GUCY2D-Associated Leber Congenital Amaurosis: A Retrospective Natural History Study in Preparation for Trials of Novel Therapies


To describe the natural history of Leber congenital amaurosis (LCA) associated with GUCY2D variants ( GUCY2D -LCA) in a cohort of children and adults, in preparation for trials of novel therapies.


Retrospective case series.


Participants : Patients with GUCY2D -LCA at a single referral center. Procedures : Review of clinical notes, retinal imaging including fundus autofluorescence (FAF) and optical coherence tomography (OCT), electroretinography (ERG), and molecular genetic testing. Main Outcome Measures : Demographic data, symptoms at presentation, visual acuity, evidence of progression, OCT and FAF findings, ERG assessment, and molecular genetics.


Twenty-one subjects with GUCY2D -LCA were included, with a mean follow-up ± standard deviation (SD) of 10 ± 11.85 years. Marked reduction in visual acuity (VA) and nystagmus was documented in all patients within the first 3 years of life. Fifty-seven percent (n = 12) exhibited photophobia and 38% (n = 8) had nyctalopia. VA was worse than hand motion in 71% of the patients (n = 15). Longitudinal assessment of VA showed stability in all patients, except 1 patient who experienced deterioration over a follow-up of 44 years. Hyperopia was reported in 13 of the 17 subjects (71%) with available refraction data. Eighteen subjects had either normal fundus appearance (n = 14) or a blond fundus (n = 3), while only 4 of the eldest subjects had mild retinal pigment epithelium (RPE) atrophy (mean, 49 years; range 40-54 years). OCT data were available for 11 subjects and 4 different grades of ellipsoid zone (EZ) integrity were identified: (1) continuous/intact EZ (n = 6), (2) focally disrupted EZ (n = 2), (3) focally disrupted with RPE changes (n = 2), and (4) diffuse EZ disruption with RPE changes (n = 1). All examined subjects had stable OCT findings over the long follow-up period. Full-field ERGs showed evidence of a severe cone-rod dystrophy in 5 of 6 patients and undetectable ERGs in 1 subject. Novel genotype-phenotype correlations are also reported.


GUCY2D -LCA is a severe early-onset retinal dystrophy associated with very poor VA from birth. Despite the severely affected photoreceptor function, the relatively preserved photoreceptor structure based on EZ integrity until late in the disease in the majority of subjects suggests a wide therapeutic window for gene therapy trials.


  • GUCY2D -associated Leber congenital amaurosis is a severe early-onset retinal dystrophy.

  • There is severe cone and rod dysfunction but with preserved photoreceptor structure evident on optical coherence tomography.

  • Stable natural history suggests a wide therapeutic window for intervention.

Leber congenital amaurosis (LCA) represents a leading cause of autosomal recessive blindness in children worldwide, affecting between 1 in 30,000 to 1 in 81,000 newborns annually. Clinically, LCA is characterized by severe visual impairment at birth or within the first months of life. Affected individuals commonly exhibit nystagmus, the oculodigital sign (eye poking), and extinguished or severely abnormal electroretinography (ERG). , , LCA accounts for the most severe form of inherited retinal disorders, and both clinically and genetically overlaps with early-onset severe retinal dystrophy (EOSRD), which comprises milder phenotypes. , EOSRD presents after infancy and before the age of 5 years. Affected individuals usually have better residual visual function than in LCA and minimal ERG signals. , LCA/EOSRD is associated with disease-causing variants in 26 genes to date. , It has been reported that certain genes are more likely to be associated with LCA, such as GUCY2D , CEP290 , NMNAT1 , and AIPL1 , while variants in other genes more frequently cause EOSRD, including RPE65 and RDH12 . The genetic variability of LCA and the rarity of the condition make detailed phenotyping in a substantial molecularly confirmed cohort of patients challenging.

GUCY2D variants commonly cause LCA/EOSRD, accounting for 10%-20% of all cases. Different sequence variants in GUCY2D are common causes of autosomal dominant (AD) cone dystrophy (COD) and cone-rod dystrophy (CORD). , GUCY2D encodes the photoreceptor enzyme guanylate cyclase 2D (GC-E), which synthesizes the intracellular messenger of photoreceptor excitation, cGMP, and is regulated by intracellular Ca 2+ -sensor proteins named guanylate cyclase–activating proteins (GCAPs). To date there are 144 identified variants in GUCY2D , with the majority reported to cause LCA/EOSRD (127 variants, 88%) and only 13 reported to cause AD-COD or AD-CORD. The AD-COD/CORD variants are all located in exon 13 (around the amino acid position 838) affecting the GC-E dimerization domain. In contrast, the variants reported to cause LCA do not have a localization hot spot but are scattered along the full length of the gene. The biochemical effect of many of the variants has been described both in vitro and in animal models. LCA/EOSRD-causing variants usually show either reduced ability or complete inability to synthesize the intracellular messenger cGMP. Moreover, some LCA/EOSRD-causing variants result in misfolding and consequent degradation of the protein in the endoplasmic reticulum. In contrast, COD/CORD-causing variants are functional but cause a shift in Ca 2+ sensitivity. Despite the rather well-characterized genetic background of GUCY2D -LCA/EOSRD, the number of detailed phenotyping studies is limited.

Previous phenotyping studies identified evidence of preserved photoreceptor structure, in contrast to the severely affected functional findings of GUCY2D -LCA/EOSRD. Reduced visual acuity is a life-long source of morbidity for patients with LCA/EOSRD, with visual impairment having been significantly associated with increased risk of mortality. Gene-based approaches to therapy are used increasingly in clinical trials, with the first Food and Drug Administration–approved gene therapy for RPE65 -LCA now available. Gene replacement therapy for GUCY2D -LCA/EOSRD has been investigated in animal studies, with considerable reported therapeutic success, using a range of vectors including recombinant adeno-associated virus serotype 2/8 (AAV2/8), adeno-associated virus serotype 5 (AAV5), and HIV1-based lentiviral vector. , , Aguirre and associates report an intact postgeniculate white matter pathway in subjects with GUCY2D -LCA/EOSRD, which provides further encouragement for the prospect of recovery of visual function with gene augmentation therapy. Jacobson and associates investigated potential outcome measures such as chromatic full-field sensitivity testing and optical coherence tomography (OCT), used to assess photoreceptor function and structure, respectively, concluding that any change in the dissociation between structure and function after intervention may serve as evidence of efficacy. Despite the planned and upcoming trials of novel therapies, a lack of longitudinal data, particularly for OCT and fundus autofluorescence (FAF) imaging, is apparent in the literature. ,

Herein, we present a retrospective natural history study in a large cohort of adults and children with variants in GUCY2D , which provides a detailed description of the genotypic and phenotypic features, with a long duration of follow-up. This information is of particular importance for improving genetic counseling and advice on prognosis, and provides a crucial step toward the design of a therapeutic clinical trial in GUCY2D -LCA/EOSRD, as well as identifying a cohort of molecularly confirmed patients who may participate in such future trials.


This retrospective study protocol adhered to the tenets of the Declaration of Helsinki and received approval from the Moorfields Eye Hospital ethics committee. Informed consent was obtained from all adult subjects, whereas informed consent and assent were obtained from parents and children, respectively.

Patient Identification

Patients were identified from the genetic retina clinics at a single tertiary referral center (Moorfields Eye Hospital, London, UK). In total, 22 patients with likely disease-causing variants in GUCY2D were ascertained for detailed phenotyping.

Molecular Diagnosis

Genomic DNA was isolated from peripheral blood lymphocytes (Gentra Puregene Blood Extraction Kit; Qiagen, Venlo, Netherlands). A combination of Sanger sequencing and next-generation sequencing, including a panel of retinal dystrophy genes, whole exome sequencing (WES), and whole genome sequencing, was used to identify variants in GUCY2D . All patients with 1 allele identified from WES were subjected to Sanger sequencing of the first coding exon of the gene to check for a second allele, owing to the lack of coverage of the GUCY2D first coding exon by WES. Mutation nomenclature was assigned in accordance with GenBank accession number NM_000180 .

Minor allele frequency for the identified variants in the general population was assessed in the Genome Aggregation Database (gnomAD) datasets ( ; accessed on December 12, 2018) ( Supplementary Table 1 ; Supplemental Material available at ). Prediction of pathogenicity was assessed using the predictive algorithms of Polymorphism Phenotyping v2 (PolyPhen2,; accessed on December 12, 2018) and Sorting Intolerant from Tolerant (SIFT,; accessed on December 12, 2018) ( Supplementary Table 1 ). Where relevant, disruption of potential splice sites was assessed using Human Splicing Finder (; accessed on December 12, 2018) ( Supplementary Table 1 ). Variants likely to affect function were assessed for segregation in available family members.

Clinical Assessment

All available clinical notes were reviewed. Visual acuity (VA), refraction, funduscopy, and slit-lamp biomicroscopy findings were extracted. All patients were seen by medical retina specialists in the genetics/medical retina clinic. Age of onset is defined as the age at which the family first noticed any symptoms and sought medical care. Age seen is the age at which the patient was first seen at our referral center.

Best-corrected logMAR visual acuity (BCVA) was assessed, monocularly, with an Early Treatment Diabetic Retinopathy Study chart. Patients were read standardized instructions. Precision Vision lightboxes were used (Precision Vision, Woodstock, Illinois, USA) and were illuminated with 2 cool daylight 20 watt fluorescent tubes, with the overhead lights turned off, so that no more than 161.4 lux should fall at the center of the chart. LogMAR values were calculated from the number of letters read, where the higher the logMAR value, the worse the BCVA. Subjective and objective refraction was undertaken by a specialist optometrist for both adults and children.

Electrophysiological Assessment

Full-field ERG and pattern electroretinography were performed using gold foil corneal electrodes and incorporated the International Society for Clinical Electrophysiology of Vision (ISCEV) standards, , except in infants and young children, who underwent ERG testing with skin electrodes without mydriasis using modified protocols.

Retinal Imaging

Color fundus imaging was obtained by conventional 35-degree fundus imaging (Topcon Great Britain Ltd, Berkshire, UK) or ultra-widefield (200-degree) confocal scanning laser imaging (Optos plc, Dunfermline, UK). FAF imaging was performed using 30-degree or 55-degree Spectralis (Heidelberg Engineering Ltd, Heidelberg, Germany), or Optos (Optos plc) imaging. Spectral-domain OCT scans (Spectralis; Heidelberg Engineering Ltd) were used to assess cross-sectional and longitudinal structural changes.



The cohort included 21 patients (female n = 11) from 19 families, with an age range at first examination in our hospital of 0-54 years. The length of follow-up ranged from 1 to 56 years (mean ± standard deviation [SD], 10 ± 11.96 years).

Molecular Genetics

Table 1 and Figure 1 summarize the molecular findings in our cohort. Two pedigrees contributed more than 1 patient (Patients P2A and P2B are siblings, as well as P8A and P8B), with the remaining 17 patients being simplex cases. All patients had 2 likely disease-causing variants in GUCY2D . Fourteen patients were compound heterozygotes and 7 patients harbored homozygous variants. The variant minor allele frequencies in the general population (gnomAD database) are reported in Supplementary Table 1 . The predicted effect of the variants identified in our cohort is summarized in Table 2 . Out of a total of 29 rare variants identified, 14 have not been previously reported in retinal dystrophies. The variants identified in our cohort were scattered throughout the full length of the gene, from exon 2 to exon 17. The majority of the variants (n = 15) are missense, in agreement with previous studies. Nine small insertions or deletions that cause a frameshift or in-frame deletion and a small number of splice site (n = 3) and premature stop codon (n = 2) variants were identified.

Table 1

Variants in the GUCY2D Cohort

P Family ID Con. Hom. variant Variant 1 Protein Effect Variant Type PUV Variant 2 Protein Effect Variant Type PUV
P1 GC12356 c.307G>A p.(Glu103Lys) Missense c.238_252delGCCGCCGCCCGCCTG p.(Ala80_Leu84del) In-frame deletion
P2A GC19319 c.307G>A p.(Glu103Lys) Missense c.1762C>T p.(Arg 588Trp) Missense
P2B GC19319 c.307G>A p.(Glu103Lys) Missense c.1762C>T p.(Arg 588Trp) Missense
P3 GC1015 c.380C>T p.(Pro127Leu) Missense c.901_908delCTTCGCAG p.(Leu301Glyfs*15) Frameshift
P4 GC17851 c.553G>C p.(Ala185Pro) Missense c.721+5G>T Splicing
P5 GC19719 c.307G>A p.(Glu103Lys) Missense c.2872A>C p.(Ser958Arg) Missense
P6 GC3264 c.652delA p.(Met218Trpfs*13) Frameshift
P7 GC22697 c.2837C>A p.(Ala946Glu) Missense c.2969G>T p.(Gly990Val) Missense
P8A GC19606 c.3056A>C p.(His1019Pro) Missense
P8B GC19606 c.3056A>C p.(His1019Pro) Missense
P9 GC16211 c.3098_3099insCGTGCTCT p.(Gly1034Valfs*15) Frameshift
P10 GC16935 c.1343C>A p.(Ser448*) Nonsense c.1958delG p.(Gly653Glufs*2) Frameshift
P11 GC16929 c.2302C>T p.(Arg768Trp) Missense c.1978C>T p.(Arg660*) Nonsense
P12 GC18677 c.2384G>A p.(Arg795Gln) Missense c.1211T>C p.(Leu404Pro) Missense
P13 GC1036 c.307G>A p.(Glu103Lys) Missense c.2849C>T p.(Ala950Val) Missense
P14 GC17418 c.c.2120T>C p.(Leu707Pro) Missense
P15 GC24539 c.3044-2A>G Splicing
P16 GC18674 c.2944+1delG Splicing c.2858C>T p.(Ser953Leu) Missense
P17 GC24284 c.1694T>C p.(Phe565Ser) missense c.2633_2636delAAGT p.(Gln878Argfs*17) Frameshift
P18 GC17645 c.129_134delTCTGCT p.(Leu44_Leu45del) In-frame deletion
P19 GC17984 c.2944delG p.(Gly982Valfs*39) Frameshift c.2291delC p.(Pro764Leufs*20) Frameshift

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Mar 14, 2020 | Posted by in OPHTHALMOLOGY | Comments Off on GUCY2D-Associated Leber Congenital Amaurosis: A Retrospective Natural History Study in Preparation for Trials of Novel Therapies

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