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Genome-wide sequencing approaches are increasingly being used in place of disease gene panel sequencing approaches. Despite the well-recognized benefits of these approaches, they also carry with them an increased burden of analyzing overwhelmingly large gene targets and an increased possibility of detecting incidental findings. We propose a novel approach for design of individualized phenotype gene panels using the set of signs and symptoms observed and selecting relevant genes on the basis of known phenotype–gene associations. We used results of diagnostic exome sequencing in 405 cases submitted to our institution to show retrospectively that using the phenotype gene panel increases the sensitivity of masked exome analysis (increase from 25.4 to 29.7% in overall diagnostic yield). We also show that such a strategy enables the possibility of masked analysis of genome-wide sequencing data in patients with poorly defined and multifaceted clinical presentations. Ultimately, we show that this approach enables control over the incidental findings rate (0.25% in phenotype gene panels). Finally, we provide a Web tool for customized phenotype panel creation (available at http://www.kimg.eu/generator). In conclusion, we present a novel approach to a phenotype-driven diagnostic process of genome scale sequencing data that harnesses the sensitivity of these approaches while restricting the analysis to genes relevant to clinical presentation in patient. Genet Med18 11, 1102–1110.

The striking similarity of disc edema without leakage on fluorescein angiography, which is pathognomonic of Leber hereditary optic neuropathy (LHON), was present in a patient with cystic fibrosis with antibiotic toxic optic neuropathy. This similarity suggested the common effect of oxidative stress on retinal ganglion cells in inherited mitochondrial and antibiotic optic neuropathies. We present the case of a patient with advanced cystic fibrosis on chronic antibiotic treatment who experienced a rapid painless bilateral visual decline over a course of a few weeks. At examination, his corrected visual acuity was reduced to 0.3 in both eyes, with dyschromatopsia and central scotoma. The appearance of the fundus resembled the typical clinical features of acute LHON with hyperemic optic discs and tortuous vessels with no dye leakage from the optic discs on fluorescein angiography. Ganglion cell layer loss was seen on optic coherence tomography, with all findings pointing to LHON. Genetic testing did not reveal any LHON-specific mutations. After extended genetic testing, a heterozygous variant c.209C>T in the OPA3 gene on chromosome 19, g.46032648G>A, classified as a variant of unknown significance, was also found. After discontinuing antibiotics and general improvements in his health, surprisingly, his visual function completely improved. Later, he also received a bilateral lung transplant that further improved his general condition, and his vision remained normal. Excluding LHON, the transient optic neuropathy in our patient could be mainly due to antibiotic toxicity of linezolid and ciprofloxacin, which have been linked to mitochondrial dysfunction and advanced cystic fibrosis with hypoxic status. We suggest the possibility that patients with cystic fibrosis may be more prone to developing mitochondrial optic neuropathy, especially with additional risk factors such as chronic antibiotic therapy, which affect mitochondrial function, and can perhaps serve as a model for LHON.

A universal biomarker panel with the potential to predict high-risk pregnancies or adverse pregnancy outcome does not exist. Transcriptome analysis is a powerful tool to capture differentially expressed genes (DEG), which can be used as biomarker-diagnostic-predictive tool for various conditions in prenatal setting. In search of biomarker set for predicting high-risk pregnancies, we performed global expression profiling to find DEG in Ts21. Subsequently, we performed targeted validation and diagnostic performance evaluation on a larger group of case and control samples. Initially, transcriptomic profiles of 10 cultivated amniocyte samples with Ts21 and 9 with normal euploid constitution were determined using expression microarrays. Datasets from Ts21 transcriptomic studies from GEO repository were incorporated. DEG were discovered using linear regression modelling and validated using RT-PCR quantification on an independent sample of 16 cases with Ts21 and 32 controls. The classification performance of Ts21 status based on expression profiling was performed using supervised machine learning algorithm and evaluated using a leave-one-out cross validation approach. Global gene expression profiling has revealed significant expression changes between normal and Ts21 samples, which in combination with data from previously performed Ts21 transcriptomic studies, were used to generate a multi-gene biomarker for Ts21, comprising of 9 gene expression profiles. In addition to biomarker's high performance in discriminating samples from global expression profiling, we were also able to show its discriminatory performance on a larger sample set 2, validated using RT-PCR experiment (AUC=0.97), while its performance on data from previously published studies reached discriminatory AUC values of 1.00. Our results show that transcriptomic changes might potentially be used to discriminate trisomy of chromosome 21 in the prenatal setting. As expressional alterations reflect both, causal and reactive cellular mechanisms, transcriptomic changes may thus have future potential in the diagnosis of a wide array of heterogeneous diseases that result from genetic disturbances.

To evaluate the impact of prenatal screening and genetic testing for trisomy 21 (T21) on the prevalence of T21 in Slovenia. Data about all prenatally and postnatally confirmed cases of T21 in Slovenia between 1981 and 2012 were collected retrospectively from all genetic laboratories in Slovenia. The expected number of babies with T21 according to maternal age was calculated. The primary outcomes measures were number of fetuses and newborn infants with T21 diagnosed prenatally and postnatally and the impact of advances in screening and genetic diagnostics on the prevalence of newborns with T21 in Slovenia. Despite a significantly increased mean maternal age from 25.4 years in year 1981 to 30.3 years in year 2012 the prevalence of newborn infants with T21 was 0.51 per 1000 births compared to 0.55 per 1000 births, respectively. The prevalence of prenatally diagnosed cases increased from 0.03 per 1000 births to 2.06 per 1000. The detection rate of T21 in year 2012 was 78,9%. The total number of prenatal invasive procedures (chorionic villous sampling and amniocenteses) carried out during that period was rising until 2002, since when it is stable at around 7%. The advancement and implementation of screening tests and prenatal diagnostic procedures in Slovenia caused an important improvement in the efficiency of the prenatal detection of T21.

The pathogenic variant p.G90D in RHO is believed to be responsible for a spectrum of phenotypes, including congenital stationary blindness (for the purpose of this study termed night blindness without degeneration; NBWD), Sector RP, Pericentral RP, and Classic RP. We present a correlation between the serum concentration of vitamin A and disease severity in patients with this variant. This prospective study involved 30 patients from 7 families (17 male; median age 46 years, range 8–73). Full ophthalmological examination including visual acuity, Goldmann perimetry, slit-lamp exam, optical coherence tomography, fundus autofluorescence, and electrophysiology was performed to determine the presenting phenotype. The serum concentration of vitamin A was determined from a fasting blood sample taken on the day of the exam, where it was found that 23.3% (7/30) of patients had NBWD, 13.3% (4/30) had Sector RP, 3.3% (1/30) had Pericentral RP, and 60% (18/30) had Classic RP. Multiple logistic regression revealed a significantly higher probability of having a milder phenotype (NBWD or Sector RP) in association with younger age (p < 0.05) and a higher concentration of vitamin A (p < 0.05). We hypothesize that vitamin A in its 11-cis-retinal form plays a role in stabilizing the constitutively active p.G90D rhodopsin and its supplementation could be a potential treatment strategy for p.G90D RHO patients.

Myotonic dystrophy 1 (DM1) is known to diminish reproductive fitness in its severe form. Since no de novo mutations are known for this disease, it has the tendency to become extinct from a population. To explain the preservation of DM1 in a population, a hypothesis that a pool of subjects for the mutated gene exists in the apparently healthy (non-DM1) population was tested. In order to determine the (CTG) repeat number, PCR was performed in 274 patients found to have primary cataract of adult onset who showed no DM1 symptoms, and were not related to DM1 patients. In four cataract patients (1.46%; 95% CI 0.5–3.7), a protomutation in the myotonin protein kinase gene was found which might lead to a complete mutation after transmission through the next generations. The number of (CTG) repeats in the remaining 270 cataract patients did not differ significantly from the control subjects in terms of the distribution of larger [(CTG) n  ≥ 19] versus smaller [(CTG) n  < 19] alleles. We consider the primary cataract patients to be the pool of DMPK protomutation from which DM1 mutation is maintained in the population.

Mutations in rhodopsin gene (RHO) are a frequent cause of retinitis pigmentosa (RP) and less often, congenital stationary night blindness (CSNB). Mutation p.G90D has previously been associated with CSNB based on the examination of one family. This study screened 60 patients. Out of these 60 patients, 32 were affected and a full characterization was conducted in 15 patients. We described the clinical characteristics of these 15 patients (12 male, median age 42 years, range 8–71) from three families including visual field (Campus Goldmann), fundus autofluorescence (FAF), optical coherence tomography (OCT) and electrophysiology. Phenotypes were classified into four categories: CSNB (N = 3, 20%) sector RP (N = 3, 20%), pericentral RP (N = 1, 6.7%) and classic RP (N = 8, 53.3% (8/15)). The phenotypes were not associated with family, sex or age (Kruskal–Wallis, p > 0.05), however, cystoid macular edema (CME) was observed only in one family. Among the subjects reporting nyctalopia, 69% (22/32) were male. The clinical characteristics of the largest p.G90D cohort so far showed a large frequency of progressive retinal degeneration with 53.3% developing RP, contrary to the previous report.

Pathogenic variants in DNA-damage regulated autophagy modulator 2 gene (DRAM2) cause a rare autosomal recessive retinal dystrophy and its disease course is not well understood. We present two Slovenian patients harboring a novel DRAM2 variant and a detailed review of all 23 other patients described to date. Whole exome and whole genome sequencing were performed in the two patients, and both underwent ophthalmological examination with a 2-year follow-up. PubMed was searched for papers with clinical descriptions of DRAM2 retinopathy. Patient 1 was homozygous for a novel variant, p.Met1?, and presented with the acute onset of photopsia and retina-wide retinopathy at the age of 35 years. The patient was first thought to have an autoimmune retinopathy and was treated with mycophenolate mofetil, which provided some symptomatic relief. Patient 2 was compound heterozygous for p.Met1? and p.Leu246Pro and presented with late-onset maculopathy at the age of 59 years. On review, patients with DRAM2 retinopathy usually present in the third decade with central visual loss, outer retinal layer loss on optical coherence tomography and a hyperautofluorescent ring on fundus autofluorescence. Either cone–rod or rod–cone dystrophy phenotype is observed on electroretinography, reflecting the importance of DRAM2 in both photoreceptor types. Non-null variants can result in milder disease.

Genetic characteristics and a long-term clinical follow-up of 18 Slovenian retinitis pigmentosa GTPase regulator (RPGR) patients from 10 families with retinitis pigmentosa (RP) or cone/cone-rod dystrophy (COD/CORD) are reported. RP (eight families) was associated with two already known (p.(Ser407Ilefs*46) and p.(Glu746Argfs*23)) and five novel variants (c.1245+704_1415-2286del, p.(Glu660*), p.(Ala153Thr), c.1506+1G>T, and p.(Arg780Serfs*54)). COD (two families) was associated with p.(Ter1153Lysext*38). The median age of onset in males with RP (N = 9) was 6 years. At the first examination (median age of 32 years), the median best corrected visual acuity (BCVA) was 0.30 logMAR, and all patients had a hyperautofluorescent ring on fundus autofluorescence (FAF) encircling preserved photoreceptors. At the last follow-up (median age of 39 years), the median BCVA was 0.48 logMAR, and FAF showed ring constriction transitioning to patch in 2/9. Among females (N = 6; median age of 40 years), two had normal/near-normal FAF, one had unilateral RP (male pattern), and three had a radial and/or focal pattern of retinal degeneration. After a median of 4 years (4–21) of follow-up, 2/6 exhibited disease progression. The median age of onset in males with COD was 25 years. At first examination (median age of 35 years), the median BCVA was 1.00 logMAR, and all patients had a hyperautofluorescent FAF ring encircling foveal photoreceptor loss. At the last follow-up (median age of 42 years), the median BCVA was 1.30 logMAR, and FAF showed ring enlargement. The majority of the identified variants (75%; 6/8) had not been previously reported in other RPGR cohorts, which suggested the presence of distinct RPGR alleles in the Slovenian population.

Variants in WDR19 (IFT144) have been implicated as another possible cause of Stargardt disease. The purpose of this study was to compare longitudinal multimodal imaging of a WDR19-Stargardt patient, harboring p.(Ser485Ile) and a novel c.(3183+1_3184-1)_(3261+1_3262-1)del variant, with 43 ABCA4-Stargardt patients. Age at onset, visual acuity, Ishihara color vision, color fundus, fundus autofluorescence (FAF), spectral-domain optical coherence tomography (OCT) images, microperimetry and electroretinography (ERG) were evaluated. First symptom of WDR19 patient was nyctalopia at the age of 5 years. After the age of 18 years, OCT showed hyper-reflectivity at the level of the external limiting membrane/outer nuclear layer. There was abnormal cone and rod photoreceptor function on ERG. Widespread fundus flecks appeared, followed by perifoveal photoreceptor atrophy. Fovea and peripapillary retina remained preserved until the latest exam at 25 years of age. ABCA4 patients had median age of onset at 16 (range 5–60) years and mostly displayed typical Stargardt triad. A total of 19% had foveal sparing. In comparison to ABCA4 patients, the WDR19 patient had a relatively large foveal preservation and severe rod photoreceptor impairment; however, it was still within the ABCA4 disease spectrum. Addition of WDR19 in the group of genes producing phenocopies of Stargardt disease underlines the importance of genetic testing and may help to understand its pathogenesis.