Genetic testing for achromatopsia

The EuroBiotech Journal TECHNICAL REPORT © 2017 European Biotechnology Thematic Network Association EBTNA UTILITY GENE TEST Genetic testing for achromatopsia Andi Abeshi1,2, Alessandra Zulian3, Tommaso Beccari4, Munis Dundar5, Benedetto Falsini6 and Matteo Bertelli2,3 Abstract We studied the scientific literature and disease guidelines in order to summarize the clinical utility of genetic testing for achromatopsia. The disease has autosomal recessive inheritance, a prevalence of 1/30000-1/50000, and is caused by mutations in the CNGB3, CNGA3, GNAT2, PDE6C, ATF6 and PDE6H genes. Clinical diagnosis is by ophthalmological examination, color vision testing and electrophysiological testing. Genetic testing is useful for confirming diagnosis and for differential diagnosis, couple risk assessment and access to clinical trials. Achromatopsia 1 MAGI Balkans, Tirana, Albania 2 MAGI’S Lab, Rovereto, Italy (other synonyms: complete or incomplete achromatopsia, pingelapese blindness, rod monochromatism, rod monochromacy, complete or incomplete color blindness) (1). 3 MAGI Euregio, Bolzano, Italy 4 Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy 5 Department of Medical Genetics, Erciyes University Medical School, Kayseri, Turkey 6 Department of Ophthalmology, Catholic University of Rome, Rome, Italy Corresponding author: M. Bertelli E-mail: info@assomagi.org Published online: 27 October 2017 doi:10.24190/ISSN2564-615X/2017/S1.03 The EuroBiotech Journal General information about the disease Achromatopsia (acronym ACHM) is a rare congenital disorder characterized by reduced visual acuity (<0.2), pendular nystagmus, eccentric fixation, increased sensitivity to light (photophobia), a small central scotoma, reduced or complete loss of color discrimination and absence of cone-mediated electroretinographic amplitudes (2,3). Most individuals have complete ACHM, affecting the function of all three types of cones. Incomplete ACHM is much less frequent and has similar but generally less severe symptoms. The estimated prevalence of ACHM is 1/30,000 (4). The diagnosis of ACHM is based on ophthalmological examination, testing of color vision and electroretinography (ERG), which shows loss of photopic but normal scotopic response. Optical coherence tomography shows progressive disruption and/or loss of the inner/outer segment junction of photoreceptors and attenuation of retinal pigmented epithelium (RPE) in the macular region. The diagnosis is confirmed by molecular genetic analysis of the responsible genes. Differential diagnosis should consider blue cone monochromatism (BCM), Leber congenital amaurosis, other cone dystrophies, hereditary red-green color vision defects, yellow-blue defects and cerebral ACHM. ACHM is a heterogeneous disorder with autosomal recessive inheritance. Pathogenic variants of CNGA3 (OMIM gene: 600053; OMIM disease: 216900), CNGB3 (OMIM gene: 605080; OMIM disease: 262300), GNAT2 (OMIM gene: 139340; OMIM disease 613856), PDE6C (OMIM gene: 600827; OMIM disease: 613093), ATF6 (OMIM gene: 605537; OMIM disease: 616517) and PDE6H (OMIM gene: 601190; OMIM disease: 610024) have been reported to be causative for autosomal recessive ACHM (5,6). CNGA3 and CNGB3 are the major causative genes of ACHM, and account for ~20-30% and 40-50% of the cases, respectively (6,7). VOLUME 1 SPECIAL ISSUE 1 | OCTOBER 2017 | 11 Pathogenic variants may include small intragenic deletions/ insertions, splice site variants, missense and nonsense variations; typically, exon or whole-gene duplications/deletions are not detected. Aims of the test • To determine the gene defect responsible for the pathology; • To confirm clinical diagnosis of the disease; • To determine carrier status for the disease. a new variation and/or without any evident pathogenic significance or with insufficient or significant conflicting 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 reassessment of pathological signs. Negative Test characteristics Experts centers/Published guidelines This test is found in the Orphanet database and is offered by 58 accredited medical genetic laboratories in the EU, and in the GTR database, offered by 10 accredited medical genetic laboratories in the US. The guidelines for clinical use of this test are described in “Clinical utility gene card” (1) and “Gene reviews” (8). Test strategy A multi-gene NGS panel is used for the detection of nucleotide variations in coding exons and flanking introns in ATF6, CNGA3, CNGB3, GNAT2, PDE6C, and PDE6H genes. Potentially causative variants and regions with low coverage are Sanger-sequenced. Sanger sequencing is also used for family segregation studies. The test identifies variations in known causative genes in patients suspected to have ACHM. To perform molecular diagnosis, a single sample of biological material is normally sufficient. This 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 interpretation of genetic variants are rapidly developing fields. It is therefore possible that genes mentioned in this note may change as new scientific data is acquired. It is also possible that genetic variants today defined as of “unknown or uncertain significance” may acquire clinical importance. Genetic test results Positive Identification of biallelic pathogenic variants in CNGB3, CNGA3, GNAT2, PDE6C, ATF6 or PDE6H genes confirms 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 significant 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 members and plan preventive and/or therapeutic measures. Inconclusive Detection of a variant of unknown or uncertain significance: 12 | VOLUME 1 SPECIAL ISSUE 1 | OCTOBER 2017 The absence of variations in the genomic regions investigated does not exclude a clinical diagnosis but suggests the possibility of: • alterations that cannot be identified by sequencing, such as large rearrangements that cause loss (deletion) or gain (duplication) of extended gene fragments; • sequence variations in gene regions not investigated by the test, such as regulatory regions (5’ and 3’ UTR) and deep intronic regions; • variations in other genes not investigated by the present test. Unexpected Unexpected results may come out from the test, for example information regarding consanguinity, absence of family correlation or the possibility of developing genetically based diseases. Risk for progeny Autosomal recessive transmission needs that both healthy carrier parents transmit their disease variant to his/her children. In this case, the probability of having an affected boy or girl is therefore 25%. Limits of the test The test is limited by current scientific knowledge regarding the genes and disease. Analytical sensitivity (proportion of positive tests when the genotype is truly present) and analytical specificity (proportion of negative tests when the genotype is not present) NGS: Analytical sensitivity: >99% (with a minimum coverage of 10X); Analytical specificity: 99.99%. SANGER: Analytical sensitivity: >99.99%; Analytical specificity: 99.99%. Clinical sensitivity (proportion of positive tests if the disease is present) and clinical specificity (proportion of negative tests if the disease is not present) Clinical sensitivity: variations in genes associated with ACHM are identified in more than 75% of cases (1). Clinical specificity is estimated at approximately 99.99% [Author’s laboratory data] (9). www.eurobiotechjournal.org Prescription appropriateness 4. The genetic test is appropriate when: a) the patient meets the diagnostic criteria for achromatopsia b) the genetic test has diagnostic sensitivity greater than or equal to other published tests (≥75% of positive tests) (1). 5. Clinical utility Clinical management Utility Confirmation of clinical diagnosis yes Differential diagnosis yes Access to clinical trial (10) yes Couple risk assessment yes 6. 7. References 1. 2. 3. Kohl S, Hamel C. Clinical utility gene card for: Achromatopsia - update 2013. Eur J Hum Genet. 2013 Nov;21(11). PubMed PMID: 23486539; PubMed Central PMCID: PMC3798849. Epub 2013/03/13. Greenberg JP, Sherman J, Zweifel SA, Chen RW, Duncker T, Kohl S, Baumann B, et al. Spectral-domain optical coherence tomography staging and autofluorescence imaging in achromatopsia. JAMA Ophthalmol. 2014 Apr 1; 132(4):437-45. PubMed PMID: 24504161; PubMed Central PMCID: PMC4423754. Pang JJ, Alexander J, Lei B, Deng W, Zhang K, Li Q, Chang B, et al. Achromatopsia as a potential candidate for gene therapy. Adv Exp Med Biol. 2010; 664:639-46. PubMed PMID: 20238068; PubMed Central PMCID: PMC3608407. The EuroBiotech Journal 8. 9. 10. Kohl S, Baumann B, Rosenberg T, Kellner U, Lorenz B, Vadalà M, Jacobson SG, et al. Mutations in the cone photoreceptor G-protein alpha-subunit gene GNAT2 in patients with achromatopsia. Am J Hum Genet. 2002 Aug; 71(2):422-5. PubMed PMID: 12077706; PubMed Central PMCID: PMC379175. Epub 2002/06/20. Ouechtati F, Merdassi A, Bouyacoub Y, Largueche L, Derouiche K, Ouragini H, Nouira S, et al. Clinical and genetic investigation of a large Tunisian family with complete achromatopsia: identification of a new nonsense mutation in GNAT2 gene. J Hum Genet. 2011 Jan;56(1):22-8. PubMed PMID: 21107338. Epub 2010/09/25. Kohl S, Varsanyi B, Antunes GA, Baumann B, Hoyng CB, Jägle H, Rosenberg T, et al. CNGB3 mutations account for 50% of all cases with autosomal recessive achromatopsia. Eur J Hum Genet. 2005 Mar; 13(3):302-8. PubMed PMID: 15657609. Wissinger B, Gamer D, Jägle H, Giorda R, Marx T, Mayer S, Tippmann S, et al. CNGA3 mutations in hereditary cone photoreceptor disorders. Am J Hum Genet. 2001 Oct; 69(4):722-37. PubMed PMID: 11536077; PubMed Central PMCID: PMC1226059. Epub 2001/08/30. Kohl S, Jägle H, Wissinger B. Achromatopsia. In: RA Pagon, MP Adam, HH Ardinger, SE Wallace, A Amemiya, LJH Bean, TD Bird et al. GeneReviews®. Seattle (WA):1993-2017. Chen B, Gagnon M, Shahangian S, Anderson NL, Howerton DA, Boone JD. Good laboratory practices for molecular genetic testing for heritable diseases and conditions. MMWR Recomm Rep. 2009 Jun 12; 58(RR-6):1-37. PubMed PMID: 19521335. Stone EM, Aldave AJ, Drack AV, Maccumber MW, Sheffield 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. Ophthalmology. 2012 Nov;119(11):2408-10. PubMed PMID: 22944025. Epub 2012/09/01. VOLUME 1 SPECIAL ISSUE 1 | OCTOBER 2017 | 13