ARTICLE
Bi-allelic variants in three genes encoding distinct subunits of the
vesicular AP-5 complex cause hereditary macular dystrophy
Graphical abstract Authors
Karolina Kaminska,
Francesca Cancellieri,
Mathieu Quinodoz, ..., Susanne Kohl,
Cristina Santos, Carlo Rivolta
Correspondence
carlo.rivolta@iob.ch
We discovered genetic changes in
three genes (AP5Z1, AP5M1, and
AP5B1) that cause macular
degeneration, affecting sharp
central vision. The proteins
encoded by these genes form aKaminska et al., 2025, The American Journal of Human Genetics 112, 1–21
April 3, 2025 
 2025 The Author(s). Published by Elsevier Inc. on behalf of
American Society of Human Genetics.
https://doi.org/10.1016/j.ajhg.2025.02.015 llcomplex that helps the cell’s
recycling system clear waste and
keep retinal cells healthy. Some
affected individuals also
experience additional symptoms
beyond vision loss.
ARTICLE
Bi-allelic variants in three genes encoding distinct
subunits of the vesicular AP-5 complex
cause hereditary macular dystrophy
Karolina Kaminska,1,2 Francesca Cancellieri,1,2 Mathieu Quinodoz,1,2,3 Abigail R. Moye,1,2
Miriam Bauwens,4,5 Siying Lin,6,7,8 Lucas Janeschitz-Kriegl,1,2 Tamar Hayman,9
Pilar Barbera´n-Martı´nez,10,11 Regina Schlaeger,12 Filip Van den Broeck,4,13,14
Almudena A´vila Ferna´ndez,15,16 Lidia Ferna´ndez-Caballero,15,16 Irene Perea-Romero,15,16
Gema Garcı´a-Garcı´a,10,11,16 David Salom,16 Pascale Mazzola,17 Theresia Zuleger,17 Karin Poths,17
Tobias B. Haack,17,18 Julie Jacob,19 Sascha Vermeer,20 Fre´de´rique Terbeek,21 Nicolas Feltgen,22
Alexandre P. Moulin,23 Louisa Koutroumanou,24
(Author list continued on next page)
Summary
Inherited retinal diseases (IRDs) are a genetically heterogeneous group of Mendelian disorders that often lead to progressive vision loss
and involve approximately 300 distinct genes. Although variants in these loci account for the majority of molecular diagnoses, other
genes associated with IRD await molecular identification. In this study, we uncover bi-allelic assortments of 23 different (22 loss-of-func-
tion) variants in AP5Z1, AP5M1, and AP5B1 as independent causes of recessive IRD in members of 19 families from nine countries.
Affected individuals, regardless of their genotypes, exhibit a specific form of macular degeneration, sometimes presenting in association
with extraocular features. All three genes encode different subunits of the vesicular fifth adaptor protein (AP-5) complex, a component of
the intracellular trafficking system involved inmaintaining cellular homeostasis and ensuring the proper functioning of lysosomal path-
ways. The retinal pigment epithelium (RPE), a cellular monolayer located posteriorly to the neural retina, is characterized by intense
lysosomal and phagocytic activity. Immunostaining of RPE cells revealed a punctate pattern of AP5Z1, AP5M1, and AP5B1 staining
and co-localizationwithmarkers of late endosomes and the Golgi, suggesting a role of AP-5 in the normal physiology of this tissue. Over-
all, the identification of independently acting variants in three distinct proteins within the same macromolecular complex reveals AP-5
as having an important function in the preservation and maintenance of normal macular functions.
Introduction
Inherited retinal diseases (IRDs) are a diverse group of ge-
netic disorders that commonly cause progressive loss of
vision due to the degeneration or dysfunction of rod and
cone photoreceptors, the light-sensitive neurons of the
eye.1 Although IRDs are monogenic conditions, their ge-
netic architecture is complex and involves alterations in
1Institute of Molecular and Clinical Ophthalmology Basel (IOB), 4031 Basel, Switzerland; 2Department of Ophthalmology, University of Basel, 4031 Basel,
Switzerland; 3Department of Genetics and Genome Biology, University of Leicester, Leicester LE1 7RH, UK; 4Center for Medical Genetics, Ghent University
Hospital, 9000 Ghent, Belgium; 5Department of Biomolecular Medicine, Ghent University, 9000 Ghent, Belgium; 6Manchester Centre for Genomic Med-
icine, Saint Mary’s Hospital, Manchester University NHS Foundation Trust, Manchester M13 9WL, UK; 7Division of Evolution, Infection and Genomics,
School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9P, UK; 8NIHR Biomedical Research
Centre, Moorfields Eye Hospital and the UCL Institute of Ophthalmology, London EC1V 9EL, UK; 9Department of Ophthalmology, HadassahMedical Cen-
ter, The Hebrew University of Jerusalem, Jerusalem, Israel; 10Molecular, Cellular, and Genomic Biomedicine Group, IIS-La Fe, 46012 Valencia, Spain; 11Joint
Unit CIPF-IIS La Fe Molecular, Cellular and Genomic Biomedicine, IIS-La Fe, 46012 Valencia, Spain; 12Department of Neurology, University Hospital Basel,
4031 Basel, Switzerland; 13Department of Head & Skin, Ghent University Hospital, 9000 Ghent, Belgium; 14Department of Ophthalmology, Ghent Uni-
versity Hospital, 9000 Ghent, Belgium; 15Department of Genetics & Genomics, Instituto de Investigacio´n Sanitaria-Fundacio´n Jime´nez Dı´az University
Hospital, Universidad Auto´noma de Madrid (IIS-FJD, UAM), 28040 Madrid, Spain; 16Center for Biomedical Network Research on Rare Diseases
(CIBERER), Instituto de Salud Carlos III, 28029 Madrid, Spain; 17Institute of Medical Genetics and Applied Genomics, University of Tu¨bingen, 72076 Tu¨-
bingen, Germany; 18Centre for Rare Diseases, University of Tu¨bingen, 72072 Tu¨bingen, Germany; 19Department of Ophthalmology, Universitair Zieken-
huis Leuven (UZ Leuven), 3000 Leuven, Belgium; 20Center for Human Genetics, University Hospitals Leuven, 3000 Leuven, Belgium; 21Department of
Neurology, Noorderhart Hospital, 3900 Pelt, Belgium; 22Department of Ophthalmology, University Hospital Basel, 4031 Basel, Switzerland; 23Jules-Gonin
Eye Hospital, Fondation Asile des Aveugles, University of Lausanne, 1004 Lausanne, Switzerland; 24Medical School, University of Crete, 715 00 Heraklion,
Greece; 25Ophthalmology Department, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP, UK; 26St. Paul’s Eye Department, Royal Liverpool Univer-
sity Hospital, Liverpool L7 8XP, UK; 27Department of Eye and Vision Sciences, Institute of Life Course and Medical Sciences, University of Liverpool, Liver-
pool L7 8TX, UK; 28Department of Ophthalmology, Lund University, 223 62 Lund, Sweden; 29Department of Medical Genetics, Hospital Santa Maria, Uni-
dade Local de Sau´de de Santa Maria, 1649-035 Lisbon, Portugal; 30Medical Genetics University Clinic, Faculty of Medicine, University of Lisbon, 1649-028
Lisbon, Portugal; 31Department of Ophthalmology, Instituto de Oftalmologia Dr Gama Pinto (IOGP), 1169-019 Lisbon, Portugal; 32University Eye Hospital,
Center for Ophthalmology, University of Tu¨bingen, 72076 Tu¨bingen, Germany; 33Institute for Ophthalmic Research, Center for Ophthalmology, Univer-
sity of Tu¨bingen, 72076 Tu¨bingen, Germany; 34Division of Ophthalmology, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA; 35UCL
(Affiliations continued on next page)
The American Journal of Human Genetics 112, 1–21, April 3, 2025 1
Please cite this article in press as: Kaminska et al., Bi-allelic variants in three genes encoding distinct subunits of the vesicular AP-5 complex
cause hereditary macular dystrophy, The American Journal of Human Genetics (2025), https://doi.org/10.1016/j.ajhg.2025.02.015

 2025 The Author(s). Published by Elsevier Inc. on behalf of American Society of Human Genetics.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
genes that are usually linked to essential processes of
various retinal cells, especially the photoreceptors and cells
of the retinal pigment epithelium (RPE).
Based on the cell types that are primarily compromised
and the pattern of retinal degeneration, IRDs can be classi-
fied into several clinical categories, including retinitis pig-
mentosa (RP), cone-rod dystrophy (CRD), or macular dys-
trophy (MD). The latter primarily affects the macula, the
central region of the retina that is rich in cone photorecep-
tors and is responsible for high-acuity vision, required for
visual tasks such as reading, watching movies, or recog-
nizing faces. Individuals with MD typically experience
initial symptoms such as central vision loss, photophobia,
metamorphopsia (visual distortion), or scotomas (blind
spots).2 Out of the
300 genes associated with IRDs and re-
ported in the RetNet database (http://retnet.org, as of
August 2024), fewer than 20 have been linked to MD and
only four to recessive MD.
The inheritance patterns of IRDs reflect their genetic
complexity and include autosomal recessive (the most
prevalent form), autosomal dominant, as well as X-linked
and mitochondrial inheritance.3 Understanding the ge-
netic etiology of IRD is crucial for the accurate diagnosis
of affected individuals, for the genetic counseling of fam-
ilies, and for the development of future targeted therapies.
The advent of next-generation sequencing technologies
and the development of novel in silico bioinformatics tools
have recently provided significant advancements in the
field of IRD molecular genetics.4 Despite this substantial
progress, a considerable proportion of individuals with
IRDs remain without a definitive genetic diagnosis, the
current molecular diagnostic rate varying between 50%
and 80%, mostly as a function of the population stud-
ied.3–9 This missing heritability can be attributed to several
factors, including technical limitations of current methods
(such as the detection of deep intronic or structural vari-
ants),10 challenges in prioritizing certain DNA changes
(such as synonymous and non-coding variants), and the
existence of still undiscovered disease-associated genes
and pathways.11–14
Lysosomal storage diseases (LSDs) result fromMendelian
variants in more than 50 different genes and are ultimately
linked to the impairment of lysosomal function and the
abnormal degradation of cellular waste products, which,
in turn, lead to the progressive accumulation of metabolic
by-products and subsequent cellular damage.15 Individuals
with LSDs typically manifest multi-organ signs and symp-
toms such as developmental delay, organomegaly, central
nervous system deficits, and, in some instances, retinop-
athy.15 Some genes implicated in LSDs, such as CLN3
(MIM: 607042),16 HGSNAT (MIM: 610453),17 and MFSD8
(MIM: 611124)18,19 have also been associated with non-
syndromic IRDs, where the buildup of autofluorescent
waste material impairs normal cellular function and con-
tributes to retinal degeneration.20
The fifth adaptor protein (AP-5) complex plays a crucial
role in intracellular trafficking, and, in particular, in the sort-
ing and transport of proteins within the late endosome-
to-Golgi retrieval network.21 This process is essential for
maintaining cellular homeostasis and ensuring the proper
functioning of lysosomal pathways.21 Moreover, the AP-5
complex is thought to be involved in the recovery of lyso-
somes from endolysosomes, a fundamental process for the
maintenance of the balance between different stages of en-
dosomal and lysosomal maturation.22 AP-5 is composed of
four subunits, zeta, mu, beta, and sigma, encoded by the
genes AP5Z1 (MIM: 613653), AP5M1 (MIM: 614368),
AP5B1 (MIM: 614367), and AP5S1 (MIM: 614824), respec-
tively.23 Notably, pathogenic variants in only one of them,
AP5Z1, have been associated with recessive hereditary spas-
tic paraplegia, type 48 (SPG48, MIM: 613647),24 a neurolog-
ical disorder characterized byprogressiveweakness and spas-
ticity of the lower limbs. This condition can be classified as a
form of LSD due to the accumulation of aberrant endolyso-
somes in cells lacking functional AP-5.25 However, the spe-
cific functionandpathophysiologyof theothercomponents
of the AP-5 complex remain elusive.
In this study, we identify bi-allelic variants in genes en-
coding three of the four subunits of the AP-5 complex
(AP5Z1, AP5M1, and AP5B1) as a previously unreported
George Papadakis,24 Andrew C. Browning,25 Savita Madhusudhan,26,27 Lotta Gra¨nse,28 Eyal Banin,9
Ana Berta Sousa,29,30 Luisa Coutinho Santos,31 Laura Kuehlewein,32 Pietro De Angeli,33
Bart P. Leroy,4,13,14,34 Omar A. Mahroo,8,35,36 Fay Sedgwick,37 James Eden,37 Maximilian Pfau,1,22
Sten Andre´asson,28 Hendrik P.N. Scholl,38,39,40 Carmen Ayuso,15,16 Jose´ M. Milla´n,10,11,16,41 Dror Sharon,9
Miltiadis K. Tsilimbaris,24 Veronika Vaclavik,23 Hoai V. Tran,23,42 Tamar Ben-Yosef,43 Elfride De Baere,4,5
Andrew R. Webster,8,35 Gavin Arno,8,35,44 Panagiotis I. Sergouniotis,6,7,45 Susanne Kohl,33
Cristina Santos,31,46 and Carlo Rivolta1,2,3,*
Institute of Ophthalmology, University College London, London EC1V 9EL, UK; 36Department of Ophthalmology, St Thomas’ Hospital, London SE1 7EH,
UK; 37Eye Team, North West Genomic Laboratory Hub, St Mary’s Hospital, Manchester M13 9WL, UK; 38Department of Clinical Pharmacology, Medical
University of Vienna, 1090 Vienna, Austria; 39Pallas Kliniken AG, Pallas Klinik Zu¨rich, 8005 Zu¨rich, Switzerland; 40European Vision Institute, 4056 Basel,
Switzerland; 41University and Polytechnic La Fe Hospital of Valencia, 46026 Valencia, Spain; 42Centre for Gene Therapy and Regenerative Medicine, King’s
College London, LondonWC2R 2LS, UK; 43The Ruth & Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel;
44Division of Research, Greenwood Genetic Center, Greenwood, SC 29646, USA; 45Manchester Royal Eye Hospital, Manchester University NHS Foundation
Trust, Manchester M13 9WL, UK; 46iNOVA4Health, NOVA Medical School, Faculdade de Cieˆncias Me´dicas, NMS, FCM, Universidade NOVA de Lisboa,
1099-085 Lisbon, Portugal
*Correspondence: carlo.rivolta@iob.ch
https://doi.org/10.1016/j.ajhg.2025.02.015.
2 The American Journal of Human Genetics 112, 1–21, April 3, 2025
Please cite this article in press as: Kaminska et al., Bi-allelic variants in three genes encoding distinct subunits of the vesicular AP-5 complex
cause hereditary macular dystrophy, The American Journal of Human Genetics (2025), https://doi.org/10.1016/j.ajhg.2025.02.015
cause of IRD, with or without extraocular features, in 22
subjects from 19 unrelated families from nine different
countries. All affected individuals exhibited a specific
form of MD, which we propose to classify as a distinct sub-
type of IRD: lysosomal macular dystrophy.
Material and methods
Families and DNA samples
This study adhered to the tenets of the Declaration of Helsinki and
was approved by the Ethics Committees of the respective Institu-
tions (Comissa˜o de E´tica para a Sau´de do Instituto de Oftalmologia
Dr. Gama Pinto, Cantonal Committee of Canton Vaud for
Research Activities on Human Subjects, Ethikkommission Nordw-
est- und Zentralschweiz, Ethics Board of theMedical Faculty of the
University Tu¨bingen, Ethical Committee for Medical Research at
Lund University, Ethic Committee of the Hadassah Medical Cen-
ter, Research Ethics Committee of the Fundacio´n Jime´nez Dı´az
Hospital, Comite´ de E´tica del Instituto de Investigacio´n Sanitaria
La Fe, Commissie voor Medische Ethiek UZ Gent, London – Cam-
den & Kings Cross Research Ethics Committee, Wales REC5, the
North West of England Research Ethics Committee, and Ethics
Committee of Technion – Israel Institute of Technology). Written
informed consent was obtained from all individuals or their legal
guardians prior to their inclusion in this study. All subjects under-
went ophthalmological evaluation. Available records including
clinical description of slit-lamp examination, best-corrected visual
acuity, perimetry, full-field electroretinography (ERG) and multi-
modal fundus imaging with retinography, autofluorescence
(FAF), optical coherence tomography (OCT), and fluorescein angi-
ography were analyzed.
DNA was obtained from whole-blood or saliva samples.
Whole-exome sequencing, whole-genome sequencing,
and data analysis
Whole-exome sequencing (WES) was performed according to pro-
tocols that were specific to each of the participating Institutions
but had nonetheless a common structure, described in detail
previously.8,26–28
Whole-genome sequencing (WGS) was performed either as part
of the Genomics England 100,000 Genomes project or the UK
NHS Genomic Medicine Service.29,30 Bioinformatics analysis and
interpretation of results were initially performed using a clinical
pipeline focusing on protein-altering variants within the R32
PanelApp Retinal Disorders gene panel.31 Additional WGS was
done at the Institute for Medical Genetics and Applied Genomics
at the University Hospital Tu¨bingen, Germany, as described
previously.9
All variants were validated using VariantValidator,32 described
in accordance with the Human Genome Variation Society
(HGVS) nomenclature,33 and classified using American College
of Medical Genetics and Genomics (ACMG) criteria.34,35 Fre-
quency of DNA variants in the general population was down-
loaded from the gnomAD database (v.2.1.1).36 We refrained from
using later versions of this database for variant filtering, since
they may contain genotypes from individuals with Mendelian
conditions (https://clinicalgenome.org/docs/clingen-guidance-to-
vceps-regarding-the-use-of-gnomad-v4, v.2.0). Homozygous calls
were confirmed to be bi-allelic by the direct analysis of BAM files,
IGV, and/or OFF-PEAK.37,38
Structural large alterations were detected in short-readWGS data
using the Canvas39 and/or Manta40 algorithms. A targeted single-
nucleotide polymorphism (SNP) array was used to validate the
M10 variant (see below). InfiniumGSA-24þ v.3.0 arrays (Illumina)
were used to test the template DNA, prepared according to the
manufacturer’s protocol. Alterations were visualized and analyzed
using the NxClinical 6.2 and FASST2 CNV calling algorithm soft-
ware (BioDiscovery).
Protein alignment and structure predictions were performed by
using T-Coffee,41 Expresso,42 and AlphaFold.43
Disease prevalence estimation
To estimate the prevalence of AP5-related diseases, we utilized the
GeniE tool (https://genie.broadinstitute.org/) by using manually
curated lists of variants extracted from gnomAD v.2.1.1.36 As per
the PVS1 (pathogenic very strong 1) criterion of the ACMG guide-
lines,34,35 we considered true loss-of-function alleles all variants
labeled as ‘‘LoF’’ by gnomAD, with the exception of those located
in the last exon of the genes (for AP5Z1 and AP5M1) or removing
more than 10% of the protein (for AP5B1 and AP5S1, which
have very large terminal exons). Additionally, the c.938A>G
(p.Tyr313Cys) variant for AP5M1 was curated manually. All
selected variants were verified to affect their relevant transcripts.
Targeted Sanger sequencing
DNA sequencing using the Sanger method was performed to vali-
date short-read sequencing findings and to perform intrafamilial
segregation analyses, where appropriate. Primer3Plus44 was used
to design primers for PCR, performed using the GoTaq polymerase
(Promega) or FIREPol DNA polymerase (Solis BioDyne) and 
2 ng
of template DNA, according to the manufacturers’ protocols. All
PCR products were treated with ExoSAP-IT (Thermo Fisher) or Il-
lustra ExoProStar (Cytiva), and Sanger sequencing was performed
by Microsynth (Balgach, Switzerland) or by StabVida (Madrid,
Spain). Sequences were visualized and compared to the human
reference sequence (Ensembl,45 GRCh37) with the CLC Genomics
Workbench 12 software (QIAGEN). Sequences of primers and PCR
conditions are available upon request.
RNA analysis
RNA analysis was performed to test the effects on splicing of the
variant c.1595G>T (GenBank: NM_014855.3) (M2) in AP5Z1, de-
tected inmultiple individuals. Three milliliters of peripheral blood
were collected in Tempus Blood RNA tubes (Applied Biosystems)
from P1, an affected person who was heterozygous for the studied
variant, as well as from a control individual. Total leukocyte RNA
was extracted with the Tempus Spin RNA Isolation Kit (Applied
Biosystems) according to the manufacturer’s instructions. RNA
from healthy retina was obtained commercially (Clontech, Ta-
kara). cDNA was synthesized from 2 mg of RNA with the
MultiScribe reverse transcriptase from the High-Capacity cDNA
Reverse Transcription Kit (Applied Biosystems) according to the
manufacturer’s instructions. A negative control for the RT-PCR
experiment (RT()) was prepared by removing reverse transcrip-
tase from one of the RNA samples from the control donor.
RT-PCR amplification was performed using GoTaq G2 DNA po-
lymerase and the provided reaction buffer (Promega) by using
primers 50-CACCTCAGCACCCTCAGATT-30 (CR-8319) and
50-CCTGGGGATCAGATCTTGG-30 (CR-8320) lying in the AP5Z1
exon 10 and the junction between exons 16 and 17, respectively,
according to standard cycling conditions. The resulting PCR
The American Journal of Human Genetics 112, 1–21, April 3, 2025 3
Please cite this article in press as: Kaminska et al., Bi-allelic variants in three genes encoding distinct subunits of the vesicular AP-5 complex
cause hereditary macular dystrophy, The American Journal of Human Genetics (2025), https://doi.org/10.1016/j.ajhg.2025.02.015
products were resolved on a 1.5% agarose gel. Amplicons were
either directly sequenced (as described above) or purified using
NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel), prior
to being subcloned into pGEM-T Easy vectors (Promega). Individ-
ual clones were then sequenced using the primers mentioned
above.
Immunostaining of human RPE explants
Human RPE flat mounts were collected and stored in Ames’ me-
dium (Sigma-Aldrich, cat. #A1420) for 4 h following enucleation
from a patient (a 67-year-old woman with uveal melanoma) and
subsequently fixed using 4% paraformaldehyde (PFA) in 13 phos-
phate-buffered saline (PBS, Merck) for 2 h. The tissue was then
incubated in 30% sucrose in PBS overnight at 4C. For immunoflu-
orescence staining, tissues were blocked for 1 h in block buffer (5%
bovine serum albumin, 10% normal goat serum, 0.4% fish skin
gelatin, and 0.1% Triton X-100 in PBS). Tissues were incubated
with primary antibodies overnight at 4C, followed by three
5-min washes in PBS, prior to a 1-h incubation with the secondary
antibodies. After three more washes in PBS, tissues were mounted
on slides with ProLong Gold Antifade Mountant (Thermo Fisher)
and #1.5 glass coverslips. Immunofluorescence staining was per-
formedwith the following primary antibodies, diluted in the block
buffer: anti-AP5Z1 (Invitrogen, cat. #PA5-57368, 1:50 dilution),
anti-AP5M1 (Invitrogen, cat. #PA5-63174, 1:30 dilution), and
anti-AP5B1 (Invitrogen, cat. #PA5-64344, 1:100 dilution). The sec-
ondary antibody used was Alexa Fluor 555 F(ab0)2 goat anti-rabbit
immunoglobulin G (IgG) (Invitrogen, cat. #A-21430, 1:500 dilu-
tion). DAPI (Thermo Fisher, cat. #D1306, 1:500 dilution) was
used for nuclear staining. Phalloidin-iFluor 647 conjugate (Abcam,
cat. #ab176759, 1:1,000 dilution) was used to stain actin fila-
ments. The specificity of the staining was confirmed by incubation
with the secondary antibody only. Imagingwas performedwith an
Olympus scanning confocalmicroscope, using a 633 oil objective.
Induced pluripotent stem cell-derived RPE cell culture
and immunostaining
Human induced pluripotent stem cell-derived RPE (iPSC-RPE) cells
were differentiated from the iPS(IMR90)-4-DL-01 line following
previously established protocols.46,47 After culturing as a mono-
layer in transwells for 1 week, cells were fixed with 4% PFA in
13 PBS for 20 min and then stored in PBS at 4C. For immunoflu-
orescence staining, membrane inserts were removed from trans-
wells, sectioned, and stained as described above for human RPE ex-
plants. The iPSC-RPE sections were then mounted on slides with
the ProLong Glass Antifade Mountant (Thermo Fisher) and
covered with #1.5 glass coverslips. Immunofluorescence staining
was performed with the following primary antibodies, diluted in
the block buffer: anti-AP5Z1 (as above), anti-AP5M1 (as above,
1:50 dilution), anti-AP5B1 (as above), anti-EEA1 (BD Biosciences,
cat. #610456, 1:100 dilution), anti-LAMP2 (Abcam, cat.
#ab25631, 1:100 dilution), anti-TfR (Invitrogen, cat. #13–6800,
1:100 dilution), anti-Rab7 (Abcam, cat. #ab50533, 1:100 dilution),
and anti-TGN46 (Sigma-Aldrich, cat. #sab4200355, 1:100 dilu-
tion). Secondary antibodies used were Alexa Fluor 488 F(ab0)2
goat anti-mouse IgG (Invitrogen, cat. #A-11017) and Alexa Fluor
594 F(ab0)2 goat anti-rabbit IgG (Invitrogen, cat. #A-21428), both
at a 1:500 dilution. DAPI (as above) was used for nuclear staining.
Phalloidin-iFluor 647 conjugate (as above) was used to stain actin
filaments. The specificity of the staining was confirmed by incuba-
tion with the secondary antibody only. Imaging was performed
with an Olympus scanning confocal microscope, using a 633 oil
objective. For higher resolution, imagingwas performed on a Leica
STELLARIS 8 Falcon point scanning confocal microscope, using an
HC PL APO 633/1.40 objective. Lightning processing was per-
formed immediately after imaging on LASX software.
Results
Clinical characteristics of the affected study subjects
A total of 22 affected individuals from 19 unrelated fam-
ilies (Figure 1) were ascertained by specialized ophthalmo-
logical examinations. In all instances, the disease appeared
to be transmitted as an autosomal recessive trait, since the
disorder manifested in individuals of both sexes, all par-
ents of affected individuals were healthy by history, and,
in some cases, probands had siblings presenting with the
same condition. All subjects exhibited ophthalmic features
characteristic of progressive MD (Tables 1 and S1), with
central vision symptoms having their onset on average
during the 5th decade of life (45 years, range 30–57 years,
Table S1). In three subjects (P14, P16, and P22), the disease
presented as flecked maculopathy without extensive atro-
phy (Figures 2A and S1). Two probands of an intermediate
stage (P5 and P18) exhibited outer retinal and RPE atrophy
alongside some remaining flecks (Figure 2B). All affected
individuals who were in the 6th decade of life and beyond
demonstrated a variable area of central chorioretinal atro-
phy extending centrifugally (Figures 2C and S1), character-
ized by complete RPE and outer retinal loss on OCT, as well
as complete loss of short-wavelength autofluorescence
signal. This atrophy was primarily macular and nasal.
Nine individuals presented a characteristic reticular
pigment clumping pattern in the peripheral retina
(Table S1), one eye showed an inactive choroidal neovascu-
lar membrane (P18, Figure 2B), and 12 individuals with
advanced disease presented outer retinal tubulations
(Table S1). Longitudinal data available for at least three
subjects documented disease progression as increasing sur-
face area of atrophic lesions over time, from the perifoveal
area toward both midperiphery and the fovea, with a
considerable enlargement over a 6- to 18-year period
(Figure S2). Despite advanced disease stages and extensive
areas of atrophy in some subjects, no individual exhibited
an extinguished ERG, indicating relative preservation of
peripheral retinal function. A large transitional zone be-
tween the most severely affected and healthy retina was
commonly observed (Table S1). Importantly, all these char-
acteristics were consistent across all individuals, regardless
of their genotypes.
Approximately half of the affected individuals exhibited
extraocular features of variable severity (Table 1). Some
manifested disorders that are rather prevalent in the gen-
eral population and were probably unrelated to their
Mendelian condition, such as thyroid dysfunction, hyper-
tension, type 2 diabetes, or ischemic stroke. Others, how-
ever, experienced symptoms that could be consistent
with the spectrum of hereditary spastic paraplegia,
4 The American Journal of Human Genetics 112, 1–21, April 3, 2025
Please cite this article in press as: Kaminska et al., Bi-allelic variants in three genes encoding distinct subunits of the vesicular AP-5 complex
cause hereditary macular dystrophy, The American Journal of Human Genetics (2025), https://doi.org/10.1016/j.ajhg.2025.02.015
although in most cases the phenotype was very mild and
non-specific, including isolated pollakiuria, polyneurop-
athy, parkinsonism, or mild intellectual disability. For
two individuals who presented with advanced neurolog-
ical symptoms (P16 and P4, Table 1), brain magnetic reso-
nance imaging (MRI) scans were conducted. MRI of P16
Figure 1. Pedigrees of the individuals analyzed
Disease-associated variants are indicated by MX; ‘‘wt’’ represents wild-type alleles. Arrows indicate probands, and square brackets indi-
cate that the phase of the provided genotype is assumed but not proven by segregation analysis, due to lack of available biological ma-
terial. The correct HGVS nomenclature for the variant in AP5Z1 ‘‘c.80_83delins’’ is ‘‘c.80_83delinsTGCTGTAAACTGTAACTGTAAA’’ and
for the variant in EYS ‘‘c.403_423delins’’ is ‘‘c.403_423delinsCTTTT.’’
The American Journal of Human Genetics 112, 1–21, April 3, 2025 5
Please cite this article in press as: Kaminska et al., Bi-allelic variants in three genes encoding distinct subunits of the vesicular AP-5 complex
cause hereditary macular dystrophy, The American Journal of Human Genetics (2025), https://doi.org/10.1016/j.ajhg.2025.02.015
Table 1. Clinical and genetic data of individuals in the study
General Information Clinical symptoms Genotype
Family ID
Affected
individual ID
Referring
center Sex
Age at
examination
(years)
Age at
onset
(years)
Initial
symptoms IRD class
Main ocular
features
Neurological
symptoms Other symptoms
Variant
1 (ID) Variant 1
Variant
2 (ID) Variant 2
AP5Z1 (GenBank: NM_014855.3)
1 P1 Portugal
(Lisbon)
M 63 50 central
vision loss
MD extensive
central
chorioretinal
atrophy
pollakiuria none reported M1 c.1836_1
839dup
(p.Leu614
TyrfsTer150)
M2 c.1595G>T
(p.Ser486_
Arg532del)
P2 Portugal
(Lisbon)
M 74 40 central
vision loss
MD extensive
central
chorioretinal
atrophy
peripheral
neuropathy
with
paraplegia
none reported M1 c.1836_
1839dup
(p.Leu614
TyrfsTer150)
M2 c.1595G>T
(p.Ser486_
Arg532del)
P3 UK
(London)
M 63 40 blurred
vision
MD extensive
central
chorioretinal
atrophy
none hearing loss (onset
at 50 years old)
M1 c.1836_
1839dup
(p.Leu614
TyrfsTer150)
M2 c.1595G>T
(p.Ser486_
Arg532del)
2 P4 Belgium
(Ghent)
M 62 50 unilateral
central vision
loss, central
scotoma,
paresthesia
MD extensive
central
chorioretinal
atrophy
paresthesia
in hands
and feet, mild
intellectual
disability,
ataxic gait
impression of
hearing loss
M2 c.1595G>T
(p.Ser486_
Arg532del)
M3 c.412C>T
(p.Arg138Ter)
3 P5 Israel
(Haifa)
M 43 43 reduced VA MD maculopathy
with atrophy
and deposits
none hearing loss (onset
at 40 years old)
M4 c.2086dup
(p.Gln696
ProfsTer67)
M4 c.2086dup
(p.Gln696Profs
Ter67)
4 P6 Israel
(Jerusalem)
F 55 53 reduced VA MD maculopathy
with atrophy
none none reported M5 c.950dup
(p.Asp317
GlufsTer93)
M6 c.1033C>T
(p.Arg345Ter)
5 P7 UK
(London)
M 56 47 central
vision loss
MD extensive
central
chorioretinal
atrophy
pollakiuria neurosensory
hearing
loss (onset at
35 years old),
hypercholesterolemia,
hypertension, asthma
M7 c.1421_
1447del
(p.Pro474_
Leu482del)
M7 c.1421_1447del
(p.Pro474_
Leu482del)
6 P8 UK
(Newcastle)
M 63 45 mostly
asymptomatic
MD extensive
central
chorioretinal
atrophy
none none reported M8 c.928C>T
(p.Arg310Ter)
M8 c.928C>T
(p.Arg310Ter)
7 P9 UK
(Liverpool)
M 63 50 central
vision loss
MD extensive
central
chorioretinal
atrophy
none hypertension,
hearing
loss (onset >50
years old)
M8 c.928C>T
(p.Arg310Ter)
M9 c.931C>T
(p.Arg311Ter)
(Continued on next page)
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Table 1. Continued
General Information Clinical symptoms Genotype
Family ID
Affected
individual ID
Referring
center Sex
Age at
examination
(years)
Age at
onset
(years)
Initial
symptoms IRD class
Main ocular
features
Neurological
symptoms Other symptoms
Variant
1 (ID) Variant 1
Variant
2 (ID) Variant 2
8 P10 UK
(Liverpool)
M 65 45 central
vision loss
MD extensive
central
chorioretinal
atrophy
none none reported M8 c.928C>T
(p.Arg310Ter)
M10 deletion
(7p22.1)
including
the AP5Z1
gene
P11 UK
(Liverpool)
F 63 late 40s central
vision loss
MD extensive
central
chorioretinal
atrophy
none hiatus hernia,
diverticulosis
and colitis,
hypertension,
bilateral
sensorineural
hearing loss
(onset in
her late 50s)
M8 c.928C>T
(p.Arg310Ter)
M10 deletion
(7p22.1)
including
the AP5Z1
gene
9 P12 Spain
(Valencia)
M 62 54 nyctalopia,
reduced VA
MD extensive
central
chorioretinal
atrophy
none impression
of hearing
loss in left ear,
hypertension,
type 2 diabetes
M11 c.1124_1132 þ
75del (p.?)
M11 c.1124_
1132 þ
75del (p.?)
10 P13 Sweden
(Lund)
M 59 40 problem with
reading
MD extensive
central
chorioretinal
atrophy
none none reported M3 c.412C>T
(p.Arg138Ter)
M12 c.180
18G>A (p.?)
11 P14 Germany
(Tu¨bingen)
F 32 31 metamorphopsias MD early
maculopathy
with deposits
none thyroid
dysfunction
M13 c.80_
83delinsTG
CTGTAA
ACTGTA
ACTGTAAA
(p.Arg27delins
LeuLeuTer)
M14 c.1852dup
(p.Leu618Profs
Ter145)
12 P15 Germany
(Tu¨bingen)
M 53 44 poor central
vision
MD extensive
central
chorioretinal
atrophy
parkinsonism none reported M15 c.824C>A
(p.Ser275Ter)
M15 c.824C>A
(p.Ser275Ter)
13 P16 Switzerland
(Basel)
F 52 46 spastic atactic
gait
MD early
maculopathy
spastic atactic
paraparesis
none reported M16 c.857_866del
(p.Leu286Profs
Ter25)
M16 c.857_866del
(p.Leu286Profs
Ter25)
14 P17 Spain
(Madrid)
F 44 40 paresthesia MD early
maculopathy
with deposits
paresthesia in
the right hand
none reported M17 c.805C>T
(p.Gln269Ter)
M17 c.805C>T
(p.Gln269Ter)
(Continued on next page)
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Table 1. Continued
General Information Clinical symptoms Genotype
Family ID
Affected
individual ID
Referring
center Sex
Age at
examination
(years)
Age at
onset
(years)
Initial
symptoms IRD class
Main ocular
features
Neurological
symptoms Other symptoms
Variant
1 (ID) Variant 1
Variant
2 (ID) Variant 2
AP5M1 (GenBank: NM_018229.4)
15 P18 Switzerland
(Lausanne)
M 46 30 reduced central
vision
MD maculopathy
with atrophy
and deposits
mild
intellectual
disability
none reported M18 c.97C>T
(p.Arg33Ter)
M18 c.97C>T
(p.Arg33Ter)
16 P19 Belgium
(Ghent)
M 59 46 Parkinson
symptoms
MD extensive
central
chorioretinal
atrophy
parkinsonism type 2 diabetes,
ischemic stroke
M19 c.1166G>A
(p.Trp389Ter)
M19 c.1166G>A
(p.Trp389Ter)
17 P20 Germany
(Tu¨bingen)
M 63 56 central
vision loss
MD extensive
central
chorioretinal
atrophy
none myocardial
infarction,
multiple allergies,
chronic anal
fistulas
M20 c.938A>G
(p.Tyr313Cys)
M20 c.938A>G
(p.Tyr313Cys)
AP5B1 (GenBank: NM_138368.5)
18 P21 Greece
(Heraklion)
M 68 55 reduced
central vision,
photophobia,
nyctalopia
MD extensive
central
chorioretinal
atrophy
polyneuropathy hearing loss
(onset
later in life)
M21 c.310del
(p.Leu104
TrpfsTer54)
M21 c.310del
(p.Leu104Trpfs
Ter54)
19 P22 UK
(London)
F 36 36 mostly
asymptomatic
MD early
maculopathy
with deposits
none none reported M22 c.463C>T
(p.Arg155Ter)
M23 c.862del
(p.Gln288Serfs
Ter29)
M, male; F, female; IRD, inherited retinal degeneration; VA, visual acuity; MD, macular dystrophy.
8
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(performed at the age of 49 years) revealed multiple small,
round-shaped hyperintense lesions in the supratentorial,
frontoparietal, and occipital white matter, as well as in
the corpus callosum. In P4, the same test (performed at
the age of 58 years) showed signs of generalized cortical
and subcortical atrophy with discrete frontal chronic
vascular leukoencephalopathy. Interestingly, six individ-
uals experienced some degree of hearing impairment;
which was confirmed not to be congenital, as it manifested
later in life, between the 4th and 6th decade. Two additional
subjects (P4 and P12) reported an impression of hearing
loss (unilateral or bilateral), which was not confirmed by
audiometry or other specialized tests (Table 1).
A degree of variability in extraocular features was also
observed in individuals from the same family, hence car-
rying the same genotype in AP-5 genes. For instance, in
family 1 the three affected brothers had similar retinal
findings but different extraocular symptoms: P1 had
pollakiuria, P2 peripheral neuropathy, and P3 hearing
loss. In family 8, one of the affected siblings (P10)
manifested non-syndromic retinal disease at his last
examination (at age 65 years), while the other sibling
(P11) developed non-specific extraocular features in her
5th and 6th decade of life, including bilateral sensori-
neural hearing loss.
Finally, since the disease manifests as a late-onset visual
impairment, it is possible that younger individuals from
these pedigrees, who were not examined or genotyped
and are reported in Figure 1 as healthy, could develop
this condition in the future.
Genetic analysis
All probands were negative for pathogenic variants in pre-
viously identified IRD-associated genes. New WES, new
A B
C
Figure 2. Multimodal retinal imaging of eight representative individuals from this study
In each panel, the top row shows color or pseudocolor fundus images, the middle row shows fundus autofluorescence images (FAF), and
the bottom row shows optical coherence tomography (OCT) scans. OCT images were not available for individual P5 from family 3. Im-
ages are ordered according to the subjects’ age. OD, right eye; OS, left eye; yo, years old.
(A) Two individuals (P14 with variants in AP5Z1, age 32; P22 with variants in AP5B1, age 36) with early-stage disease presenting with
flecks without atrophy.
(B) Two individuals (P5 with variants in AP5Z1, age 43; P18 with variants in AP5M1, age 46) with limited atrophy of the RPE and outer
retina and some deposits.
(C) Four individuals with advanced disease (P19 with variants in AP5M1, age 59; P4 with variants in AP5Z1, age 62; P3 with variants in
AP5Z1, age 53; and P21 with variants in AP5B1, age 70) with variable degree of central chorioretinal atrophy.
The American Journal of Human Genetics 112, 1–21, April 3, 2025 9
Please cite this article in press as: Kaminska et al., Bi-allelic variants in three genes encoding distinct subunits of the vesicular AP-5 complex
cause hereditary macular dystrophy, The American Journal of Human Genetics (2025), https://doi.org/10.1016/j.ajhg.2025.02.015
WGS screening, or a reanalysis of previous sequencing
data by using a customized analytical pipeline led to the
identification of assortments of bi-allelic variants in
AP5Z1 (14 families), AP5M1 (three families), and AP5B1
(two families) (Table 1 and Figure 1). These three genes
encode different subunits of the AP-5 complex, namely
zeta, mu, and beta, respectively, and share structural
similarity with corresponding subunits of other AP
complexes.25
In total, we detected 23 distinct variants: 17 in AP5Z1,
three in AP5M1, and three in AP5B1 (Figure 3), the fre-
quency of which in gnomAD36 ranged from absent to
4.4 3 105. Twenty-two out of these 23 changes were pre-
sumed loss-of-function (LoF) variants: nine nonsense,
eight frameshift, one splice-site alteration, one partial
and one full gene deletion, one non-frameshift deletion,
and one single-nucleotide substitution creating a
missense but predicted to alter pre-mRNA splicing and
resulting in the ablation of an entire exon (M2, see
below). The remaining DNA change was a missense (Ta-
bles 1 and S2). Most variants (17 out of 23) were not re-
ported previously in ClinVar48 or in the biomedical liter-
ature (Table S2).
We also estimated the putative prevalence of AP-5-
related disorders by analyzing the frequency values of
LoF alleles in the general population, as reported by gno-
mAD.36 According to these data, 1 in 471,000 individuals
should carry bi-allelic LoF variants in AP5Z1, making
AP5Z1-associated disease the most prevalent one. AP5B1-
related disease would have a prevalence of 1 in 5.9 million,
and disorders linked to AP5M1 variants would be present
in 1 in 6.5 million people. If deleterious variants in
AP5S1were also associated with a pathological phenotype,
the prevalence of such disease would be rather rare, i.e., 1
in 105 million. Overall, the combined prevalence across
all AP-5-related conditions was estimated at 1 in 407,000
people, translating to approximately 20,000 potentially
affected individuals in the world—comprising 17,400
AP5Z1 cases, 1,400 AP5B1 cases, 1,200 AP5M1 cases, and
only 80 AP5S1 cases. As a reference, late-onset MDs have
a collective prevalence of roughly 1 in 10,000 people, as as-
certained by integrating existing data on individuals with
ABCA4 (late-onset Stargardt disease, MIM: 248200),
PRPH2 (central areolar choroidal dystrophy, MIM:
613105), and BEST1 (late-onset MD, MIM: 153700) patho-
genic variants.49–53
Figure 3. Schematic representation of the genetic variation spectrum of AP5Z1, AP5M1, and AP5B1, with respect to macular dystro-
phy and spastic paraplegia
The exons of each gene are indicated by numbered boxes, whereas introns are shown by horizontal lines. Their sizes with respect to
actual genomic sequence, using canonical transcripts, are roughly proportional, although not to scale. 50 UTRs and 30 UTRs are omitted.
The protein size, in number of amino acids (aa), is also indicated. Variants associatedwith an ocular phenotype (MD,macular dystrophy)
and detected in this study (M1 to M23) are shown in various colors above the gene of interest, while variants previously reported in as-
sociation with spastic paraplegia type 48 (SPG48) are indicated below the gene (only for AP5Z1). These latter DNA changes represent
pathogenic or likely pathogenic variants reported in ClinVar as of July 2024, documented in the literature and/or linked to an affected
individual. M10, a large deletion affecting the whole AP5Z1 sequence, is indicated by a horizontal line. No clear genotype-phenotype
correlation within or across conditions could be detected. Of note, all disease-causing variants identified are presumed loss-of-function
alleles, with the exception of M20 in AP5M1, which is a missense (see text for details). The correct HGVS nomenclature for the variant in
AP5Z1 ‘‘c.80_83delins’’ is ‘‘c.80_83delinsTGCTGTAAACTGTAACTGTAAA.’’
10 The American Journal of Human Genetics 112, 1–21, April 3, 2025
Please cite this article in press as: Kaminska et al., Bi-allelic variants in three genes encoding distinct subunits of the vesicular AP-5 complex
cause hereditary macular dystrophy, The American Journal of Human Genetics (2025), https://doi.org/10.1016/j.ajhg.2025.02.015
Variants in AP5Z1
Family 1 was from Portugal and included three affected in-
dividuals (P1, P2, and P3), born to healthy parents. The
proband, P1, was found to carry two heterozygous variants
in AP5Z1: c.1836_1839dup (p.Leu614TyrfsTer150) (M1,
reference sequences for AP5Z1 mRNA and protein: Gen-
Bank: NM_014855.3 and NP_055670.1, respectively) and
c.1595G>T (p.Ser486_Arg532del) (M2, an SNV leading to
aberrant splicing; see below). Molecular analysis of his
two affected brothers and his two unaffected nephews
confirmed these variants to be in trans and to co-segregate
with the disease in a recessive fashion (Figure 1). Proband
P4 from family 2 was a simplex case with MD from
Belgium, born to unaffected parents and with unaffected
offspring. He was also found to carry M2, in a compound
heterozygous state with the nonsense variant c.412C>T
(p.Arg138Ter), or M3. Co-segregation analysis confirmed
the bi-allelic nature of his genotype (Figure 1).
In family 3, an Israeli pedigree of North African Jewish
descent, the proband (P5) presented with MD and late-
onset hearing loss. He carried a homozygous frameshift
variant (c.2086dup [p.Gln696ProfsTer67], M4). Intrigu-
ingly, he had three relatives with a different retinal disease,
RP, who did not share the AP5Z1 genotype. Molecular
analysis confirmed that these individuals had a different
etiology for their disorder, a homozygous known patho-
genic frameshift variant in EYS (c.403_423delinsCTTTT
[GenBank: NM_001142800.1] [p.Thr135LeufsTer26],
ClinVar: 812320). This latter variant, previously identified
as a founder mutation for autosomal recessive RP (MIM:
602772) in the Israeli Moroccan Jewish subpopulation,54
aligned with the family’s origin. All variants segregated
with the respective diseases within the family and were
confirmed to be inherited from the unaffected carrier par-
ents (Figure 1). Individual P6 from family 4, a pedigree of
Ashkenazi Jewish origin, was also from Israel. She was a
simplex case with MD, born to unaffected parents and
with three unaffected children. She was found to carry
two LoF variants in AP5Z1, c.950dup (p.Asp317Gluf-
sTer93) (M5) and c.1033C>T (p.Arg345Ter) (M6). Co-
segregation analysis was not possible, due to the lack of
biological material from family members, but gnomAD
reported these two alleles to belong to two different
haplotypes (100% estimated likelihood, with no individ-
uals carrying both variants), strongly supporting the bi-
allelic nature of the detected genotype.
Families 5, 6, 7, and 8 were all from the United Kingdom.
Family 5 was a consanguineous pedigree of Pakistani
descent. The proband, P7, presented with MD and late-
onset hearing loss (Table 1). Molecular analysis revealed
a homozygous non-frameshift deletion in AP5Z1
(c.1421_1447del [p.Pro474_Leu482del], or M7), involving
the ablation of 9 amino acid residues highly conserved
across vertebrates. He was also positive for a rare homozy-
gous missense (c.22G>C [GenBank: NM_173477.5]
[p.Ala8Pro]), classified as a variant of uncertain significance
(VUS) in ClinVar: 1687494, in USH1G, a gene linked to
Usher syndrome type I (MIM: 606943). This variant is un-
likely to be contributing to the retinopathy observed in
this individual because USH1G is associated with a
different condition, characterized by congenital profound
hearing loss and early-onset RP.55 Of note, this individual
had significant homozygous regions across the genome
(more than 700 Mb,56 overall, for which more than 20%
of all genomic variants appear in the homozygous state).
Families 6, 7, and 8 included individuals of British
ancestry, again all presenting with MD. P8, the proband
of family 6, was homozygous for the nonsense variant
c.928C>T (p.Arg310Ter), or M8. The same DNA change
was present in P9, from family 7, as well as in P10 and
P11, two siblings from family 8, in a compound heterozy-
gous state with two other variants (Figure 1). Based on the
visualization of sequencing data,37 in P9 M8 was detected
in trans with another nearby nonsense variant, c.931C>T
(p.Arg311Ter) (M9), whereas in P10 and P11 it was in trans
with a large deletion (M10) on chromosome 7p22.1, which
included the full sequences of AP5Z1, RADIL, and PAPOLB,
as well as parts of the genes FOXK1 and MMD2
(g.4808369_4950979del [GenBank: NC_000007.13]).
Subject P12 from family 9 was a simplex case from
Spain, with signs and symptoms consistent with MD.
He was born to unaffected parents and had an unaffected
daughter and two unaffected siblings (Figure 1). Molecu-
lar analysis revealed the presence of another deletion in
AP5Z1, in a homozygous state, involving the end of
exon 9 and part of intron 9 (c.1124_1132þ75del [p.?],
or M11). In silico tools predicted this DNA change to result
in the skipping of the full exon and a shift of the reading
frame (p.Ala323GlyfsTer52) (Table S2).
Families 10, 11, 12, 13, and 14 were from Sweden, Ger-
many, Germany (of Turkish ancestry), Switzerland, and
Spain, respectively, and were all ascertained as pedigrees
composed of affected individuals with unaffected parents
who did not provide biological material for further ge-
netic testing. All subjects were positive for pathogenic
variants in AP5Z1 (Figure 1). Individual P13 from family
10 was found to carry two heterozygous variants,
c.412C>T (p.Arg138Ter) (M3), mentioned previously,
and c.18018G>A (p.?) (M12). This latter change was
predicted by SpliceAI57 to alter canonical splicing by
creating a new strong acceptor site, in turn leading to
the shift of the reading frame and a premature stop codon
(p.Arg60HisfsTer41) (Table S2). Although co-segregation
analysis was not possible, data from gnomAD indicated
that the M3 and M12 changes are likely to be in trans,
as they occur individually in presumably different sub-
jects. Two heterozygous variants, c.80_83delinsTGCTGT
AAACTGTAACTGTAAA (p.Arg27delinsLeuLeuTer) (M13)
and c.1852dup (p.Leu618ProfsTer145) (M14), were de-
tected in P14 from family 11. Again, due to the absence
of DNA from parents or unaffected siblings, we could
not prove the bi-allelic nature of these LoF variants. How-
ever, M13 was detected previously in two French siblings
with spastic paraplegia,58 suggesting that it belongs to a
The American Journal of Human Genetics 112, 1–21, April 3, 2025 11
Please cite this article in press as: Kaminska et al., Bi-allelic variants in three genes encoding distinct subunits of the vesicular AP-5 complex
cause hereditary macular dystrophy, The American Journal of Human Genetics (2025), https://doi.org/10.1016/j.ajhg.2025.02.015
different haplotype with respect to M14. Probands from
Families 12, 13, and 14 were all homozygotes for LoF
alleles: c.824C>A (p.Ser275Ter) (M15), c.857_866del
(p.Leu286ProfsTer25) (M16), and c.805C>T (p.Gln269
Ter) (M17), respectively.
Variants in AP5M1
Probands from three additional pedigrees, families 15, 16,
and 17, presented with ophthalmic features similar to
those displayed by individuals with pathogenic variants
in AP5Z1, described above (Table 1). All of them were
simplex cases, born to unaffected parents (Figure 1). P18,
from family 15, was a male of Portuguese origin from
Switzerland. His parents were genetically related. Molecu-
lar analysis revealed the presence of a homozygous
nonsense variant in AP5M1, c.97C>T (p.Arg33Ter), or
M18 (reference sequences for mRNA and protein: Gen-
Bank: NM_018229.4 and NP_060699.3, respectively). P19
from family 16 was from Belgium and was found to be ho-
mozygous for another nonsense DNA change, c.1166G>A
(p.Trp389Ter), or M19. Finally, P20 from family 17, an
individual of Turkish ancestry ascertained in Germany,
carried a homozygous missense variant, c.938A>G
(p.Tyr313Cys) (M20). This DNA change affects an amino
acid residue that is conserved across all vertebrates, as
well as all AP5M1 human paralogs (AP1M1, AP2M1,
AP3M1, and AP4M1; Figure S3), had a MutScore value of
0.924 (maximum ¼ 1.000)59 and was predicted to be dele-
terious by Franklin (https://franklin.genoox.com), with
an aggregated in silico score from multiple tools of 0.84
(maximum ¼ 1.00). Notably, M20 was the only true
missense change among all of the causative variants de-
tected in this study and, interestingly, a missense affecting
Tyr284 of AP4M1, the corresponding amino acid residue of
Tyr313 in AP5M1, is linked to spastic paraplegia type 50
(MIM: 612936).60
Variants in AP5B1
Individual P21 belongs to family 18, from Greece, while
individual P22 was from a pedigree of Afghani origin
from the United Kingdom (family 19). Both probands
were simplex cases, presenting with signs of MD
(Figure 1). They both carried variants in AP5B1, encoding
a third component of the AP-5 complex. P21 was
found to be homozygous for the LoF change c.310del
(p.Leu104TrpfsTer54), or M21 (GenBank: NM_138368.5
and NP_612377.4, respectively), whereas P22 was a
likely compound heterozygote for two other LoF
variants, c.463C>T (p.Arg155Ter), or M22, and c.862del
(p.Gln288SerfsTer29), or M23. The bi-allelic nature of
these variants in P22 was very likely, since gnomAD re-
ported the presence of M22 independently from M23 in
two heterozygous Asian individuals, but this could not
be proven experimentally owing to the non-availability
of additional family members. Even though the three var-
iants described above are located in the last exon of
AP5B1 (Figure 3), they all remove more than two-thirds
of the protein’s full sequence and are therefore extremely
likely to represent full LoF alleles.
Functional validation of M2
The M2 change, c.1595G>T (p.Ser486_Arg532del), occur-
ring in the last base of exon 12 of AP5Z1, was the second
most frequent variant detected in our cohort. Although
this base substitution would theoretically result in the
missense p.Arg532Met, it was predicted to affect pre-
mRNA splicing by four in silico tools, SpliceAI57 (score:
D0.76 out of 1.00, donor loss), dbSNV61 (0.99 out of
1.00), SQUIRLS62 (0.99 out of 1.00), and MaxEntScan63
(D5.72, strong),64 and therefore likely to result in an LoF
allele. To validate these predictions, we analyzed the RNA
(cDNA) of leukocytes from individual P1 (carrying M2 het-
erozygously) and a control individual, as well as the RNA
from a healthy human retina sample. The analysis revealed
that M2 leads to an aberrant splicing event resulting in the
skipping of exon 12 of AP5Z1 and causing an in-frame
deletion of 47 amino acid residues, r.1455_1593del
(p.Ser486_Arg532del). This aberrant transcript was absent
in both the control sample and in cDNA from the retina
(Figure 4), allowing us to classify M2 as a likely pathogenic
splice-altering variant (Table S2).34
Localization of AP5Z1, AP5M1, and AP5B1 in the human
RPE and in iPSC-RPE monolayers
Previous immunofluorescence microscopy in fibroblasts
derived from individuals with spastic paraplegia type
48,65 as well as studies on cellular25 and animal66 models
of AP5Z1 knockdown, highlighted the importance of the
AP-5 complex in tissues with high endolysosomal activity.
We therefore reasoned that, for a retinal phenotype, pri-
mary defects linked to AP-5 deficiency would likely occur
in the RPE, a tissue for which intracellular vesicular activity
is elevated. Such activity is needed to maintain cellular ho-
meostasis as well as the continuous phagocytosis of frag-
ments of photoreceptors’ outer segments. We thus assessed
the presence and localization of the three components of
the AP-5 complex studied here by immunofluorescence
on human RPE flat-mount sections. Confocal microscopy
imaging revealed punctate labeling of AP5Z1, AP5M1,
and AP5B1 in the cytoplasm of RPE cells, with the highest
abundance for AP5B1 and the lowest for AP5M1 (within
the limits of protein quantification by immunofluores-
cence) (Figure 5).
We then used human iPSC-RPE monolayers to further
investigate the localization of these AP-5 components
with respect to markers of distinct cellular compartments
involved in the endolysosomal trafficking as well as their
various vesicular maturation stages (Figure S4A). Immuno-
fluorescence imaging of those sections confirmed the
punctate staining patterns of AP5Z1, AP5M1, and AP5B1,
revealing their strongest co-localization with Rab7, a
marker of late endosomes, and partial overlap with
TGN46-positive Golgi-derived vesicles (Figures S4B and
S6). Conversely, no significant co-localization was detected
12 The American Journal of Human Genetics 112, 1–21, April 3, 2025
Please cite this article in press as: Kaminska et al., Bi-allelic variants in three genes encoding distinct subunits of the vesicular AP-5 complex
cause hereditary macular dystrophy, The American Journal of Human Genetics (2025), https://doi.org/10.1016/j.ajhg.2025.02.015
with LAMP2 (lysosomal marker), EEA1 (early endosomal
marker), or TfR (a marker of recycling endosomes)
(Figure S4B). High-resolution imaging provided additional
insights into the subcellular distribution of these AP-5 pro-
teins, predominantly observed on the apical side of the
iPSC-RPE monolayer, with no overlap with the nuclear re-
gion, as shown in cross-sectional views (Figure 6). In partic-
ular, within the apical cytoplasm, they seemed to be orga-
nized in tubular structures, potentially extending through
the microvilli region, although the functional significance
of this arrangement in vivo remains unclear (Figure S5 and
Video S1).
These results were consistent with previous reports
investigating the presence of active AP-5 complexes in
other cells,67,68 supporting the hypothesis that AP-5 has
a key role in the maintenance of correct lysosomal path-
ways by facilitating the sorting step between late endo-
somes and the trans-Golgi network, also in the RPE.
Discussion
In this study, we investigated 22 individuals affected with
recessive MD from 19 unrelated families, mostly of Euro-
pean descent. All study subjects exhibited a consistent
retinal phenotype, with the median age of symptom onset
being in their 5th decade. Based on the available clinical
data, the disease seems to include flecks at an early stage,
incomplete retinal atrophy at an intermediate stage, and
complete chorioretinal atrophy extending from the mac-
ula toward the periphery at later stages. This pattern was
observed in all individuals, regardless of their genotypes,
and was supported by longitudinal data from at least three
subjects. Fleck-like deposits are common at early and inter-
mediate stages of IRDs with RPE involvement, such as Star-
gardt disease, Stargardt-like diseases, pattern dystrophies,
and central areolar choroidal dystrophy.2,69 Features dis-
tinguishing ocular findings in this study from ABCA4-asso-
ciated retinopathy are foveal sparing, early peripapillary
involvement, and reticular pigment clumping in the pe-
ripheral retina.
All affected individuals carried variants in any of three
genes encoding subunits of the AP-5 complex: AP5Z1 (pre-
viously associated with spastic paraplegia type 48, SPG48),
AP5M1, and AP5B1. Specifically, we identified 23 unique
variants, most of which (74%) were not described previ-
ously. Overall, the distribution of these pathogenic vari-
ants across the different AP-5 genes aligned well with the
relative prevalence of potentially pathogenic bi-allelic
changes in the general population. AP5Z1 cases are pre-
dicted to be themost common, followed by pathogenic ge-
notypes in AP5B1 and AP5M1. While it is still unclear
whether deleterious variants in AP5S1 are associated with
disease, population-based estimates suggest that such
A
B
D
C
Figure 4. Effect of the M2 variant in AP5Z1 on pre-mRNA splicing
(A) Schematic representation of the last bases of exon 12 and first bases of intron 12 of AP5Z1, corresponding to the electropherogram of
the PCR product obtained from the genomic DNA (gDNA) of individual P1. The arrow indicates the position of the variant M2, a single-
nucleotide change of guanine into thymine (G>T).
(B) Diagram of exons 11–13 of AP5Z1, including the position of the M2 variant and the aberrant splicing event detected. The canonical
mRNA transcript is indicated by ‘‘wt.’’
(C) Electrophoresis of RT-PCR products from healthy retina, leukocytes of a control individual (ctrl), and leukocytes of the affected in-
dividual P1, carrying M2 heterozygously, in trans with M1. The amplification product of the canonical transcript measures 885 bp in
length, while the aberrant one has a size of 744 bp, corresponding to the skipping of the whole exon 12. RT(), non-reverse transcriptase
control reaction; water, negative control of amplification.
(D) Electropherograms representing the PCR products shown in (C). The retina and the control sample contain only the canonical tran-
script, while P1 presents both wt (corresponding to the upper band on the electrophoresis gel) and aberrant (corresponding to the lower
band) transcripts.
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cause hereditary macular dystrophy, The American Journal of Human Genetics (2025), https://doi.org/10.1016/j.ajhg.2025.02.015
events would be extremely rare. Interestingly, most of the
DNA changes detected (22 out of 23) were predicted to
be LoF variants, supporting their pathogenic role and
clearly indicating that the retinal phenotype observed clin-
ically is a consequence of the lack of AP-5 components at
the cellular level. Interestingly, one of these variants
(M2), expected to result in a single amino acid substitu-
tion, was demonstrated through a functional assay to
cause the skipping of the entire exon 12 of AP5Z1 and
was classified as a splice-disrupting change. Like for M7,
M2 resulted in the in-frame deletion of several amino
acid residues of AP5Z1, in a region that is phylogenetically
conserved. Moreover, most variants were detected only in
one family, highlighting the allelic heterogeneity and
rarity of this AP-5-related class of IRDs. Some DNA changes
(M2, M3, and M8) seemed to have occurred early in
the history of the European population and be relatively
prevalent in this broad continental group, as they
mostly occurred in a compound heterozygous state in fam-
ilies from Portugal, Belgium, Sweden, and the United
Kingdom. Altogether, these data suggest common underly-
ing disease mechanisms and, potentially, shared therapeu-
tic approaches.
In the context of SPG48 due to variants in AP5Z1, visual
impairment has been noted as a non-specific clinical
feature of the disease spectrum, albeit only in five syn-
dromic individuals from a single study and from a review
paper, out of all the cases described so far.65,70 These sub-
jects primarily exhibited spastic paraparesis, sensory and
motor neuropathy, ataxia, dystonia, myoclonus, and
parkinsonism.While the brief ophthalmic description pro-
vided did not clearly associate defects in this gene with
IRDs, the only fundus image available from one of these
subjects closely resembles the chorioretinal atrophy
observed in all the individuals from our study with
advanced disease.70 Ocular abnormalities, such as pigmen-
tary retinal degeneration, ophthalmoplegia, optic atrophy,
A
B
Figure 5. AP5Z1, AP5M1, and AP5B1
display punctate localization in human
RPE explants
(A) En-face imaging of flat-mount sections
is presented, showing the localization of
AP5Z1, AP5M1, and AP5B1 (green), in
the cytoplasm of RPE cells. Phalloidin
(magenta) stains actin filaments, revealing
individual cells’ plasma membranes, which
are arranged in a typical hexagonal mosaic
pattern within this tissue. DAPI (4,6-diami-
dino-2-phenylindole; gray) stains nuclei.
Scale bars, 10 mm.
(B) Magnified images of selected regions,
focusing on individual RPE cells, demon-
strating the punctate labeling of AP5Z1,
AP5M1, and AP5B1, respectively. Scale
bars, 5 mm.
cataracts, and nystagmus, are com-
mon in other forms of spastic para-
plegia, such as SPG7 (MIM: 607259),
SPG35 (MIM: 612319), or SPG45 (MIM: 613162), not
linked to AP5Z1, where ophthalmic changes may precede
extra-ocular motor symptoms.71 Conversely, AP5M1 and
AP5B1 were not previously associated with any human he-
reditary disease.
In our cohort, all affected individuals exhibited a consis-
tent ophthalmic phenotype characterized by progressive
MD, as described above, with a variable range and severity
of extraocular symptoms. A subset of them had comorbid-
ities that are relatively prevalent in the general population
and are probably unrelated to the defects in the AP-5 genes
detected in their genome. However, ten individuals pre-
sented neurological features that could be compatible
with the disease spectrum of spastic paraplegia. Interest-
ingly, eight subjects experienced some degree of late-onset
hearing impairment (either confirmed sensorineural loss
or a subjective feeling of hearing deterioration), which
aligns with sporadic reports of hearing impairment in indi-
viduals with SPG48.65,72 Importantly, however, most
affected individuals did not report significant extraocular
symptoms (not even at advanced ages), suggesting that
variants in AP5Z1, AP5M1, and AP5B1, including bi-allelic
LoFs likely resulting in no protein products, mostly cause a
non-syndromic IRD phenotype. Indeed, it is not uncom-
mon for LSDs to manifest as an isolated retinal condition,
which can be variant specific (e.g., for mutations in
HGSNAT73) or variant-independent (e.g., CLN3-associated
diseases, where the same DNA change can cause syn-
dromic or non-syndromic phenotypes).74 Our analysis
did not reveal any clear genotype-phenotype correlations
between the specific AP-5 genes bearing pathogenic vari-
ants, their alternative transcripts, and the range and
severity of symptoms manifested by affected individuals.
The same was true for a possible relationship between
the position of a given variant with respect to the protein
primary sequence and the ascertained phenotypes, also
considering known SPG48 pathogenic variants in AP5Z1.
14 The American Journal of Human Genetics 112, 1–21, April 3, 2025
Please cite this article in press as: Kaminska et al., Bi-allelic variants in three genes encoding distinct subunits of the vesicular AP-5 complex
cause hereditary macular dystrophy, The American Journal of Human Genetics (2025), https://doi.org/10.1016/j.ajhg.2025.02.015
M6 (previously associated with SPG4870) in our cohort led
to non-syndromic ocular disease in individual P6. Simi-
larly, M8, associated in ClinVar with SPG48, was identified
in three unrelated families from our study, with no subjects
displaying manifest neurological symptoms. In particular,
one of the probands, P8, carried it homozygously and
yet displayed non-syndromic retinal deficit, which was
confirmed by a normal MRI of the brain and the spinal
cord, performed at the age of 63 years. It is possible, how-
ever, that the limited number of considered cases does not
allow any statistically meaningful analysis and that further
studies are needed to unambiguously prove or refute any
notable genotype-phenotype relationship.
In humans, there are five adaptor protein complexes
(AP-1 to AP-5), all involved in distinct pathways of intracel-
lular vesicular trafficking. In general, they are involved in
the formation of vesicles that transport cargo proteins to
various cellular compartments, such as the trans-Golgi
network, endosomes, lysosomes, and the plasma mem-
brane.75–77 Mutations in components of all five AP com-
plexes have been found to have consequences for human
health, leading to the so-called adaptinopathies, a class
of neurodevelopmental and neurometabolic disorders of
membrane trafficking.25,78 While this term provides a rela-
tively broad clinical and molecular definition, specific
studies of the AP-5 complex have shown that its depletion
Figure 6. AP5Z1, AP5M1, and AP5B1 co-localize with markers of late endosomes and the trans-Golgi network in human iPSC-RPE cell
lines
Immunofluorescence of an iPSC-derived RPE monolayer shows co-staining of all AP-5 components studied here (cyan) with Rab7
(magenta), a marker of late endosomes (left) or TGN46 (magenta), a marker of the trans-Golgi network structures (right side of each
panel). White asterisks on higher-magnification images highlight regions of co-localization, revealed either by white areas (indicative
of magenta and cyan overlap) or by magenta areas engulfing cyan spots. En-face and cross-sectional views of the same images are shown
on the left of each panel. Phalloidin (yellow) stains actin filaments and plasma membranes, and DAPI (blue) stains nuclei. Scale bars,
3 mm (images with phalloidin staining) and 1 mm (images without phalloidin staining).
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cause hereditary macular dystrophy, The American Journal of Human Genetics (2025), https://doi.org/10.1016/j.ajhg.2025.02.015
results in the accumulation of aberrant endolysosomes,
which exhibit multi-lamellar structures filled with storage
material similar to those observed in LSDs.21 AP-5 defi-
ciency also impairs the retrieval of several proteins from
late endosomes back to the Golgi apparatus, suggesting
that AP-5 facilitates a late-acting retrieval pathway that
contributes to normal lysosomal homeostasis.21 Further-
more, the absence of functional AP-5 leads to a notable
increase in the size and number of LAMP1-positive puncta
(revealing late endosomes, lysosomes, and endolyso-
somes), in turn indicating lysosomal dysfunction and sug-
gesting a specific role for the AP-5 complex in the recycling
of lysosomal enzymes and membrane proteins.25 Interest-
ingly, the loss of AP5Z1 and AP5B1 causes a concomitant
reduction in levels of AP5M1, likely due to a decreased
structural stability of the resulting aberrant AP-5 com-
plexes.23,25 Additionally, depletion of either AP5B1 or
AP5M1 causes the relocalization of lysosomal membrane
and retromer proteins as well as an increase in the size of
multivesicular bodies near the Golgi apparatus.23 Taken
together, these observations suggest that the AP-5 complex
is involved in late endosomal sorting and in lysosomal
maintenance and that its disruption impairs lysosomal
function and endosome trafficking, leading to a previously
unrecognized type of LSD.25 The AP-5 complex also inter-
acts with spatacsin (SPG11, gene: SPG11, MIM: 610844)
and spastizin (SPG15, gene: ZFYVE26, MIM: 612012),
two proteins involved in the endosomal-lysosomal
pathway.68 These proteins are in turn associated with spas-
tic paraplegia types 11 (MIM: 604360) and 15 (MIM:
270700), respectively, two conditions that include retinal
phenotypes bearing similarities with those observed in
the subjects of our study.71,79 In particular, the mild to
moderate flecked maculopathy with limited atrophy, pre-
dominantly observed in younger individuals, closely
resembled ocular features of Kjellin syndrome, a late-onset
neuro-ophthalmologic disorder caused by pathogenic var-
iants in SPG11 and ZFYVE26.80–82 Previous studies have
also shown that fibroblasts derived from individuals car-
rying mutations in SPG11 and ZFYVE26, as well as RPE
cell lines depleted for these genes, exhibit significantly
enlarged lysosomes and endolysosomes in addition to an
accumulation of autolysosomes.83,84 These data strongly
suggest that the retinal abnormalities observed in individ-
uals carrying AP-5 variants could be the consequence of
RPE dysfunction, as is the case in many other IRD
types.85–89
The RPEmonolayer, located dorsally to the neuroretina, is
crucial for maintaining retinal health and function. One of
its primary roles is the daily phagocytosis of fragments of
photoreceptorouter segments (POSs),where lysosomes facil-
itate the degradation of such internalized structures within
fused phagolysosomes. Its impairment in the RPE results in
the buildup of harmful aggregates that lead to damage of
the outer retina, a process observed in conditions such as
Stargardt disease and choroideremia,90 as well as in LSD
caused by CLN3 pathogenic variants, where patient-derived
iPSC-RPE lines exhibit disrupted POS phagocytosis.20 In
addition to heterophagy (breakdown of extracellular mate-
rial), the RPE also relies heavily on autophagy, which
involves the formation of autophagosomes that fuse with
lysosomes to degrade damaged cellular components. This is
vital for maintaining cellular homeostasis and is regulated
by various signaling pathways, which influence lysosomal
biogenesis and function.91,92 A previous study using animal
models suggested that the AP-5 complex can also play a sig-
nificant role in regulating the autophagic flux and that its
impairment leads to the accumulation of undegraded auto-
phagic cargoes, contributing to cellular stress and degenera-
tion.66 Recent research has also underscored the importance
of maintaining correct autophagic flux and lysosomal integ-
rity in the RPE to prevent retinal degeneration.92,93 Thera-
peutic strategies targeting these pathways, such as enhance-
ment of degradative enzyme activity or activation of the
AMPK pathway, have demonstrated potential in restoring
lysosomal clearance and protecting the RPE from degenera-
tion in the context of Stargardt disease.94,95 Additionally,
the rescue of lysosomal function by several lysosome-
enhancing compounds has emerged as a promising thera-
peutic strategy for treating LSDs, including SPG15.96 These
data further emphasize the critical role of lysosomes in the
proper functioning of the RPE and open the possibility of
developing treatments for AP-5-associated disorders.
To the best of our knowledge, the localization and role of
the AP-5 complex in retinal tissues have not been studied
previously. Our data on human RPE explants and wild-
type iPSC-derived RPE monolayers show punctate labeling
of all three AP5Z1, AP5M1, and AP5B1 proteins on the api-
cal side of the cell. Furthermore, we demonstrate co-local-
ization of these AP-5 subunits with markers of late endo-
somes and the trans-Golgi network, suggesting that the
AP-5 complex is primarily associated with these compart-
ments in RPE cells, consistent with findings from other
cellular models.68,97 Interestingly, we did not observe sig-
nificant co-staining of any of the studied AP-5 proteins
with LAMP2, a lysosomal marker, whereas prior studies
have shown partial localization of AP5Z1 to lyso-
somes.23,68 This may be due to differences in the studied
cell types, variations in experimental conditions, or the dy-
namic nature of vesicular trafficking, highlighting the
need for further exploration of the roles of the AP-5 com-
plex in these intracellular processes. Although we clearly
reveal the presence of the AP-5 complex in the RPE, sup-
porting the potential role of this tissue in early events of
pathogenesis for the disorder described, in this work we
do not demonstrate any direct link between this tissue
and macular degeneration. Further experimental research,
building on the genetic findings presented here, is needed
to establish the precise molecular etiology of AP-5-associ-
ated vision loss.
In summary, through a large multi-national collabora-
tive effort involving multiple centers, we have identified
pathogenic variants in the genes AP5Z1, AP5M1, and
AP5B1 as an independent cause of a recessive form of
16 The American Journal of Human Genetics 112, 1–21, April 3, 2025
Please cite this article in press as: Kaminska et al., Bi-allelic variants in three genes encoding distinct subunits of the vesicular AP-5 complex
cause hereditary macular dystrophy, The American Journal of Human Genetics (2025), https://doi.org/10.1016/j.ajhg.2025.02.015
retinal disease characterized by the progressive loss of
macular photoreceptors. This clinical phenotype likely re-
sults from defects of the AP-5 complex as a whole at the
cellular level, thus broadening the spectrum of previously
described adaptinopathies. It is plausible that future
studies will identify causative variants in the fourth
component of the AP-5 complex, AP5S1, and associate it
with a similar human phenotype. Due to the particular
clinical andmolecular features observed, we propose desig-
nating this phenotype as a distinct clinical subtype of IRD,
termed ‘‘lysosomal macular dystrophy.’’ This specific group
of disorders may share molecular pathology mechanisms
with other LSDs, adaptinopathies, and potentially other
forms of retinal blindness, suggesting the possibility of
adopting common therapeutic strategies in the future.
Data and code availability
All variants identified in this study have been submitted to the
ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/).
Acknowledgments
We would like to acknowledge all participants and their families
for their involvement in this study. We extend our deepest grati-
tude to Patricia Galliker for her exceptional support in culturing
the iPSC-RPE cells, which was instrumental to this work. We are
also grateful to Virginie G. Peter, Patricia Spa¨ni, Raquel Rodrigues,
Nils Schaerer, Daniela Hauenstein, Petra Rossouw, Chrysoula Gab-
rani, Dhryata Kamdar, Pierre Balmer, Mattias Van Heetvelde, Mar-
ieke De Bruyne, Inmaculada Martin-Merida, Lidia Rodrı´guez Pen˜a,
Luisa Quintanilla Mata, Esther Simarro Rueda, and Vasileia Mani-
adi for their support in collecting the data and to Sitta Fo¨hr for
finalizing the submission of the manuscript.
We thank the Imaging Core Facility (IMCF, Biozentrum, Univer-
sity of Basel), and in particular Alexia Loynton-Ferrand, for the
technical assistance provided on the Stellaris 8 Falcon point scan-
ning confocal microscope.
This research was made possible through access to data in the
National Genomic Research Library, which is managed by Geno-
mics England Limited (a wholly owned company of the Depart-
ment of Health and Social Care). The National Genomic Research
Library holds data provided by patients and collected by the NHS
as part of their care and data collected as part of their participation
in research. The National Genomic Research Library is funded by
the National Institute for Health Research and NHS England. The
Wellcome Trust, Cancer Research UK, and the Medical Research
Council have also funded research infrastructure.
Part of Figure S4 and the graphical abstract were created with
BioRender (www.biorender.com).
Funding sources are listed as supplemental information.
Author contributions
K.K., M.Q., and C.R. designed the study. K.K. and A.R.M. generated
wet-lab experimental data. A.P.M. and A.R.M. collected human
RPE tissue. K.K., F.C., M.Q., M.B., S.L., T.H., P.B.-M., A.A.F.,
L.F.-C., I.P.-R., G.G.G., D.S., P.M., T.Z., K.S., T.B.H., C.A., J.M.M.,
D.S., T.B.-Y., E.D.B., G.A., P.I.S., S.K., and C.R. were involved in
the genetic data generation and analysis. M.Q. was responsible
for computer-assisted analyses. L.J.-K., L. Kuehlewein, P.D.A.,
R.S., F.V.d.B., J.J., S.V., F.T., N.F., L. Koutroumanou, G.P., A.C.B.,
S.M., L.G., E.B., D.S., A.B.S., L.C.S., B.P.L., S.L., O.A.M., S.A.,
H.P.N.S., M.P., M.K.T., V.V., H.V.T., A.R.W., and C.S. contributed
with collection and evaluation of clinical data. K.K., C.S., and
C.R. wrote the original draft. All authors reviewed and approved
the manuscript.
Declaration of interests
The authors declare no competing financial or non-financial
interests.
Declaration of generative AI and AI-assisted
technologies in the writing process
AI technology was used to check for spelling mistakes and, in gen-
eral, copy-edit parts of our manuscript draft. No new text was
generated by using AI tools.
Supplemental information
Supplemental information can be found online at https://doi.org/
10.1016/j.ajhg.2025.02.015.
Web resources
ClinVar, https://www.ncbi.nlm.nih.gov/clinvar/
Franklin, https://franklin.genoox.com
GeniE, https://genie.broadinstitute.org/
gnomAD, https://gnomad.broadinstitute.org/
OMIM, https://omim.org/
RetNet, http://retnet.org
Received: September 12, 2024
Accepted: February 13, 2025
Published: March 12, 2025
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