Developing a diagnostic service for Stargardt disease – a feasibility study

Emily Packham

Oxford Regional Molecular Genetics Laboratory

Introduction

Inherited eye disorders Services currently available for some of the

AD and X-linked conditions Limited services currently for AR conditions

(Asper Ophthalmics offer commercial genotyping of some genes)

Why? Significant clinical overlap Genetically heterogeneous

Stargardt disease may be feasible

Stargardt disease

Autosomal recessive juvenile macular degeneration Prevalence of 1 in 10,000

Stargardt disease/Fundus Flavimaculatus (STGD/FF)

Characterised by yellow-white flecks and atrophy

STGD FF

Symptoms

Age of onset varies from early childhood to twenties

Early stages – difficulty reading, watching TV, missing patches in vision, photophobia, slow dark adaption

Later stages – always disturbance of central vision and sometimes: peripheral disturbance, increasing photophobia or problems with dark vision

Diagnosis

Clinical diagnosis Sophisticated imaging but dependent on tests

performed, experience and stage of disorder Late stage shows clinical overlap

Genetic diagnosis Support or confirm diagnosis Provide prognosis information Aid genetic counselling Therapeutic intervention

Genetics of Stargardts disease

ABCA4 (1p13-p22) 50 exons (6819bp ORF)

Highly polymorphic No mutation hotspots 500+ variants identified

Most common seen in ~ <10% of patients Many missense variants

ABCA4 protein (ABCR / Rim)

ATP-binding cassette (ABC) transporter superfamily Transmembrane proteins involved in

transportation of compounds across cell membranes

2273 amino acid protein expressed in cones and rods

ABCA4 function and disease pathology

Actively ‘flips’ Ret-PE across disc rim membrane Enables retinal signalling to continue

Loss-of-function mutations Loss of/reduction in ABCA4 function

results in accumulation of toxic lipofuscin deposits

Destroys retinal pigment epithelium and rod and cone cells, resulting in visual loss

ABCA4 and other retinopathies

Stargardt disease AR cone-rod dystrophy AR retinitis pigmentosa Age-related macular degeneration?

Genotype/phenotype correlation model based on residual activity of protein

Screening strategy

30 patients selected for testing

Highly polymorphic, 50 exon gene with no particular hotspots

Bi-directional sequencing Robotics approach –5 patients per batch Pathogenicity investigations performed

MLPA

Results

37 different potential pathogenic variants detected in 26 patients

13/20 patients with two or more variants had all of them classified as either

likely or highly likely Most common seen in 4 patients

No. of variants No. of patients

0 4

1 6

2 18

3 2

Results

Results

Extensive published data

MLPA normal in all 10 patients tested

Variant classification Number detected

Highly likely pathogenic 17

Likely pathogenic 10

Intermediate 10

Total 37

Feasibility

Clinical sensitivity 67% or 43% (+/- intermediate variants) Higher than literature

Different screening methods and patient selection

Clinical utility Able to interpret most variants Supports clinical diagnosis, aids counselling and

therapy Improves equity of access

What next?

Report our 30 patients Determine if variants are in trans Submit gene dossier Collaborate with BRC retinal research project

Evaluating use of high throughput sequencing to test numerous inherited retinal conditions

NHS lab BRC

Acknowledgements

Oxford Molecular Genetics Laboratory Anneke Seller Treena Cranston Tina Bedenham, Louise Williams, Kate Gibson

Oxford Clinical Genetics and BRC Andrea Nemeth

Oxford eye hospital Susan Downes