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Genetic testing for Stargardt macular dystrophy



We studied the scientific literature and disease guidelines in order to summarize the clinical utility of genetic testing for Stargardt macular dystrophy (STGD). STGD is mostly inherited in an autosomal recessive manner and rarely in an autosomal dominant manner, with an overall prevalence of 1-5 per 10 000 live births. It is caused by variations in the ABCA4, CNGB3, ELOVL4, PRPH2 and PROM1 genes. Clinical diagnosis is based on ophthalmological examination, fluorescein angiography, electroretinography, visual field testing, optical coherence tomography and color testing. The genetic test is useful for confirming diagnosis, and for differential diagnosis, couple risk assessment and access to clinical trials.
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VOLUME 1 SPECIAL ISSUE 1 | OCTOBER 2017 | 105e EuroBiotech Journal
We studied the scientic literature and disease guidelines in order to summarize the clinical utility of genetic testing for Star-
gardt macular dystrophy (STGD). STGD is mostly inherited in an autosomal recessive manner and rarely in an autosomal
dominant manner, with an overall prevalence of 1-5 per 10 000 live births. It is caused by variations in the ABCA4, CNGB3,
ELOVL4, PRPH2 and PROM1 genes. Clinical diagnosis is based on ophthalmological examination, uorescein angiography,
electroretinography, visual eld testing, optical coherence tomography and color testing. e genetic test is useful for conrm-
ing diagnosis, and for dierential diagnosis, couple risk assessment and access to clinical trials.
Stargardt macular dystrophy
(other synonyms: Stargardt macular degeneration, juvenile macular degeneration, STGD)
(Retrieved from
General information about the disease
Stargardt macular dystrophy (STGD) is a rare congenital group of disorders including
Stargardt disease/fundus avimaculatus and Stargardt-like macular dystrophy. Onset is
typically in the rst two decades of life and characterized by loss of photoreceptors and
retinal pigment epithelium cells, decreased visual acuity (20/200), progressive loss of cen-
tral vision, scotoma, photophobia and loss of color vision.
STGD accounts for 7 % of all retinal dystrophies and its estimated prevalence is 1-5 per
10000 live births (1).
Diagnosis of STGD is based on clinical ndings, ophthalmological examination, u-
orescein angiography, autouorescence, electroretinography, visual eld testing, optical
coherence tomography and color testing. It is conrmed by detection of pathogenic vari-
ants in causative genes.
Dierential diagnosis should consider other retinal dystrophies such as central areolar
choroidal dystrophy (CACD), achromatopsia (ACHM) and cone-rod dystrophy (CORD).
STGD is most oen associated with variations in ABCA4 (OMIM gene: 601691;
OMIM disease: 248200), CNGB3 (OMIM gene: 605080; OMIM disease: 248200),
ELOVL4 (OMIM gene: 605512; OMIM disease: 600110), PRPH2 (OMIM gene: 179605)
and PROM1 (OMIM gene: 604365; OMIM disease: 603786) genes.
Pathogenic variants may include small intragenic deletions/insertions, as well as splice-
site, missense, nonsense and deep intronic variants. Partial or whole gene deletions/dupli-
cations are also reported in ABCA4 and PRPH2 genes.
1MAGI Balkans, Tirana, Albania
2MAGI’S Lab, Rovereto, Italy
3MAGI Euregio, Bolzano, Italy
4Department of Pharmaceutical Sciences,
University of Perugia, Perugia, Italy
5Department of Medical Genetics, Erciyes
University Medical School, Kayseri, Turkey
6Head and Neck Department, School of
Medicine and Surgery, University of Naples
“Federico II”, Italy
7ICORG (Imperial College Ophthalmology
Research Group), Western Eye Hospital,
London, United Kingdom
Corresponding author: M. Bertelli
Published online: 27 October 2017
Genetic testing for Stargardt macular dystrophy
Andi Abeshi1,2, Alessandra Zulian3, Tommaso Beccari4, Munis Dundar5, Fabiana D’Esposito3,6,7
and Matteo Bertelli2,3
Aims of the test
• To determine the gene defect responsible for the pathology;
• To conrm clinical diagnosis of the disease;
• To determine carrier status for the disease.
Test characteristics
Experts centers/Published guidelines
e test is listed in the Orphanet database and is oered by 25
accredited medical genetic laboratories in the EU, and in the
GTR database, oered by 12 accredited medical genetic labo-
ratories in the US.
e guidelines for clinical use of the test are described in
“Genetics home reference” (
Test strategy
A multi-gene NGS panel is used for the detection of nucleo-
tide variations in coding exons and anking introns in ABCA4,
CNGB3, ELOVL4, PRPH2 and PROM1 genes. Potentially caus-
ative variants and regions with low coverage are Sanger-se-
quenced. MLPA is used for detection of duplications and de-
letions in ABCA4 and PRPH2 genes. Sanger sequencing is also
used for family segregation studies
e test identies variations in known causative genes in pa-
tients suspected to have STGD. To perform molecular diagno-
sis, a single sample of biological material is normally sucient.
is may be 1 ml blood in a sterile tube with 0.5 ml K3EDTA or
1 ml saliva in a sterile tube with 0.5 ml ethanol 95%. Sampling
rarely has to be repeated. Gene-disease associations and the in-
terpretation of genetic variants are rapidly developing elds. It
is therefore possible that the genes mentioned in this note may
change as new scientic data is acquired. It is also possible that
genetic variants today dened as of “unknown or uncertain sig-
nicance” may acquire clinical importance.
Genetic test results
Identication of pathogenic variants in ABCA4, CNGB3,
ELOVL4, PRPH2 or PROM1 conrms the clinical diagnosis
and is an indication for family studies.
A pathogenic variant is known to be causative for a given
genetic disorder based on previous reports or predicted to be
causative based on the loss of protein function or expected sig-
nicant damage to protein or protein/protein interactions. In
this way it is possible to obtain a molecular diagnosis in new/
other subjects, establish the risk of recurrence in family mem-
bers and plan preventive and/or therapeutic measures.
Detection of a variant of unknown or uncertain signicance:
a new variation and/or without any evident pathogenic signif-
icance or with insucient or signicant conicting evidence
to indicate it is likely benign or likely pathogenic for a given
genetic disorder. In these cases, it is advisable to extend testing
to the patient’s relatives in order to assess variant segregation
and clarify its contribution. In some cases it could be necessary
to perform further examinations/tests or to do a clinical reas-
sessment of pathological signs.
Unexpected results may come out from the test, for example in-
formation regarding consanguinity; absence of family correla-
tion or the possibility of developing genetically based diseases.
e absence of variations in the genomic regions investigated does
not exclude a clinical diagnosis but suggests the possibility of:
• alterations that cannot be identied by sequencing, such as
large rearrangements that cause loss (deletion) or gain (du-
plication) of extended gene fragments;
• sequence variations in gene regions not investigated by this
test, such as regulatory regions (5’ and 3’ UTR) and deep
intronic regions;
• variations in other genes not investigated by the present test.
Risk for progeny
Autosomal recessive transmission needs that both healthy car-
rier parents transmit their disease variant to his/her children.
In this case, the probability of having an aected boy or girl is
therefore 25%.
In autosomal dominant transmission, the probability that a
carrier transmits the disease variant to his/her children is 50%
in any pregnancy, independently of the sex of the conceived.
Limits of the test
e test is limited by current scientic knowledge regarding the
genes and disease.
Analytical sensitivity (proportion of positive tests
when the genotype is truly present) and analytical
specicity (proportion of negative tests when the
genotype is not present)
NGS: Analytical sensitivity: >99% (with a minimum coverage
of 10X); Analytical specicity: 99.99%.
SANGER: Analytical sensitivity: >99.99%; Analytical specic-
ity: 99.99%.
MLPA: Analytical sensitivity: >99.99%; Analytical specicity:
Clinical sensitivity (proportion of positive tests
if the disease is present) and clinical specicity
(proportion of negative tests if the disease is not
Clinical sensitivity: two Italian studies suggest that a mutant
allele of the ABCA4 gene can be identied in 95.7% to 100%
patients with Stargardt’s disease, while biallelic variations, re-
sponsible for the phenotype, are present in 75% to 78% of pa-
tients (2,3).
VOLUME 1 SPECIAL ISSUE 1 | OCTOBER 2017 | 107e EuroBiotech Journal
Clinical specicity: is estimated at approximately 99.99%
[Author’s laboratory data] (4).
Prescription appropriateness
e genetic test is appropriate when:
a) the patient meets the diagnostic criteria for the disease;
b) the genetic test has diagnostic sensitivity greater than or
equal to other published tests.
Clinical utility
Clinical management Ulity
Conrmaon of clinical diagnosis yes
Dierenal diagnosis yes
Access to clinical trial (5) yes
Couple risk assessment yes
1. Rotenstreich Y, Fishman GA, Anderson RJ. Visual acuity loss and
clinical observations in a large series of patients with Stargardt
disease. Ophthalmology. 2003 Jun;110(6):1151-8. PubMed PMID:
2. Passerini I, Sodi A, Giambene B, Mariottini A, Menchini U, Torricelli
F. Novel mutations in of the ABCR gene in Italian patients with
Stargardt disease. Eye (Lond). 2010 Jan;24(1):158-64. doi: 10.1038/
eye.2009.35. PubMed PMID: 19265867. Epub 2009/03/06.
3. Oldani M, Marchi S, Giani A, Cecchin S, Rigoni E, Persi A, Podavini
D, et al. Clinical and molecular genetic study of 12 Italian families
with autosomal recessive Stargardt disease. Genet Mol Res. 2012
Dec 17;11(4):4342-50. PubMed PMID: 23096905.
4. Chen B, Gagnon M, Shahangian S, Anderson NL, Howerton DA,
Boone JD. Good laboratory practices for molecular genetic test-
ing for heritable diseases and conditions. MMWR Recomm Rep.
2009 Jun 12;58(RR-6):1-37. PubMed PMID: 19521335.
5. Stone EM, Aldave AJ, Drack AV, Maccumber MW, Sheeld VC,
Traboulsi E, Weleber RG. Recommendations for genetic testing
of inherited eye diseases: report of the American Academy of
Ophthalmology task force on genetic testing. Ophthalmolo-
gy. 2012 Nov;119(11):2408-10. PubMed PMID: 22944025. Epub
ResearchGate has not been able to resolve any citations for this publication.
Full-text available
Stargardt disease was diagnosed in 12 patients from 12 families using complete ophthalmologic examination, fundus photography, fundus autofluorescence, and spectral-domain optical coherence tomography. DNA was extracted for polymerase chain reaction (PCR) and direct DNA sequencing (ABCA4 gene). Genetic counseling and eye examination were offered to 16 additional family members. Various patterns of presentation were observed in patients with clinical diagnoses of Stargardt disease. The genetic study identified 2 mutations in 75% of families (9/12); a second mutation could not be found in the remaining 25% of families (3/12). The most frequent mutation was G1961E, found in 17% of families (2/12). This finding is similar to that of a previous analysis report of an Italian patient series. Four new mutations were also identified: Tyr1858Asp, Leu1195fsX1196, p.Thr850Cys, and p.Thr959Ala. Our results suggest that PCR and direct DNA sequencing are the most appropriate techniques for the analysis of the ABCA4 gene. However, this method requires supplementation with specific PCR analysis to diagnose large deletions. The study of the families identified healthy carriers and affected subjects in presymptomatic stages and was also useful for evaluating the risk of transmission to progeny. Combined ophthalmologic and genetic evaluation enabled better clinical management of these families.
Full-text available
Under the Clinical Laboratory Improvement Amendments of 1988 (CLIA) regulations, laboratory testing is categorized as waived (from routine regulatory oversight) or nonwaived based on the complexity of the tests; tests of moderate and high complexity are nonwaived tests. Laboratories that perform molecular genetic testing are subject to the general CLIA quality systems requirements for nonwaived testing and the CLIA personnel requirements for tests of high complexity. Although many laboratories that perform molecular genetic testing comply with applicable regulatory requirements and adhere to professional practice guidelines,specific guidelines for quality assurance are needed to ensure the quality of test performance. To enhance the oversight of genetic testing under the CLIA framework,CDC and the Centers for Medicare & Medicaid Services (CMS) have taken practical steps to address the quality management concerns in molecular genetic testing,including working with the Clinical Laboratory Improvement Advisory Committee (CLIAC). This report provides CLIAC recommendations for good laboratory practices for ensuring the quality of molecular genetic testing for heritable diseases and conditions. The recommended practices address the total testing process (including the preanalytic,analytic,and postanalytic phases),laboratory responsibilities regarding authorized persons,confidentiality of patient information,personnel competency,considerations before introducing molecular genetic testing or offering new molecular genetic tests,and the quality management system approach to molecular genetic testing. These recommendations are intended for laboratories that perform molecular genetic testing for heritable diseases and conditions and for medical and public health professionals who evaluate laboratory practices and policies to improve the quality of molecular genetic laboratory services. This report also is intended to be a resource for users of laboratory services to aid in their use of molecular genetic tests and test results in health assessment and care. Improvements in the quality and use of genetic laboratory services should improve the quality of health care and health outcomes for patients and families of patients.
Genetic testing can make a very positive impact on individuals and families affected with inherited eye disease in a number of ways. When properly performed, interpreted, and acted on, genetic tests can improve the accuracy of diagnoses and prognoses, can improve the accuracy of genetic counseling, can reduce the risk of disease occurrence or recurrence in families at risk, and can facilitate the development and delivery of mechanism-specific care. However, like all medical interventions, genetic testing has some specific risks that vary from patient to patient. For example, the results of a genetic test can affect a patient's plans to have children, can create a sense of anxiety or guilt, and can even perturb a patient's relationships with other family members. For these reasons, skilled counseling should be provided to all individuals who undergo genetic testing to maximize the benefits and minimize the risks associated with each test.
Stargardt disease (STGD) is the most prevalent juvenile macular dystrophy, and it has been associated with mutations in the ABCR gene, encoding a photoreceptor-specific transport protein. In this study, we determined the mutation spectrum in the ABCR gene in a group of Italian STGD patients. The DNA samples of 71 Italian patients (from 62 independent pedigrees), affected with autosomal recessive STGD, were analysed for mutations in all 50 exons of the ABCR gene by the DHPLC approach (with optimization of the DHPLC conditions for mutation analysis) and direct sequencing techniques. In our group of STGD patients, 71 mutations were identified in 68 patients with a detection rate of 95.7%. Forty-three mutations had been already reported in the literature, whereas 28 mutations had not been previously described and were not detected in 150 unaffected control individuals of Italian origin. Missense mutations represented the most frequent finding (59.2%); G1961E was the most common mutation and it was associated with phenotypes in various degrees of severity. Some novel mutations in the ABCR gene were reported in a group of Italian STGD patients confirming the extensive allelic heterogeneity of this gene-probably related to the vast number of exons that favours rearrangements in the DNA sequence.
To assess visual acuity impairment in Stargardt disease. Retrospective clinic-based cross-sectional study. Three-hundred sixty-one patients with Stargardt disease. Clinical findings in 361 patients were analyzed as part of a cross-sectional evaluation. Visual acuity at their most recent visit, fundus photographs, and electroretinographic findings were reviewed, and patients were categorized into four clinical phenotypes. Seventy-three patients with 20/40 or better vision and 38 patients with 20/50 to 20/100 vision in the better seeing eye at their initial visit who were followed for at least 1 year were included in a survival analysis. For analysis purposes, these latter patients were categorized into four 20-year age groups according to their age at initial visit. Best-corrected visual acuity from the eye with better vision on the most recent visit was used in the cross-sectional analysis. For the survival analysis, best-corrected visual acuity was used from the eye with better vision on the initial visit. Eighty-two of the 361 patients (23%) had 20/40 or better acuity in at least one eye, 64 (18%) 20/50 to 20/100, and 199 (55%) 20/200 to 20/400, whereas 16 (4%) had worse than 20/400 in each eye at their most recent visit. In the patients with visual acuity of 20/40 or better, 59 (72%) had foveal sparing visible on ophthalmoscopic examination. The median time to develop visual acuity of 20/200 or worse was 22 years for the patients with 20/40 or better visual acuity at their initial visit. Those seen initially in the first two decades of life with this level of acuity showed a median time of 7 years to reach a visual acuity of 20/200 or worse compared with 22 years and 29 years for those who were initially seen at ages 21 to 40 or 41 to 60, respectively. Analyzing by the four 20-year age groups, the log rank statistic indicated significant differences in the survival experience among the four groups (P = 0.004). The median time to develop 20/200 vision or worse was 6 years for the patients with 20/50 to 20/100 visual acuity at their initial visit, and this result, based on the log rank statistic, was independent of age group at initial visit (P = 0.852). In a large cohort of Stargardt patients, a cross-sectional analysis showed that almost a quarter had vision of 20/40 or better, whereas 4% had acuity of worse than 20/400. The presence of foveal sparing ophthalmoscopically was associated with a higher prevalence of 20/40 or better visual acuity. Survival analysis showed that the prognosis of patients who initially were seen with visual acuity of 20/40 or better is related to age at initial visit.