Usher syndrome patients.
    Full-text · Article · Nov 2014
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