Homozygous stop mutation in AHR causes autosomal recessive foveal hypoplasia and infantile nystagmus

Abstract

Herein we present a consanguineous family with three children affected by foveal hypoplasia with infantile nystagmus, following an autosomal recessive mode of inheritance. The patients showed normal electroretinography responses, no signs of albinism, and no anterior segment or brain abnormalities. Upon whole exome sequencing (WES), we identified a homozygous mutation (c.1861C>T;p.Q621*) in the aryl hydrocarbon receptor (AHR) gene that perfectly co-segregated with the disease in the larger family. The aryl hydrocarbon receptor is a ligand-activated transcription factor that has been intensively studied in xenobiotic-induced toxicity. It was further shown to play a physiological role under normal cellular conditions, such as in immunity, inflammatory response and neurogenesis. Notably, knockout of the Ahr gene in the mouse impairs optic nerve myelin sheath formation and results in oculomotor deficits sharing many features with our patients: the eye movement disorder in the Ahr^-/- mice appears early in development and presents as conjugate horizontal pendular nystagmus (Chevallier et al., 2013; Juricek et al., 2017). We therefore propose AHR to be a novel disease gene for a new, recessively inherited disorder in humans, characterized by infantile nystagmus and foveal hypoplasia.

Introduction

Nystagmus presents as involuntary rhythmic eye movements that can have a genetic origin or be acquired during infancy or later in life. Its prevalence is estimated to be 2.4 in 1,000 with Europeans being slightly more often affected than other ethnic groups (Sarvananthan et al., 2009). The causes of acquired nystagmus include neurological disorders and drug toxicity (Choudhuri et al., 2007), while infantile nystagmus can be idiopathic or accompanying hereditary ophthalmic diseases like achromatopsia, Leber congenital amaurosis or ocular albinism (Papageorgiou et al., 2014). Nystagmus is often one of the first signs identified by the parents, indicating that their child suffers from vision loss.

In infantile nystagmus, the oscillations of the eyes are conjugate and predominantly of a horizontal jerk waveform characterized by a slow drift of the eyes followed by a corrective fast eye movement that is responsible for realigning the fovea to the object of interest (Thomas et al., 2011a). Initially, the nystagmus can also be of a pendular (sinusoidal-like) waveform, changing later in life to the jerk waveform (Hertle and Dell'Osso, 1999). Patients with infantile nystagmus usually do not suffer from oscillopsia (the illusion of constant movement of the surroundings) as patients with acquired nystagmus tend to do (Papageorgiou et al., 2014). Infantile nystagmus may be sub-categorized into idiopathic infantile nystagmus (IIN) without obvious retinal dysfunction or dysmorphology and forms of infantile nystagmus associated with signs of ocular mal-development featuring foveal hypoplasia as in PAX6-linked Aniridia (Azuma et al., 1996) or FHONDA (foveal hypoplasia, optic nerve decussation and outer segment dysgenesis) due to mutations in SLC38A8 (Poulter et al., 2013). These patients typically show mildly reduced visual acuity and an abnormal optokinetic response (OKR) (Thomas et al., 2008; Thomas et al., 2011a; Self et al., 2007). However the distinction between IIN and IN with foveal hyploplasia may be artifical given findings of a recent study by Thomas and colleagues who showed for the first time that patients with FRMD7-related IN can have mild foveal hypoplasia and optic nerve changes, indicating that an abnormal afferent system development could underlie the childhood nystagmus (Thomas et al., 2014). This is further evidenced by abnormalities of OKR and retinal circuitry detected in Frmd7 knockout mice (Yonehara et al., 2016).

IIN mostly follows an X-linked mode of inheritance. Three X-linked forms have been localized to Xq26.2 (NYS1; FRMD7) (Tarpey et al., 2006), Xp11.4-p11.3 (NYS5; Cabot et al., 1999) (CASK, Hackett et al., 2010), and Xp22.2 (NYS6; GPR143) (Bassi et al., 1995). However, CASK mutations are associated with mental retardation (Hackett et al., 2010), and GPR143 mutations cause ocular albinism (Bassi et al., 1995). Autosomal dominant forms of IIN have been mapped to four different loci on chromosome 6p12 (NYS2; Kerrison et al., 1996), 7p11.2 (NYS3; Klein et al., 1998), 13q31-q33 (NYS4; Ragge et al., 2003), and 1q31.3-q32.1 (NYS7; Xiao et al., 2012, Li et al., 2012), but no genes have been associated with these forms yet.

Here we present the clinical findings of a consanguineous Christian Arab family from Northern Israel with three children affected by a new autosomal recessive disease characterized by infantile nystagmus and foveal hypoplasia with no signs of albinism. Whole exome sequencing (WES) revealed the presence of a homozygous nonsense mutation in the aryl hydrocarbon receptor gene (AHR; MIM 600253) that perfectly segregates in the family. Our data suggest AHR to be the first gene causing an autosomal recessive IIN-related disease in humans.

Materials and Methods

Patients and DNA samples

The family under investigation was ascertained from the Triangle Regional Research and Development Center, Kfar Qari’, Israel, for a comprehensive genetic testing. For candidate gene screening, further patients diagnosed with IIN with or without foveal hypoplasia were recruited at several collaborating centers (Cleveland Clinic, Cole Eye Institute, Cleveland, OH, USA, n=6; Department of Ophthalmology, Justus-Liebig-University Giessen, Giessen, Germany, n=10; Department of Pediatrics and Medical Genetics, Ghent University, Ghent, Belgium, n=53; Department of Neuroscience, Psychology and Behaviour, University of Leicester, Leicester, UK, n=156) specialized in inherited eye diseases and infantile nystagmus. The study was performed according to the tenets of the Declaration of Helsinki, approved by the local ethics committee, and written informed consent was obtained from each study participant or from the parents in case of minor study subjects.

Genomic DNA was extracted from venous blood samples or oral mucosa cells following standard procedures.

Clinical examination

Patients underwent comprehensive ophthalmological assessment including slit lamp biomicroscopy, fundoscopy, colour vision testing (Ishihara pseudoisochromatic plates), optical coherence tomography (OCT) and electrodiagnostic testing (full-field electroretinography and flash visual evoked potential (VEP). Visual acuity was assessed with habitual refractive correction using a Lea chart. Binocular visual acuity was examined and then each eye was tested independently with the opposite eye being occluded. OCT was performed using the Topcon 3D OCT-2000 (Topcon, Tokyo, Japan). Three-dimensional cube scans centered at the fovea were obtained. The location of the foveal scan was determined based on features of foveal specialization as previously described (Thomas et al., 2011b). Magnetic resonance imaging of the brain was carried out to assess cerebellar integrity. Eye movement video recordings were done to determine the predominant nystagmus waveform and analyzed by subjective clinical judgement of an experienced neuro-ophthalmologist (YC). General and neurological examinations were carried out in addition to assessment of developmental milestones.

Exome sequencing in family TR 16

Whole exome sequencing was carried out using genomic DNA from patient IV:5 of family TR 16. Exonic regions were enriched applying Agilent’s SureSelectXT Human All Exon V5 system and sequenced on a NextSeq500 (300 cycle chemistry) platform (Illumina, San Diego, USA) in paired-end mode according to the manufacturer’s protocol. The bioinformatics analysis was done using an in-house analysis pipeline based on tools that are freely available. Variants with a minor allele frequency of less than 0.01 were kept; synonymous variants and variants with > 20 observations in our in-house database were removed. Segregation analysis of the identified premature stop variant in the AHR gene was performed by PCR and Sanger sequencing using internal primers for exon 10 (forward: 5’-ACGAGAATGGCTTCAACACC-3’; reverse: 5’-GTTCATGAGCAGCGAAGTCA-3’).

Candidate gene screening by Sanger sequencing

All coding exons and flanking intronic and untranslated region sequences of the AHR gene (RefSeq: NM_001621.4, GRCh38/hg38) were amplified from genomic DNA by PCR using standard PCR amplification protocols (Supplementary Table 1). PCR fragments were purified by treatment with ExoSAP-IT (GE Healthcare, Freiburg, Germany) and sequenced with BigDye Termination chemistry (Applied Biosystems, Darmstadt, Germany). The products were run on a capillary sequencer (ABI 3130, Applied Biosystems) and sequencing data were analyzed with the Sequencing Analysis software (version 5.2, Applied Biosystems) and sequence trace alignment software (SeqMan, DNASTAR, Madison, USA).

Candidate gene screening by NGS approaches

We carried out AHR mutation screening in 53 unrelated families (53 index patients, 35 males, 18 females) from a Belgian cohort with IIN and no identified coding mutations or copy number variations in FRMD7 and GPR143. Genomic DNA of the patients was amplified for regions covering the entire coding sequence and splice junctions of AHR (NM_001621.4) using targeted next generation sequencing (NGS) on an Illumina MiSeq platform as previously described (De Leeneer et al., 2015).

Another 156 patients with infantile nystagmus with normal electroretinograms and VEPs were screened by performing chip-based targeted NGS covering all know nystagmus and retinal dystrophy genes (Thomas et al., 2017) as well as the candidate gene AHR.

Results

Clinical phenotype of the family under investigation

The pedigree of the family is shown in Fig. 1. The three affected siblings (Patients IV:5, IV;6 and IV:7) were diagnosed with infantile nystagmus at birth. They were born to a consanguineous Christian-Arabic couple (Subjects III:4 and III:5; third degree cousins). All three children (1, 6 and 11 years old) were born at term after normal pregnancy and delivery. There was no significant medical or family history. Investigations which included MRI, EEG, metabolic workup and electroretinograms were normal in the two older affected subjects (IV:5 and IV:6). Both general and neurological examinations were normal with attainment of normal developmental milestones and no evidence of any dysmorphic features. Of minor note, all three affected siblings showed head circumferences at the 90^th−95^th percentile.

Figure 1. Pedigree of family TR 16 (NYS 12) and electropherograms of the identified AHR mutation.

Whole-exome sequencing was performed for individual IV:6 and revealed a homozygous nonsense mutation (c.1861C>T;p.Q621*) in the AHR gene. Segregation analysis was performed with all available DNA samples (genotypes underneath; plus sign = wild-type allele). Representative sequences are shown underneath. WT, wild-type; Het, heterozygous; Hom, homozygous.

The clinical features are summarized in the Supplementary Table 2. Ophthalmic examination revealed reduced visual acuity, nystagmus and foveal hypoplasia. Colour vision was normal. All three siblings had a partially accommodative esotropia with anomalous head postures noted in IV:5 (right head turn) and IV:6 (chin up). Both older siblings (Patients IV:5 and IV:6) underwent strabismus surgery at a younger age. Fundoscopic examination revealed absent foveal light reflexes in all three patients, while the anterior segment was unremarkable. Specifically, no iris transillumination defects were noted. The nystagmus was conjugate and horizontal with significant intrafamilial variability (Supplementary Material, videos 1-3). Patient IV:5 had horizontal pendular nystagmus in primary position, while patient IV:6 had right beating jerk nystagmus with comparatively higher intensity in primary position. The youngest affected patient (Patient IV:7) had horizontal conjugate pendular nystagmus with large amplitude and low frequency. VEP (performed in IV:5) revealed a prolonged peak time (latency) for both eyes. OCT was obtained in two (Patients IV:5 and IV:6) of the three affected patients. Both individuals had grade 3 foveal hypoplasia (Thomas et al., 2011b), characterized by (1) lack of a foveal pit (fovea plana), (2) lack of outer segment lengthening, (3) persistence of inner retinal layers at the location of the putative fovea, and (4) some outer nuclear layer widening (Fig. 2).

Figure 2. Optical coherence tomogram through the fovea in individuals IV:5 and IV:6.

Features consistent with grade 3 foveal hypoplasia are seen. These include: lack of a foveal pit (1), incursion of inner retinal layers (2), outer nuclear layer widening (3) and lack of outer segment lengthening (4).

Genetic analysis

WES of Patient IV:5 revealed a homozygous nonsense mutation (c.1861C>T;p.Q621*) in AHR. AHR maps to chromosome 7p21.1 and is 47.53 kb in length comprising 11 exons, which encode a protein of 848 residues (NM_001621.4). The identified AHR mutation may result in an abnormally shortened protein lacking a significant proportion of the Q-rich subdomain required for transcriptional activation of target genes. However, it is likely that at least a major fraction of mutant transcripts will undergo nonsense-mediated mRNA decay. The mutation showed concordant segregation with the phenotype in the larger family compatible with an autosomal recessive mode of inheritance (Fig. 1). By applying the guidelines for variant interpretation (Richards et al., 2015) the c.1861C>T;p.Q621* mutation was scored to be pathogenic (class 5; Supplementary Table 3). Further, the mutation is not listed in the Genome Aggregation Database (gnomAD) and only a single nonsense variant (c.499C>T;p.R167*) observed at an ultra-low frequency (4.078e-6; 1 of 245,206 analyzed chromosomes; no homozygotes) is listed in gnomAD.

We have analyzed 16 nystagmus patients that were recruited at the Cleveland Clinic and Justus-Liebig-University Giessen for potential mutations in AHR by performing PCR and Sanger sequencing of all coding exons. Further 209 cases were screened in collaborating centers by targeted NGS technologies. However, this candidate gene screening of AHR in 225 patients with (idiopathic) infantile nystagmus presumably not following an X-linked trait did not reveal a second case. Patients with nystagmus attributed to mutations in AHR therefore seem to be extremely rare.

Discussion

In this study, we have shown that a homozygous loss of function (LOF) mutation in the aryl hydrocarbon receptor (AHR) gene is associated with autosomal recessively inherited foveal hypoplasia with infantile nystagmus. Although intrafamilial variability of the nystagmus characteristics was seen in our patients, it could represent the natural evolution of infantile nystagmus as described previously (Gottlob 1997; Hertle and Dell'Osso, 1999).

Foveal hypoplasia typically occurs in hereditary developmental retinal disorders such as albinism, PAX6-related phenotypes and achromatopsia, with infantile nystagmus being a common feature of these conditions (Azuma et al., 1996; Thomas et al., 2011b). By ultrahigh-resolution OCT imaging it has recently been shown that patients primarily diagnosed with IIN can have retinal and optic nerve changes, as FRMD7 mutations can present as isolated foveal hypoplasia (Thomas et al., 2014). Therefore, abnormal afferent system development in association with FRMD7 mutations is an important etiological factor in the development of infantile nystagmus (Thomas et al., 2014; Yonehara et al., 2016). Similarly, we hypothesize that impaired retinal development associated with the AHR mutation leads to development of infantile nystagmus.

Interestingly, constitutive absence of Ahr in adult mice (Ahr^-/-) leads to abnormal eye movements in the form of a spontaneous pendular horizontal nystagmus (Chevallier et al., 2013). Its origin was shown to be neither vestibular nor cerebellar, but OKR was less effective in Ahr^-/- mice, suggesting a deficit in the visual or visuo-motor circuitry. It was further shown by (Chevallier et al., 2013) that Ahr is expressed in retinal ganglion cells during embryonic development. The phenotype of Ahr^-/- mice shares many features with IIN and albino patients: the nystagmus appeared early in development (<4 weeks) with conjugate and purely horizontal oscillations. It increased with fixation attempt and was greater in light than in the dark. With age, the nystagmus observed in Ahr^-/- mice evolved from pendular to jerk-like waveforms following a similar natural history as seen in humans with infantile nystagmus (Chevallier et al., 2013). However, it is important to recognize the difference between mice and human retinae, the lack of a fovea, which raises the interesting possibility that abnormal retinal circuitry could underlie this disorder.

Accordingly, the same group (Juricek et al., 2017) demonstrate that the infantile nystagmus in Ahr^-/- mice developed together with deficits in the early processing of visual information. Ahr^-/- mice were shown to have normal projections of the optic nerves to the thalamus, but the recorded VEPs from the primary visual cortex showed decreased amplitudes compared to wild-type animals. While this indicated a deficit in the integration of visual information, the conduction velocity was shown to be preserved in Ahr^-/- mice. In addition, the presentation of different visual stimuli showed a decrease in the sensitivity to contrast, but not in the visual acuity in the knockout animals. Further, (Juricek et al., 2017) showed that Ahr deficiency led to an altered optic nerve myelin sheath. Ahr therefore is proposed to play a role in the myelination of the visual and optomotor pathways, and its disruption as a putative cause for infantile nystagmus (Juricek et al., 2017).

AHR acts as a ligand-activated transcription factor. A transactivation domain located at the carboxyl terminus was shown to be composed of three subdomains (the acidic subdomain, residues 500-600; the Q-rich subdomain, residues 600-713; and the P/S/T-rich subdomain, residues 713-848) and to interact with several transcriptional co-activators (Rowlands et al., 1996). Deletion analysis of the Q-rich subdomain revealed a critical 23 amino acid stretch between residues 666 and 688 of human AHR, which is required for transcriptional activation of target genes (Kumar et al., 2001). A schematic structure of the human AHR protein is shown in Supplementary Fig. 1. The premature stop mutation at amino acid position glutamine-621 in our family TR 16 formally results in a protein lacking a significant proportion of the Q-rich domain most likely leading to complete absence of the trans-activating activity. Recently, valine-647 and an adjacent motif that comprises residues 650-661 within the Q-rich domain were demonstrated to regulate intracellular trafficking of AHR in context of both nucleocytoplasmic shuttling and receptor activation (Tkachenko et al., 2016). However, we rather propose that the premature stop codon induces nonsense-mediated mRNA decay (Frischmeyer and Dietz 1999), therefore representing a null (true LOF) allele.

Considering the observed (n=1) and expected (n=29.7) LOF variant counts from the Exome Aggregation Consortium (http://exac.broadinstitute.org/) database for AHR, calculation of a pLI score (the probability of being LOF-intolerant) of 1 indicates AHR be to extremely intolerant of LOF variants (Lek et al., 2016), indicating a strong natural selection against LOF variants. Recently a homozygous splice mutation in AHR has been described to be associated with retinitis pigmentosa (RP) (Zhou et al., 2018). This was identified in two individuals in the same family. Interestingly, the variant list from the whole exome sequencing of the index patient also revealed a homozygous BBS2 mutation (c.G209A:p.S70N), which has previously been described to be pathogenic (Katsanis et al., 2001;Zhagloul et al., 2010). We hypothesize that the BBS2 mutation is causative of the RP phenotype. In our family, there was no evidence of pigmentary changes on fundoscopy, the full field ERG was normal and the OCT only showed foveal hypoplasia with no outer retinal changes. Thus we conclude that the AHR homozygous stop mutation is causative of foveal hypoplasia and infantile nystagmus”.

In summary, the data generated in a knockout mouse model system together with the observations in our patients being homozygous for a LOF mutation indicate that AHR deficiency causes foveal hypoplasia with infantile nystagmus in humans.

Abbreviations

AHR

aryl hydrocarbon receptor

gnomAD

Genome Aggregation Database

IIN

idiopathic infantile nystagmus

LOF

loss of function

NGS

next generation sequencing

OCT

optical coherence tomography

OKR

optokinetic response

VEP

visual evoked potential

WES

whole exome sequencing