# Mutation spectrum and clinical investigation of achromatopsia patients with mutations in the GNAT2 gene

Julia Felden, Britta Baumann, Manir Ali, Isabelle Audo, Carmen Ayuso, Béatrice Bocquet, Ingele Casteels, Blanca Garcia-Sandoval, Samuel Jacobson, Bernhard Jurklies, et al.

► To cite this version:

Julia Felden, Britta Baumann, Manir Ali, Isabelle Audo, Carmen Ayuso, et al. Mutation spectrum and clinical investigation of achromatopsia patients with mutations in the GNAT2 gene. Human Mutation, 2019, 41 (8), pp.humu.23768. 10.1002/humu.23768. hal-04958958

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# Mutation spectrum and clinical investigation of achromatopsia patients with mutations in the GNAT2 gene

Julia Felden<fnref n="1" /> ID

Britta Baumann<fnref n="1" />

Manir Ali<fnref n="2" /> ID

Isabelle Audo<fnref n="3" /> ID

Carmen Ayuso<fnref n="4" />

Beatrice Bocquet<fnref n="5" />

Ingele Casteels<fnref n="6" />

Blanca Garcia-Sandoval<fnref n="7" />

Samuel G. Jacobson<fnref n="8" />

Bernhard Jurklies<fnref n="9" />

Ulrich Kellner<fnref n="10" /> ID

Line Kessel<fnref n="11" />,<fnref n="12" />

Birgit Lorenz<fnref n="13" />

Martin McKibbin<fnref n="2" />

Isabelle Meunier<fnref n="5" />

Thomy deRavel<fnref n="14" />

Thomas Rosenberg<fnref n="11" />,<fnref n="12" />

Klaus Rüther<fnref n="15" />

Maria Vadala<fnref n="16" />

Bernd Wissinger<fnref n="1" />

Katarina Stingl<fnref n="17" />

Susanne Kohl<fnref n="1" />

**Correspondence**

Dr. Susanne Kohl, Molecular Genetics
Laboratory, Institute for Ophthalmic
Research, Centre for Ophthalmology,
University of Tuebingen, Elfriede-Aulhorn-
Strasse 5-7, 72076 Tuebingen, Germany.

Email: susanne.kohl@uni-tuebingen.de

**Funding information**

Bundesministerium für Bildung und
Forschung, Grant/Award Numbers:
01GM0850, 01GM1108A; Raregenomics
(Consejería de Educación de la Comunidad de
Madrid); Medical Faculty of the University of
Tuebingen; The European Reference Network
dedicated to Rare Eye Diseases (ERN-EYE);
Deutsche Forschungsgemeinschaft, Grant/
Award Numbers: SFB430, KFO134; FEDER

**Abstract**

Achromatopsia (ACHM) is a hereditary cone photoreceptor disorder characterized by the inability to discriminate colors, nystagmus, photophobia, and low-visual acuity. Six genes have been associated with this rare autosomal recessively inherited disease, including the GNAT2 gene encoding the catalytic α-subunit of the G-protein transducin which is expressed in the cone photoreceptor outer segment. Out of a cohort of 1,116 independent families diagnosed with a primary clinical diagnosis of ACHM, we identified 23 patients with ACHM from 19 independent families with likely causative mutations in GNAT2, representing 1.7% of our large ACHM cohort. In total 22 different potentially disease-causing variants, of which 12 are novel, were identified. The mutation spectrum also includes a novel copy number variation, a heterozygous duplication of exon 4, of which the

<footnote n="1">¹Institute for Ophthalmic Research, Centre for Ophthalmology, University Tuebingen, Tuebingen, Germany</footnote>

<footnote n="2">²Section of Ophthalmology and Neuroscience, Leeds Institute of Medical Research at St. James's University Hospital, University of Leeds, Leeds, England</footnote>

<footnote n="3">³Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institute de la Vision/CHNO des Quinze-Vingts, DHU Sight Restore, INSERM-DHOS, Paris, France</footnote>

<footnote n="4">⁴University Hospital Fundación Jiménez Díaz/Center for Biomedical Network Research on Rare Diseases (CIBERER), ISCIII, Madrid, Spain</footnote>

<footnote n="5">⁵Centre de Référence Maladies Sensorielles Génétiques, Hôpital Gui de Chauliac; Montpellier University and INSERM U1051, Institute for Neurosciences of Montpellier, Montpellier, France</footnote>

<footnote n="6">⁶Department of Ophthalmology, University Hospitals Leuven, Leuven, Belgium</footnote>

<footnote n="7">⁷Department of Ophthalmology, Fundación Jimenez Diaz University Hospital, Madrid, Spain</footnote>

<footnote n="8">⁸Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania, USA</footnote>

<footnote n="9">⁹Helios Clinics Wuppertal, Wuppertal, Germany</footnote>

<footnote n="10">¹⁰Rare Retinal Disease Center, AugenZentrum Siegburg, MVZ ADTC Siegburg GmbH, Europaplatz 3, Siegburg, Germany</footnote>

<footnote n="11">¹¹The National Eye Clinic, Rigshospitalet, Kennedy Center, Glostrup, Denmark</footnote>

<footnote n="12">¹²Department of Clinical Medicine, University of Copenhagen, Denmark</footnote>

<footnote n="13">¹³Department of Ophthalmology, Justus-Liebig-University Giessen, Giessen, Germany</footnote>

<footnote n="14">¹⁴Center for Human Genetics, University Hospitals Leuven, University of Leuven, Leuven, Belgium</footnote>

<footnote n="15">¹⁵Augenarztpraxis, Dorotheenstrasse 56, Berlin, Germany</footnote>

<footnote n="16">¹⁶Ophthalmology Institute, Dipartimento di Biomedicina, Neuroscienze e Diagnostica Avanzata (BiND), Università degli Studi di Palermo</footnote>

<footnote n="17">¹⁷University Eye Hospital, Center for Ophthalmology, University of Tübingen, Germany</footnote>
(European Regional Development Fund);
CIBERER, Grant/Award Number: 06/07/0036;
FIS, Grant/Award Number: PI16/00425; PRO
RETINA foundation

breakpoint matches exactly that of the previously reported exon 4 deletion. Two patients
carry just a single heterozygous variant. In addition to our previous study on GNAT2-
ACHM, we also present detailed clinical data of these patients.

KEYWORDS

achromatopsia, copy number variations, GNAT2, mutations, transducin

**1** | INTRODUCTION

Achromatopsia (ACHM), also called complete color blindness or rod monochromatism, is a rare stationary congenital hereditary cone photoreceptor disorder with a prevalence of 1:30,000–1:50,000 (Sharpe & Nordby, 1990; Sharpe, Stockman, Jagle & Nathans, 1999). Affected individuals usually present first with pendular nystagmus and/or severe photophobia shortly after birth or during their first months of life. Complete ACHM is furthermore characterized by impaired visual acuity (< 0.1 or 20/200) and the inability to discriminate colors. Examination of the fundus appears normal but electroretinographic recordings (ERG) show an absent cone photo-receptor (photopic) and a normal rod photoreceptor (scotopic) response in affected individuals. Some individuals show residual color vision and slightly better visual acuity leading to a clinical diagnosis of incomplete ACHM. Although ACHM was originally thought to be a stationary disease, retinal degenerative changes in the central retina have been reported, suggesting a progressive disease on the cellular level (Aboshiha et al., 2014; Genead et al., 2011; Thiadens et al., 2010; Thomas, Kumar, Kohl, Proudlock, & Gottlob, 2011; Thomas, McLean, Kohl, Sheth, & Gottlob, 2012).

ACHM is an autosomal recessively inherited and genetically heterogenous disease. To date, mutations in six genes have been associated with ACHM. The most frequently associated genes, accounting for ~70-90% of cases, are CNGA3 (MIM# 600053, Kohl et al., 1998) and CNGB3 (MIM# 605080, Kohl et al., 2000; Sundin et al., 2000), which encode for the α- and β subunit of the cyclic nucleotide gated (CNG) channel, respectively, which is expressed in the cone photoreceptor outer segments. Less frequently mutated genes include the GNAT2 gene (MIM# 139340, Kohl et al., 2002; Aligianis et al., 2002), which encodes the catalytic α-subunit of the G-protein transducin in the cone outer segments as well as PDE6C (MIM# 600827, Chang et al., 2009; Thiadens et al., 2009), and PDE6H (MIM# 601190, Kohl et al., 2012) encoding the catalytic α'- and inhibitory γ- subunits of the cone photoreceptor-specific phosphodiesterase, respectively. In addition, we identified mutations in ATF6 (MIM# 605537, Kohl et al., 2015) as a new disease gene for ACHM. ATF6 is a ubiquitously expressed gene encoding a transcription factor which is known to be a key player of the unfolded protein response (UPR) and endoplasmic reticulum homeostasis (Walter & Ron, 2011).

In 2002, we reported five families with patients affected by
ACHM caused by mutations in GNAT2 (Kohl et al., 2002; Rosenberg
et al., 2004). Since then only few reports of single families have been

published (Aligianis et al., 2002; Piña, Baumert, Loyer, & Koenekoop, 2004; Ouechtati et al., 2011; Langlo et al., 2016, Taylor et al., 2017; Carss et al., 2017; Ueno et al., 2017). Herein we report the results of our genetic investigation of our complete ACHM cohort over a period of 16 years, in total identifying 23 affected individuals from 19 independent families carrying likely disease-causing mutations in GNAT2, of which 12 mutations have never been reported before. This study almost doubles the GNAT2 mutation spectrum and provides, due to the large number of patients and families, a rather reliable estimation of the prevalence of GNAT2 mutations in patients with ACHM of Europe or European descent. Furthermore, we were able to characterize a novel copy number variation (CNV) hereby identifying the elusive second allele in one of our GNAT2 single heterozygous patients which escaped prior genetic routine screenings by exon-focused polymerase chain reaction (PCR) and Sanger sequencing.

**2** | MATERIAL AND METHODS

**2.1** | Patient cohort and clinical examination

Patients diagnosed with ACHM were recruited over a period of 21 years
in several ophthalmic centers mainly in Europe (Italy, Denmark, Spain,
Belgium, France, Great Britain, and Germany) as well as in the USA, and
blood or DNA samples were sent to the Institute for Ophthalmic
Research in Tuebingen for genetic investigation. The clinical diagnosis
was established using standard clinical ophthalmologic examinations
including assessment of visual acuity and color vision, funduscopy and, if
available, electroretinography (ERG) and optical coherence tomography
(OCT). Clinical data were summarized retro- and prospectively.

Peripheral blood was collected from all ACHM diagnosed
patients and available family members who gave written informed
consent approved by the respective local research and ethical boards
or dependent on the local regulatory bodies. Specifically the study
was approved by the Ethnics Board of the University of Tuebingen
under the study no. 116/2015BO2. Whole genomic DNA was
extracted using standard procedures and stored at the Institute for
Ophthalmic Research in Tuebingen. In total we have collected and
analyzed a cohort of 1,116 families with a suspected clinical diagnosis
or differential diagnosis of ACHM (n = 1,020), blue cone monochro-
matism (BCM; n = 20), or other cone-related retinal diseases, for
example, cone-/cone-rod dystrophy (CD/CRD), oligo-cone trichro-
macy (OT), macular dystrophy, and stationary cone dysfunction
(n = 76).
## 2.2 | Mutation detection, segregation analysis, and variant assessment

Mutation screening in GNAT2 was performed as previously described (Kohl et al., 2000). Segregation analysis to prove the presence and independent inheritance of two biallelic GNAT2 mutations was performed by PCR and Sanger sequencing in those cases for which DNA from additional family members was available (Figure 1).

All variants were denoted based on the NCBI reference sequence for GNAT2 (NM_005272.3; GRCh38). Possible effects of missense, nonsense, and (canonical) splice site variants as well as smaller and larger indels on transcript splicing and protein function were assessed using the following computational *in silico* tools: Mutation-Taster2 (http://www.mutationtaster.org; Schwarz, Cooper, Schuelke & Seelow, 2014), SIFT (http://sift.jvic.org; Kumar, Henikoff & Ng, 2009); PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/; Adzhubei et al., 2010), and/or Human Splice Finder (HSF 3.0) applying the integrated MaxEnt algorithm (Desmet et al., 2009). In addition, splice site predictions were performed by the integrated software Alamut Genova (http://www.interactive-biosoftware.com) using default settings. To classify the variants, the standards and guidelines provided by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology were applied (http://www.medschool.umaryland.edu/genetic_variant_interpretation_tool2.html/; Richards et al., 2015) to score for possible or likely pathogenicity. All variants have been submitted to ClinVar (http://www.ncbi.nlm.nih.gov/clinvar/; Landrum et al., 2014) with accession codes from SCV000891375.1 to SCV000891396.1

## 2.3 | Quantitative Real-Time PCR (qRT-PCR)

Allele quantification for GNAT2 was performed by a quantitative real-time PCR (qRT-PCR) based assay applying the QuantiTec SYBR Green PCR Kit (QIAGEN, Hilden, Germany) for all eight coding exons (for primers see Table S1) in patients carrying only a single heterozygous GNAT2 variant to search for larger deletions or duplications (i.e., copy number variations). The assay was performed as described for CNGB3 (Mayer et al., 2017). In short, for each reaction, 40 ng of genomic DNA in a total reaction volume of 20 µl were analyzed on an Applied Biosystems 7500 Real-Time PCR system using autos settings (cycle threshold (Ct) = 0.2). The obtained data were normalized against a reference gene (SDC4; De Preter et al., 2002; Pattyn, Speleman, De Paepe, & Vandesompele, 2003). To each run, a control sample was added. All samples were measured as triplicates and for data analysis the ΔΔCt value and the standard deviation were calculated.

## 2.4 | Characterization and segregation analysis of copy number variants

### 2.4.1 | Breakpoint characterization.

The breakpoint characterization was carried out for CHRO946 by long-range PCR using the Long Amp DNA Taq (New England Biolabs).

Potential breakpoint primers were chosen dependent on the most likely physical breakpoint position referring to the rise in the log₂ ratios of the qRT-PCR evaluation assuming a tandem duplication event. To determine amplicons which were unique to the CNV carrier, a sample from a healthy control was added to the long-range PCR run. The successfully amplified PCR product was then sequenced to define the definite insertion point of the duplicated exon.

### 2.4.2 | Segregation analysis.

The primer designed to define the insertion point by long-range PCR were specific to the exon duplication, meaning the amplicon is specific for the CNV and its carriers. Available family members were analyzed using the unique long-range PCR to determine possible other CNV carriers.

# 3 | RESULTS

## 3.1 | GNAT2 mutation spectrum in patients with ACHM

We screened for ACHM associated mutations in a large cohort of 1,116 independent families. The majority of those families (n = 1,020, 91.4%) were clinically diagnosed as ACHM or rod monchromatism, and 8.6% with other cone photoreceptor-related diseases (i.e., BCM, OT, CD, and CRD). Nineteen families (1.7%) could be identified carrying potential ACHM associated mutations in GNAT2 (Figure 1, Table S2), five of these families (CHRO22, CHRO53, CHRO68, CHRO87, and CHRO94) have previously been published (Kohl et al., 2002; Rosenberg et al., 2004). Twelve index patients harbored homozygous mutations whereas seven index patients carried two heterozygous mutations in GNAT2 potentially explaining the ACHM phenotype. In two families (CHRO1094 and BCM274) only a single heterozygous variant could be identified using both standard screening techniques and qPCR-based CNV analysis. The nonsense mutation c.481 C > T;p.(Arg161*) was seen in three families, in two families homozygously (CHRO73 Danish and CHRO330 Turkish), and once compound heterozygously (CHRO752, French). Two families carry the previously reported exon 4 deletion (CHRO22 and CHRO752), the first homozygously, the latter compound heterozygously. The mutation c.811_813delAAG was seen in two Spanish families (CHRO469 and CHRO555). Although CHRO469 carries this mutation homozygously, CHRO555 is compound heterozygous for this mutation. The pedigrees including genotypes for all analyzed families are depicted in Figure 1.

To confirm the presence and independent inheritance of two mutant GNAT2 alleles, segregation analysis was performed in both parents for nine index patients, and in other family members or siblings for four index patients (see Figure 1). Moreover, parental consanguinity was reported for two of the analyzed families. For six cases, no family members were available for segregation analysis.
<figure><img src="image_2.png" /><figcaption>FIGURE 1 Continued.</figcaption></figure>
The disease-related sequence alterations which were identified comprise four nonsense mutations (two novel), four small deletions, and two small duplications (Fig. S1; Table 1). They either directly generate a premature stop codon or cause a frame-shift resulting in a premature termination codon (PTC) with subsequent translation termination in most instances leading to a severely truncated protein if the transcript is translated. Nevertheless most transcripts are predicted to undergo nonsense-mediated decay (NMD). Furthermore three (one novel) putative splice site mutations were found and are predicted to lead to aberrant splicing. The novel variant (c.591-2A > C) as well as the previously reported variant c.720+2T > C affect the canonical splice sites of exon 5 and exon 6, and are therefore likely to have a major effect on splicing. *In silico* assessment using four algorithms embedded in the Alamut software indeed predicted the virtual abolishment of the respective splice sites (Table S3; SSF-like score for c.591-2A > C: 88.57; SSF-like score for c.720+2T > C: 84.07). In addition, eight (all novel) missense mutations which are predicted to probably affect protein function, and one CNV comprising a duplication of the complete exon 4, and a CNV deleting exon 4 (Kohl et al., 2002) were identified. Taking possible biallelism into account, one index patient (CHRO589, 18189, no notion of parental consanguinity) habouring a homozygous missense mutation in exon 4 of GNAT2 was additionally tested for the recurrent exon 4 deletion with a negative outcome. The complete mutation spectrum including their positions on the genomic, transcript and protein level of all GNAT2 mutations in our cohort and presented in this study (both known and novel) is graphically summarized in Figure 2 and Table 1. The percentage of each type of mutation comprised in our cohort is depicted in Fig. S1.

According to the ACMG guidelines 11 out of these 22 variants were classified as pathogenic, four as likely pathogenic and seven as variants of uncertain significance (VUS). Two of those VUS (c.724 C > T;p.(Arg242Cys), c.943 G > A;p.(Asp315Asn)) were unique to single patients and observed only in a heterozygous state without a second mutant GNAT2 allele. It remains unknown whether these variants are related to the disease in these patients.

## 3.2 | Detection of a novel copy number variation (CNV) in GNAT2

In three out of 19 index patients only a single rare heterozygous variant, that was likely ACHM associated, was identified by standard screening procedures. As previously described for CNGB3 (Mayer et al., 2017), and as a deletion of exon 4 had already been known, we assume that the second disease-causing mutation could be a structural variant. To detect such a structural aberration on the second allele we applied qPCR based copy number variation (CNV)

quantification for all eight coding exons of GNAT2. With this assay it was possible to detect a duplication of exon 4 in one (CHRO946) of the three cases (Figure 3a). Furthermore segregation of this duplication in the mother of the patient (Figure 3b) was successfully proven. Unfortunately, no CNV could be identified for the other two index patients carrying solely a single heterozygous variant, and they remain unsolved (Fig. S2).

Subsequently, breakpoint mapping was performed using a long-range PCR specific for the duplication of exon 4, and completed by the ensuing Sanger sequencing of the junction fragments (Figure 3c). Analysis of the sequences showed that exon 4 was duplicated in *tandem* and that this duplication comprises 2,018 bp (NC_000001.11: g.109,607,657_109,609,675dup). Interestingly, these 2,018 bp comprise the very same fragment which is deleted in families CHRO22 and CHRO752 (Figure 3d). To identify the underlying mutational mechanism, bioinformatic analyses were performed revealing mammalian-wide interspersed repeat (MIR2b) elements which flank the identified duplication at both sites and allowed both structural genomic rearrangements of exon 4 in GNAT2 (Figure 3c,d).

## 3.3 | Clinical presentation

The ACHM patient cohort with GNAT2 mutations consisted of 19 index patients and four affected relatives (15 males and 8 females). All available clinical characteristics are depicted in Table S4.

The age of the patients at examination was 31.5 years on average (±21 years) ranging from 3 months up to 79 years. Diagnoses of all patients except for patient CHRO53-II:2, for whom no clinical data are available, are compatible with a clinical diagnosis of ACHM. In seven patients the incomplete type of ACHM was diagnosed. In one patient (CHRO798-II:1) the diagnosis could not be differentiated with certainty from CD. In one patient (CHRO946-II:1) the diagnosis of OT was chosen.

The first signs reported by the patients were typical for ACHM: Color vision problems, poor vision, and photophobia, most patients had nystagmus and one case also had strabismus. If known, the age of onset of first symptoms was in the preschool age in all patients except for family CHRO73: both siblings have been examined only at the age of 10 and 11 years, respectively; the real ages of onset of symptoms are unknown.

Only one patient (CHRO87-III:2) denied photophobia as a symptom. The majority of patients (16/23) had nystagmus. In one of the patients (CHRO87-III:2) the nystagmus was reported only on the right eye without further details available; four patients had no nystagmus (CHRO68-II:1, CHRO555-II:1, ZD80-II:1, and BCM274-II:1); in three patients no documentation was available regarding

**FIGURE 1** Pedigrees, genotypes and segregation analysis for all families of the ACHM cohort carrying mutations in GNAT2. Affected individuals are given with filled symbols; unaffected individuals are represented with open symbols and individuals with a milder phenotype are given with gray symbols. Arrows indicate the index patient. DNA numbers and genotypes for individuals with available DNA are given below each symbol. Proven compound-heterozygosity is indicated by square brackets. CHRO designates patients initially diagnosed with ACHM, ZD patients originally diagnosed with cone dystrophy and BCM patients diagnosed with blue cone monochromacy. ACHM, achromatopsia; BCM, blue cone monochromatism; CD, cone dystrophy
<table><caption>TABLE 1 Complete mutation spectrum in GNAT2 of this as well as all previously published studiesª</caption><thead><tr><th>Variant</th><th>Polypeptide</th><th>HGMD accession no.</th><th>Diagnosis</th><th>Literature</th></tr></thead><tbody><tr><td colspan="5"><strong>Missense/nonsense mutations</strong></td></tr><tr><td>c.107 T > G</td><td>p.(Leu36Arg)</td><td></td><td>ACHM</td><td>This study</td></tr><tr><td>c.139 A > G</td><td>p.(Ser47Gly)</td><td></td><td>ACHM/Cone dysfunction</td><td>This study</td></tr><tr><td>c.145 A > G</td><td>p.(Ile49Val)</td><td></td><td></td><td>LOVD</td></tr><tr><td>c.235 C > T</td><td>p.(Gln79*)</td><td>CM021611</td><td>ACHM</td><td>Kohl et al. (2002)</td></tr><tr><td>c.257 G > A</td><td>p.(Arg86Gln)</td><td></td><td></td><td>LOVD</td></tr><tr><td>c.313 C > T</td><td>p.(Arg105*)</td><td></td><td>ACHM</td><td>This study</td></tr><tr><td>c.370 G > A</td><td>p.(Val124Met)</td><td>CM1612168</td><td>ACHM</td><td>Langlo et al. (2016)</td></tr><tr><td>c.481 C > T</td><td>p.(Arg161*)</td><td></td><td>ACHM</td><td>This study</td></tr><tr><td>c.593 T > A</td><td>p.(Met198Lys)</td><td></td><td>ACHM</td><td>This study</td></tr><tr><td>c.605 G > A</td><td>p.(Gly202Glu)</td><td>CM178845</td><td>Cone dysfunction disorder</td><td>Taylor et al. (2017)</td></tr><tr><td>c.620 A > T</td><td>p.(Glu207Val)</td><td></td><td>CD</td><td>This study</td></tr><tr><td>c.623 G > C</td><td>p.(Arg208Thr)</td><td></td><td>BCM</td><td>This study</td></tr><tr><td>c.724 C > T</td><td>p.(Arg242Cys)</td><td></td><td>ACHM</td><td>This study</td></tr><tr><td>c.886 C > T</td><td>p.(Tyr296His)</td><td></td><td>CD</td><td>This study</td></tr><tr><td>c.896 C > A</td><td>p.(Ala299Glu)</td><td>CM184433</td><td>ACHM</td><td>Bryant et al. (2018)</td></tr><tr><td>c.906 C > A</td><td>p.(Tyr302*)</td><td>CM175547</td><td>Retinal disease</td><td>Carss et al. (2017)</td></tr><tr><td>c.937 C > T</td><td>p.(Arg313*)</td><td>CM110693</td><td>ACHM</td><td>Ouechtati et al. (2011)</td></tr><tr><td>c.943 G > A</td><td>p.(Asp315Asn)</td><td></td><td>ACHM</td><td>This study</td></tr><tr><td colspan="5"><strong>Splice site mutations</strong></td></tr><tr><td>c.461 + 24 G > A</td><td>-</td><td>CS045213</td><td>ACHM</td><td>Rosenberg et al. (2004)</td></tr><tr><td>c.591-2 A > C</td><td>-</td><td></td><td>ACHM</td><td>This study</td></tr><tr><td>c.720 + 2 T > C</td><td>-</td><td>CS184437</td><td>ACHM</td><td>Bryant, Lozynska, Maguire, Aleman, and Bennett (2018)</td></tr><tr><td colspan="5"><strong>Small deletions</strong></td></tr><tr><td>c.730_743del</td><td>p.(His244Serfs*7)</td><td>CD175910</td><td>ACHM</td><td>Ueno et al. (2017)</td></tr><tr><td>c.811_813del</td><td>p.(Lys271del)</td><td>CD044786</td><td>Cone-rod degeneration</td><td>Pina, Baumert, Loyer, and Koenekoop (2004)</td></tr><tr><td>c.955del</td><td>P.(Ile319Serfs*5)</td><td>CD021829</td><td>ACHM</td><td>Kohl et al. (2002)</td></tr><tr><td colspan="5"><strong>Small insertions</strong></td></tr><tr><td>c.503dup</td><td>-</td><td>CI021912</td><td>ACHM</td><td>Kohl et al. (2002)</td></tr><tr><td>c.803_806dup</td><td>-</td><td>CI021913</td><td>ACHM</td><td>Kohl et al. (2002)</td></tr><tr><td>c.840_843dup</td><td>-</td><td>CI022183</td><td>ACHM</td><td>Aligianis et al. (2002)</td></tr><tr><td colspan="5"><strong>Large insertions/deletions</strong></td></tr><tr><td>c.(303 + 365)_(461 + 974)del</td><td>Exon 4 deletion</td><td>CG024902</td><td>ACHM</td><td>Kohl et al. (2002)</td></tr><tr><td>c.(303 + 365)_(461 + 974)del</td><td>Exon 4 duplication</td><td></td><td>ACHM</td><td>This study</td></tr><tr><td colspan="5"><strong>Small indels</strong></td></tr><tr><td>c.285delTGCTGAA_insCTGTAT</td><td>p.(Ala96Cysfs*61)</td><td></td><td>Cone dysfunction</td><td>Kohl et al. (2002)</td></tr></tbody></table>

Note: ACHM, achromatopsia; BCM, Blue cone monochromatism.

nystagmus (CHRO53-II:2, CHRO330-II:1, and CHRO684-II:1). None of patients had a clearly progressive disease.

The functional examinations showed reduced visual acuity of 20/400 to 20/50. Color vision was abnormal in all patients; different methods were used to document this (i.e., anomaloscope, Ishihara plates, Farnsworth-Munsell 15-hue, or 100-hue test). In patient

CHRO946-II:1 the color vision test showed a nearly normal result, in three patients a predominant blue color disturbance was found (CHRO1094-II:1, CHRO752-II:1, and BCM274-II:1). Atypical cases were both members of family CHRO87, who both showed clear abnormalities in the anomaloscope testing (ACHM and deuteromaly), however, naming of colors and 100 hue Farnsworth-Munsell

<footnote>ªMutations were retrieved from this study, literature, HGMD and LOVD.</footnote>
<figure><img src="image_3.png" /><figcaption>**FIGURE 2** Mutation spectrum and localization of mutations associated with ACHM in GNAT2 presented in this study. Depicted from top to bottom are the gene locus of GNAT2 with its eight coding exons, the transcript and the polypeptide. GTP binding domains are shown in dark gray. All novel nonsense, missense and splice site mutations as well as indels identified in this study are depicted in black, all previously published mutations of our cohort (Kohl et al., 2002) are shown in light gray below the protein and gene locus scheme, all other previously reported mutations are depicted in light gray above the protein and gene locus scheme. Underlined missense mutations are predicted to also potentially alter splicing (compare Table S3). The gross duplication and gross deletion are depicted above and splice site mutations below the gene locus. aa, amino acids; ACHM, achromatopsia; ATG, denotes the translation start codon; CNV, copy number variation; COOH, carboxyterminus; GTP, guanosine triphosphate; NH₂, amino terminus; UTR, untranslated region</figcaption></figure>

test gave results almost in the normal ranges. The full-field ERG recordings, if available, were typical for ACHM: No measurable photopic responses (or severely reduced in two cases), and mostly normal or slightly reduced scotopic responses.

Retinal imaging showed usually normal or nearly normal findings. Funduscopy and retinal photography revealed normal fundus appearance in the majority of the cases; in some patients altered central funduscopic reflexes were described. Similarly, the fundus autofluorescence retinal imaging revealed mostly normal findings. OCT scans, if available, were reported as normal in five cases, and in six further cases there were central retinal abnormalities (see Table S4).

## 4 | DISCUSSION

ACHM is a rare autosomal recessive cone photoreceptor disease. *CNGB3* and *CNGA3* are the most frequently reported disease-causing genes for ACHM and we now report that mutations in *GNAT2* represent 1.7% of our cohort of patients with ACHM.

GNAT2 shows a broad spectrum of different disease-causing mutations (Table S3). Most frequently we observed nonsense mutations resulting in PTC, and the mutant mRNA transcripts are predicted to undergo NMD. Out of the four different nonsense mutations, two were novel (c.313 C > T;p.[Arg105*] and c.481 C > T;p.[Arg161*]). Similar to the two already published nonsense

mutations (Kohl et al., 2002; Ouechtati et al., 2011), the mutations will result in the loss of important functional domains, if the mutant transcripts are translated.

Furthermore we were able to identify eight novel missense mutations which occur either in a homozygous (c.139 A > G;p.(Ser47Gly), c.593 T > A;p.(Met198Lys)), or in a compound heterozygous state (c.107 T > G;p.(Leu36Arg), c.620 A > T;p.(Glu207Val), c.623 G > C;p.(Arg208Thr), c.724 C > T;p.(Arg242Cys), c.886 T > C;p.(Tyr296His), and c.943 G > A;p.(Asp315Asn)). All novel missense variants affect amino acid residues which are evolutionary highly or even completely conserved, not only in GNAT2 proteins, but also GNAT1, the rod photoreceptor transducin (Fig. S3). Furthermore the missense mutation c.139 A > G;p.(Ser47Gly) affects the last amino acid residue of the first nucleotide binding domain of GNAT2 probably inhibiting the binding mechanism of GTP and thereby disturbing the function of the α-subunit of transducin. Of note, the missense mutation c.943 G > A;p.(Asp315Asn) occurred only once in our cohort in combination with a wild-type allele further weakening its association with the disease.

Moreover, we were able to identify one novel canonical splice site mutation (c.591-2 A > C) which is predicted to result in missplicing and to be pathogenic due to destruction of an exonic splicing enhancer site (Table S3). In addition it is predicted to activate an exonic cryptic acceptor site. The previously reported splice site mutation c.720 + 2 T > C (Bryant, Lozynska, Maguire, Aleman, & Bennett, 2018) was also predicted to have a major effect on the
<figure><img src="image_4.png" /></figure>

**FIGURE 3** qRT-PCR analysis for CNV detection, CNV segregation for family CHRO946, breakpoint mapping and MIR2b mediated deletion and duplication of exon 4. (a) Results of the qRT-PCR analysis for CNV detection are depicted. Each of the eight coding and analyzed exons was either screened alone or in combination with its neighboring exon. Exons without indication of any structural variations show a ΔΔCt value of approximately 0 whereas exon 4 which is duplicated in this patient shows a decrease in the ΔΔCt value (gray box). (*) indicates a false positive deletion result for exons 7/8. (b) Segregation analysis of the duplication of exon 4 in family CHRO946. A breakpoint PCR was carried out for the index patient and the mother, as well as a healthy control (c). DNA of the father was unavailable. The PCR product specifically amplifies the breakpoint. The breakpoint PCR analysis shows that the duplication of exon 4 segregates from the mother to the index patient. (c) Sequencing data of the junction fragment for the duplication of exon 4 is shown. The electropherogram was obtained by PCR amplification of the insertion point fragment and subsequent Sanger sequencing. Black letters represent the mutant allele as it was observed in the patient. The reference sequence is represented in gray letters above for the 5'- and below for the 3'-sequence of the insertion point. The continued reference sequence is given in red on each side. Homologous regions are partly marked in yellow. The homologous regions consist of two 228 bp repeats flanking the duplicated region. The repeats show a homology rate of 99.1%. A scheme for the duplication of exon 4 is given underneath the electropherogram. (d) MIR2b mediated deletion and duplication of exon 4. Exon 4 is flanked by two MIR2b repeats. The repeats result in a recombination of an identical fragment including exon 4 leading to a deletion (top, CHRO22, CHRO752) or duplication (bottom, CHRO946) of exon 4. CNV, copy number variation; qRT-PCR, quantitative real-time polymerase chain reaction

splicing process. However, the appearance of a GC splice donor site instead of a GT splice donor site is not uncommon and holds true for approximately 1% of all splice donor sites (Burset, Seledtsov, & Solovyev, 2000).

We detected only one 1 bp duplication c.503dup (Kohl et al., 2002) leading to a premature stop codon and the loss of the majority of the protein. All allele compositions of GNAT2 of our study cohort are summarized in Table S2.

In three out of our 19 index patients we found only a single heterozygous variant in GNAT2 with our standard screening procedures. Therefore we hypothesized that they may carry another disease-causing alteration, a CNV, on the other allele, as we could already show for the CNGB3 gene (Mayer et al., 2017), and by our previous data from our cohort (Kohl et al., 2002). We screened those patients for CNVs in all eight coding exons of GNAT2 using a qRT-PCR-based assay. With this approach we successfully solved one of three cases. We found that the index patient of family CHRO946 carries in addition to a heterozygous nonsense mutation

(c.937 C > T; p.(Arg313*)) a structural variation, namely, a duplication of exon 4, on the counter allele. This duplication comprises 2,018 bp and is flanked by MIR2b repeats. Interestingly the breakpoint of the duplication is identical to the one described in the deletion of exon 4. During cell division, the MIR2b repeats can lead to chromosomal rearrangements finally resulting in either the duplication or deletion of exon 4 and parts of the flanking intronic regions (Figure 3d). If during the maturation of the mRNA the CNV also results in a duplicated exon 4 in the mature mRNA, this would lead to a frameshift resulting very likely in a PTC, and represent a *null* allele.

In 2002, we had reported mutations in GNAT2 as a rare cause of ACHM (Kohl et al., 2002). At that time, five families had been identified and reported. In this study, we enlarged our previous GNAT2 cohort (Kohl et al., 2002) and present data on a total of 22 (12 novel), likely disease associated mutations including four nonsense mutations, eight missense, five indels including small deletions, insertion and duplication, as well as three splice site mutations. In addition, we describe two intragenic CNVs, a deletion
of exon 4 (Kohl et al., 2002) in three patients of two independent families, and a novel duplication of exon 4 in one patient. These genomic rearrangements are most probably triggered by the use of MIR2b elements flanking exon 4 and the adjacent introns of GNAT2. To complete and complement this study the mutation spectrum of all published mutations in GNAT2 is given in Table 1.

The clinical phenotypes of all patients are compatible with ACHM, both complete and incomplete cases were observed. The most consistent findings over the whole cohort are a childhood onset of symptoms with poor vision, color vision disturbances, and non-recordable photopic full-field ERG with a normal scotopic full-field ERG. Nystagmus and photophobia were present in most of the cases, but not all of them. In none of the patients there was clear evidence of progression.

Besides a generally unremarkable fundus appearance, fundus autofluorescence (FAF) and high-resolution OCT (optical coherence tomography) imaging revealed notable changes in a few patients, all were limited to the foveal region. Unfortunately, many of the clinical centers did not have the same imaging instruments and a complete data set with this modality is missing from this report. It also has to be noted that high-resolution retinal imaging such as OCT or FAF presents a challenge in patients with nystagmus. If images are obtainable, they are frequently of low quality, thus, very subtle changes cannot be always reported. Both patients with FAF abnormalities also showed a pathologic foveal finding in the OCT: The hypofluorescent fovea in BCM274 corresponded to granular ellipsoid zone (EZ) and foveal thinning, and the perifoveal hyper-fluorescent ring in CHRO1094 corresponded to a hyporeflective EZ. These subtle, but evident changes in these few patients resemble foveal changes also seen in CNGA3 or CNGB3 achromats, which occur usually later in life (Zobor et al., 2017). However, the morphological foveal findings tend to be more subtle in GNAT2 achromats than those reported in CNGA3 achromats and were found in a mix of younger patients aged 9, 10, 15, and 31 years, rather than in the older patients of our population.

Nevertheless, the phenotypes of the GNAT2 associated ACHM we describe here do not show a clear pattern of findings that would allow to clinically differentiate these from other genotypes of ACHM. Thus, sequencing of the GNAT2 should be a part of molecular genetic diagnostics of ACHM.

Patient ZD80 had normal foveal FAF, however, her images showed a single flavimaculatus-like spot between the fovea and optic nerve head on the right eye only. The patient suffered from sarcoidosis, arterial hypertension, and was a smoker. This lesion corresponds in OCT to disturbances of the photoreceptor outer segments. The left eye did not present with this finding, but had drusen in the optic nerve head. To what extent these findings are associated with the GNAT2 mutations is unclear.

Interestingly, a new mouse model also carrying a CNV, namely a 7,654 bp deletion spanning exons 2-9 of the mouse Gnat2 gene, revealed in comparison with our patients no obvious cone photo-receptor loss, neither a disruption of the cone mosaic nor changes in the retinal morphology up to an age of 9 month (Ronning et al., 2018).

However, similar to the patients, no a-wave could be detected in ERG measurements and cone phototransduction was completely abolished (Ronning et al., 2018). Two other GNAT2 mouse models, the *cpfl3* mouse model harboring a missense mutation (c.598 G > A; p.(Asp200Asn)) in exon 6 of *Gnat2* (Chang et al., 2006) as well as another naturally occurring mouse carrying another missense mutation in exon 6 (c.518 A > G; p.[Asp173Gly]), Jobling, Vessey, Waugh, Mills, & Fletcher, 2013) showed a slowly progressing degeneration of the cone photoreceptor response. Both models display the hallmarks of ACHM including opsin mislocalization and poor cone-mediated ERG responses making the *cpfl3* mouse as well as the second "missense-mouse-model" suitable mouse models to study GNAT2-associated ACHM.

In conclusion our study underlines the role of GNAT2 as a rare but still important disease-causing gene for ACHM. Furthermore, it shows and extends the GNAT2 mutation spectrum considerably, and implies that CNVs affecting exon 4 of GNAT2 should be added to the genetic testing approach. As mutations in GNAT2 are sufficient to cause ACHM, and animal model data show that the cone photo-receptor degeneration is moderate to slow, prospectively, this gene would be another very good candidate gene for a gene replacement therapy approach.

## ACKNOWLEDGMENTS

The authors would like to thank all patients and family members for taking part in this study. This study was funded by grants of the Deutsche Forschungsgemeinschaft (DFG; SFB430 and KFO134 to BW and SK), the Bundesministerium für Bildung und Forschung (BMBF; HOPE and HOPE2, grant no. 01GM0850 and 01GM1108A to BW and SK), the PRO RETINA foundation and funds of the Medical Faculty of the University of Tuebingen, as well as supported by grants from the Instituto de Salud Carlos III (ISCIII) from the Spanish Ministry of Health, including CIBERER (06/07/0036), FIS (PI16/00425), Raregenomics (Consejeria de Educacion de la Comunidad de Madrid) and FEDER (European Regional Development Fund). Isabelle Audo, Line Kessel, Birgit Lorenz, Isabelle Meunier, Katarina Stingl and Susanne Kohl are members of the European Reference Network dedicated to Rare Eye Diseases (ERN-EYE).

## ORCID

Julia Felden http://orcid.org/0000-0002-8888-6824

Manir Ali http://orcid.org/0000-0003-3204-3788

Isabelle Audo http://orcid.org/0000-0003-0698-5309

Ulrich Kellner http://orcid.org/0000-0001-5221-4631

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## SUPPORTING INFORMATION

Additional supporting information may be found online in the Supporting Information section at the end of the article.

**How to cite this article:** Felden J, Baumann B, Ali M, et al. Mutation spectrum and clinical investigation of achromatopsia patients with mutations in the GNAT2 gene. *Human Mutation*. 2019;40:1145-1155.

https://doi.org/10.1002/humu.23768