Skorczyk‑Werner et al. Orphanet J Rare Dis (2020) 15:345 https://doi.org/10.1186/s13023‑020‑01634‑y RESEARCH Open Access Novel gene variants in Polish patients with Leber congenital amaurosis (LCA) Anna Skorczyk‑Werner1* , Zuzanna Niedziela1,2, Marcin Stopa2 and Maciej Robert Krawczyński1,3 Abstract Background: Leber congenital amaurosis (LCA) is a rare retinal disease that is the most frequent cause of congenital blindness in children and the most severe form of inherited retinal dystrophies. To date, 25 genes have been impli‑ cated in the pathogenesis of LCA. As gene therapy is becoming available, the identifcation of potential treatment candidates is crucial. The aim of the study was to report the molecular basis of Leber congenital amaurosis in 22 Polish families. Methods: Single Nucleotide Polymorphism‑microarray for LCA genes or Next Generation Sequencing diagnostic panel for LCA genes (or both tests) were performed to identify potentially pathogenic variants. Bidirectional Sanger sequencing was carried out for validation and segregation analysis of the variants identifed within the families. Results: The molecular background was established in 22 families. From a total of 24 identifed variants, 23 were pre‑ dicted to afect protein‑coding or splicing, including 10 novel variants. The variants were identifed in 7 genes: CEP290, GUCY2D, RPE65, NMNAT1, CRB1, RPGRIP1, and CRX. More than one‑third of the patients, with clinical LCA diagnosis con‑ frmed by the results of molecular analysis, appeared to be afected with a severe form of the disease: LCA10 caused by the CEP290 gene variants. Intronic mutation c.2991 1655A>G in the CEP290 gene was the most frequent variant identifed in the studied group. + Conclusions: This study provides the frst molecular genetic characteristics of patients with Leber congenital amau‑ rosis from the previously unexplored Polish population. Our study expands the mutational spectrum as we report 10 novel variants identifed in LCA genes. The fact that the most frequent causes of the disease in the studied group of Polish patients are mutations in one out of three genes that are currently the targets for gene therapy (CEP290, GUCY2D, and RPE65) strongly emphasizes the importance of the molecular background analyses of LCA in Polish patients. Keywords: Leber congenital amaurosis (LCA), Novel variants, SNP‑microarray for LCA genes, Targeted NGS panel for LCA genes Background IRDs. Te disease typically becomes evident in the frst Leber congenital amaurosis (LCA) is a rare retinal disease year of life, and it is estimated that about 20% of chil- that is the most frequent cause of inherited blindness in dren with visual impairment in specialized schools are children. LCA is the most severe form of all inherited afected by LCA [1]. Te prevalence of the disorder is retinal dystrophies (IRD) and accounts for about 5% of all estimated to be 1 in 30,000 [2], and there can be about 1000–1200 patients sufering from LCA in Poland. To date, the following 25 genes have been implicated in the *Correspondence: [email protected] pathogenesis of LCA: AIPL1, CABP4, CCT2, CEP290, 1 Department of Medical Genetics, Poznan University of Medical Sciences, CLUAP1, CRB1, CRX, DTHD1, GDF6, GUCY2D, IFT140, 8, Rokietnicka St, 60‑806 Poznan, Poland Full list of author information is available at the end of the article IMPDH1, IQCB1, KCNJ13, LCA5, LRAT, NMNAT1, © The Author(s) 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Skorczyk‑Werner et al. Orphanet J Rare Dis (2020) 15:345 Page 2 of 19 OTX2, PRPH2, RD3, RDH12, RPE65, RPGRIP1, SPATA7, Review Board. Written informed consent was obtained TULP1 [3]. LCA is most often inherited in an autoso- from all participants or their legal guardians. mal recessive manner, but in rare cases, the disease may also be transmitted as an autosomal dominant trait. Clinical diagnosis Te symptoms usually include severe and early visual A total of 28 patients from 22 unrelated families living in impairment, Franceschetti’s oculo-digital sign (compris- Poland (21 families of Polish ethnicity and 1 of Romany ing eye-poking, pressing, and rubbing), nystagmus, and origin) with a clinical diagnosis of LCA confrmed by sluggish or near-absent pupillary responses. Other typi- molecular analysis results were evaluated in this study. cal ophthalmological features associated with LCA are Ophthalmologic examinations, including best-corrected photophobia, refraction defects, nyctalopia, keratoconus, visual acuity (BCVA) and funduscopy, were performed and cataract. Te electroretinogram (ERG) is character- in all the probands. Electroretinography (ERG) was per- istically "nondetectable" or severely subnormal [2]. LCA’s formed in 24 patients (it was not performed in patients: clinical symptoms are very often similar to these of dif- 4–11, 11–36, 15–44, 17–57). Spectral-Domain Optical ferent retinal dystrophies, so the accurate clinical diag- Coherent Tomography (SD-OCT) retinal scans (Opto- nosis, especially in infants, sometimes cannot be made vue, Fremont, CA, USA) were obtained in four families: at the frst visit or has to be revised once the molecular Family no. 6 (patients 6–18, 6–19, 6–52), Family 10 ( analysis is performed. Terefore, genetic-molecular test- 10–33), Family 13 (patients 13–53 and 13–54) and Family ing is necessary to obtain a defnitive diagnosis of LCA 17 (patient no.17–57). Fluorescein angiography (FA) was through pathogenic variant identifcation. Unraveling the done in three patients: 10–33, 13–53, and 17–57. In four molecular background of the disease contributes to the patients, we conducted perimetry: 10–33, 13–53, 17–57, identifcation of the potential treatment gene candidates and 22–50. Magnetic resonance imaging (MRI) of the and the development of therapeutic approaches. Te aim head and eye orbits were obtained during early infancy of the study was to report the molecular basis of Leber in seven subjects: 3–7, 5–15, 8–25, 9–29, 12–39, 16–56, congenital amaurosis in 22 families living in Poland. 20–59. Some patients, especially older ones, do not remember whether they underwent MRI. Te symptoms Material and methods observed in our study group are listed in Table 1. Patients, ethic statements 51 Polish families with a clinical diagnosis of LCA were Molecular genetic analysis referred to our genetic clinic in 2010–2019, and 44 of Blood samples from the afected individuals, their them had the molecular analyses. Some of these patients healthy parents, and their unafected siblings (in families: did not decide to order the genetic tests as they are quite 3, 7, 8, 12, and 19) were obtained for genetic examination expensive and not funded by the National Health Fund in (the total number of samples analyzed was 60). Genomic Poland. Moreover, until 2016 only LCA SNP microarray DNA was extracted from peripheral blood leukocytes based on the Arrayed Primer Extension (APEX) approach using standard protocols. DNA samples of one proband analysis (Asper Ophthalmics, Asper Biotech Ltd., Tartu, from each family with LCA were subjected to either an Estonia) was performed that did not allow to iden- LCA mutation chip based on the APEX approach or tify novel variants. Among patients who had LCA SNP targeted NGS diagnostic panel for LCA genes (Asper microarray test only (35 patients), there is a group with- Biogene, Asper Biotech Ltd., Tartu, Estonia) or both of out any mutation found (16 patients), with one heterozy- these tests. Searching for the molecular background of gous variant identifed in autosomal recessive LCA genes LCA in patients referred to the genetic clinic until 2016 (6 patients) and the group of patients with the molecu- (13 families; see the last column in Table 2) was per- larly confrmed LCA diagnosis (13 patients). In this formed based on SNP microarray test, while in those study, we focused on a group of 22 families (28 patients) who came to the clinic in 2017–2019—the NGS-LCA with LCA diagnosis fully confrmed by the results of panel was carried out (9 families; Table 2).NGS on the molecular analysis based on SNP microarray and NGS- diagnostic panel for LCA genes was also performed in 4 LCA panel. Patients were numbered with Patient ID, families who did not show any known variants detected where the frst digit indicated the Family number and the by the SNP microarray.Te SNP-chip for LCA was used next digit after the hyphen was the individual’s laboratory to screen the total of 780 mutations in the following 15 number. Tis study was conducted in accordance with genes: AIPL1, CEP290, CRB1, CRX, GUCY2D, IQCB1, the tenets of the Declaration of Helsinki and the Associa- LCA5, LRAT, MERTK, RD3, RDH12, RPE65, RPGRIP1, tion for Research in Vision and Ophthalmology (ARVO) SPATA7, TULP1. Te NGS diagnostic panel captured the statement on human subjects.