Blueprint Genetics (Finland)

PurposeWhile cancer phenotypes in carriers of a single mutant allele in most major cancer susceptibility genes are well-established, there is a paucity of data on the phenotype of carriers of two pathogenic variants—double heterozygotes (DH) or homozygous carriers. Here, we describe the phenotype of carriers of homozygous and DH pathogenic sequence variants (PSVs) in major cancer susceptibility genes.MethodsIndividuals referred for multigene panel testing at Blueprint Genetics laboratory were included. Ethically approved comparison of cancer type and age at diagnosis between DH, homozygous, and single PSV carriers was performed per gene.ResultsOf 6,685 eligible participants, 928 (13.9%) were single heterozygous PSV carriers, 6 (0.09%) were homozygous PSV carriers, and 17 (0.25%) were DH PSV carriers. Mean age at diagnosis of any cancer among single PSV age was 46.8 ± 14.9 years and among DH PSV carriers 37.6 ± 13.0 years (P < 0.0001). Notably, age at diagnosis for breast cancer among single BRCA1 PSV carriers (n = 59) was 43.8 ± 8.7 years (p = 0.7606), among single BRCA2 PSV carriers (n = 52)—47.9 ± 13.0 years (p = 0.2274) compared with 42.3 ± 13.0 years among DH PSV carriers (n = 10- 9 of whom were carriers of either BRCA1 or BRCA2).ConclusionDH for PSV in two cancer susceptibility genes is a rare event, and the mean age at cancer diagnosis is younger in DH PSV carriers compared with single PSV carriers.

Retinitis pigmentosa (RP), a heterogenous group of inherited retinal disorder causes slow progressive vision loss with no effective treatments available. Mutations in the rhodopsin gene (RHO), account for ∼25% cases of autosomal dominant RP (adRP). In this study, we describe the disease characteristics of the first ever reported mono-allelic copy number variation (CNV) in RHO as a novel cause of adRP. We (1) show advanced retinal degeneration in a male patient (60-70 year old) harboring four transcriptionally active intact copies of rhodopsin, (2) recapitulated the clinical phenotypes using retinal organoids, and (3) assessed the utilization of a small molecule, Photoregulin3 (PR3), as a clinically viable strategy to target and modify disease progression in RP patients associated with RHO-CNV. Patient retinal organoids showed photoreceptors dysgenesis, with rod photoreceptors displaying stunted outer segments with occasional elongated cilia-like projections (microscopy); increased RHO mRNA expression (qRT-PCR and bulk RNA-sequencing); and elevated levels and mislocalization of rhodopsin protein (RHO) within the cell body of rod photoreceptors (western blotting and immunohistochemistry) over the extended (300-days) culture time period when compared against control organoids. Lastly, we utilized PR3 to target NR2E3, an upstream regulator of RHO, to alter RHO expression and observed a partial rescue of RHO protein localization from the cell body to the inner/outer segments of rod photoreceptors in patient organoids. These results provide a proof-of-principle for personalized medicine and suggest that RHO expression requires precise control. Taken together, this study supports the clinical data indicating that adRP due to RHO-CNV develops due protein overexpression overloading the photoreceptor post-translational modification machinery.

Abstract Retinitis pigmentosa (RP), a heterogenous group of inherited retinal disorder causes slow progressive vision loss with no effective treatments available. Mutations in the rhodopsin gene (RHO), account for ∼40% cases of autosomal dominant RP (adRP). In this study, we describe the disease characteristics of the first ever reported mono-allelic copy number variation (CNV) in RHO as a novel cause of adRP. We (1) show advanced retinal degeneration in a male patient (late 60s) harboring four transcriptionally active intact copies of rhodopsin, (2) recapitulated the clinical phenotypes using retinal organoids, and (3) assessed the utilization of a small-drug like molecule, Photoregulin3 (PR3), as a clinically viable strategy to target and modify disease progression in RP patient associated with RHO-CNV. Patient retinal organoids showed the survival of photoreceptors with rudimentary outer segments, where rod photoreceptors displayed stunted outer segments with semi-occasional elongated cilia-like projections (microscopy); increased RHO mRNA expression (qRT-PCR and bulk RNA-sequencing); along with elevated levels and mislocalization of rhodopsin protein (RHO) within the cell body of rod photoreceptors (western blotting and immunohistochemistry) over the extended (300-days) culture time period. Lastly, we utilized PR3 to target NR2E3, an upstream regulator of RHO, to effectively alter the RHO expression and observed a partial rescue of RHO protein localization from the cell body to the inner/outer segments of rod photoreceptors in patient organoids. These results provided a proof-of-principle for personalized medicine and suggest that RHO expression requires a precise control. Taken together, this study supports the clinical data indicating that adRP due to RHO-CNV develops due to a dominant negative gain of function.

To characterize the phenotype observed in a case series with macular disease and determine the cause. Multicenter case series. Six families (7 patients) with sporadic or multiplex macular disease with onset at 20 to 78 years, and 1 patient with age-related macular degeneration. Patients underwent ophthalmic examination; exome, genome, or targeted sequencing; and/or polymerase chain reaction (PCR) amplification of the breakpoint, followed by cloning and Sanger sequencing or direct Sanger sequencing. Clinical phenotypes, genomic findings, and a hypothesis explaining the mechanism underlying disease in these patients. All 8 cases carried the same deletion encompassing the genes TPRX1, CRX, and SULT2A1, which was absent from 382 control individuals screened by breakpoint PCR and 13 096 Clinical Genetics patients with a range of other inherited conditions screened by array comparative genomic hybridization. Microsatellite genotypes showed that these 7 families are not closely related, but genotypes immediately adjacent to the deletion breakpoints suggest they may share a distant common ancestor. Previous studies had found that carriers for a single defective CRX allele that was predicted to produce no functional CRX protein had a normal ocular phenotype. Here, we show that CRX whole-gene deletion in fact does cause a dominant late-onset macular disease.

Background Modern clinical genetic tests utilize next-generation sequencing (NGS) approaches to comprehensively analyze genetic variants from patients. Out of these millions of variants, clinically relevant variants that match the patient's phenotype need to be identified accurately within a rapid timeframe that facilitates clinical action. As manual evaluation of variants is not a feasible option for meeting the speed and volume requirements of clinical genetic testing, automated solutions are needed. Various machine learning (ML), artificial intelligence (AI), and in silico variant pathogenicity predictors have been developed to solve this challenge. These solutions rely on the comprehensiveness of the available data and struggle with the sparse nature of genetic variant data. Therefore, careful treatment of missing data is necessary, and the selected methods may have a huge impact on the accuracy, reliability, speed and associated computational costs. Results We present an open-source framework called AMISS that can be used to evaluate performance of different methods for handling missing genetic variant data in the context of variant pathogenicity prediction. Using AMISS, we evaluated 14 methods for handling missing values. The performance of these methods varied substantially in terms of precision, computational costs, and other attributes. Overall, simpler imputation methods and specifically mean imputation performed best. Conclusions Selection of the missing data handling method is crucial for AI/ML-based classification of genetic variants. We show that utilizing sophisticated imputation methods is not worth the cost when used in the context of genetic variant pathogenicity classification.

ANKRD17 is an ankyrin repeat-containing protein thought to play a role in cell cycle progression, whose ortholog in Drosophila functions in the Hippo pathway as a co-factor of Yorkie. Here, we delineate a neurodevelopmental disorder caused by de novo heterozygous ANKRD17 variants. The mutational spectrum of this cohort of 34 individuals from 32 families is highly suggestive of haploinsufficiency as the underlying mechanism of disease, with 21 truncating or essential splice site variants, 9 missense variants, 1 in-frame insertion-deletion, and 1 microdeletion (1.16 Mb). Consequently, our data indicate that loss of ANKRD17 is likely the main cause of phenotypes previously associated with large multi-gene chromosomal aberrations of the 4q13.3 region. Protein modeling suggests that most of the missense variants disrupt the stability of the ankyrin repeats through alteration of core structural residues. The major phenotypic characteristic of our cohort is a variable degree of developmental delay/intellectual disability, particularly affecting speech, while additional features include growth failure, feeding difficulties, non-specific MRI abnormalities, epilepsy and/or abnormal EEG, predisposition to recurrent infections (mostly bacterial), ophthalmological abnormalities, gait/balance disturbance, and joint hypermobility. Moreover, many individuals shared similar dysmorphic facial features. Analysis of single-cell RNA-seq data from the developing human telencephalon indicated ANKRD17 expression at multiple stages of neurogenesis, adding further evidence to the assertion that damaging ANKRD17 variants cause a neurodevelopmental disorder.

ObjectivesThe study aims to generate the whole genome sequence of L. monocytogenes strain S2542 and to compare it to the genomes of strains RO15 and ScottA. In addition, we aimed to compare gene expression profiles of L. monocytogenes strains S2542, ScottA and RO15 after high-pressure processing (HPP) using ddPCR.ResultsThe whole genome sequence of L. monocytogenes S2542 indicates that this strain belongs to serotype 4b, in contrast to the previously reported serotype 1/2a. Strain S2542 appears to be more susceptible to the treatment at 400 MPa compared to RO15 and ScottA strains. In contrast to RO15 and ScottA strains, viable cell counts of strain S2542 were below the limit of detection after HPP (400 MPa/8 min) when stored at 8 °C for 24 and 48 h. The transcriptional response of all three strains to HPP was not significantly different.

AimsCardiomyopathies comprise a heterogeneous group of diseases, often of genetic origin. We assessed the current practice of genetic counselling and testing in the prospective European Society of Cardiology EURObservational Research Programme Cardiomyopathy Registry.Methods and resultsA total of 3208 adult patients from 69 centres in 18 countries were enrolled. Genetic counselling was performed in 60.8% of all patients [75.4% in hypertrophic cardiomyopathy (HCM), 39.2% in dilated cardiomyopathy (DCM), 70.8% in arrhythmogenic right ventricular cardiomyopathy (ARVC), and 49.2% in restrictive cardiomyopathy (RCM), P < 0.001]. Comparing European geographical areas, genetic counselling was performed from 42.4% to 83.3% (P < 0.001). It was provided by a cardiologist (85.3%), geneticist (15.1%), genetic counsellor (11.3%), or a nurse (7.5%) (P < 0.001). Genetic testing was performed in 37.3% of all patients (48.8% in HCM, 18.6% in DCM, 55.6% in ARVC, and 43.6% in RCM, P < 0.001). Index patients with genetic testing were younger at diagnosis and had more familial disease, family history of sudden cardiac death, or implanted cardioverter defibrillators but less co‐morbidities than those not tested (P < 0.001 for each comparison). At least one disease‐causing variant was found in 41.7% of index patients with genetic testing (43.3% in HCM, 33.3% in DCM, 51.4% in ARVC, and 42.9% in RCM, P = 0.13).ConclusionsThis is the first detailed report on the real‐life practice of genetic counselling and testing in cardiomyopathies in Europe. Genetic counselling and testing were performed in a substantial proportion of patients but less often than recommended by European guidelines and much less in DCM than in HCM and ARVC, despite evidence for genetic background.

Pathogenic variants in the gene HGSNAT (heparan-α-glucosaminide N-acetyltransferase) have been reported to underlie two distinct recessive conditions, depending on the specific genotype, mucopolysaccharidosis type IIIC (MPSIIIC)—a severe childhood-onset lysosomal storage disorder, and adult-onset nonsyndromic retinitis pigmentosa (RP). Here we describe the largest cohort to-date of HGSNAT-associated nonsyndromic RP patients, and describe their retinal phenotype, leukocyte enzymatic activity, and likely pathogenic genotypes. We identified biallelic HGSNAT variants in 17 individuals (15 families) as the likely cause of their RP. None showed any other symptoms of MPSIIIC. All had a mild but significant reduction of HGSNAT enzyme activity in leukocytes. The retinal condition was generally of late-onset, showing progressive degeneration of a concentric area of paramacular retina, with preservation but reduced electroretinogram responses. Symptoms, electrophysiology, and imaging suggest the rod photoreceptor to be the cell initially compromised. HGSNAT enzymatic testing was useful in resolving diagnostic dilemmas in compatible patients. We identified seven novel sequence variants [p.(Arg239Cys); p.(Ser296Leu); p.(Phe428Cys); p.(Gly248Ala); p.(Gly418Arg), c.1543–2A>C; c.1708delA], three of which were considered to be retina-disease-specific alleles. The most prevalent retina-disease-specific allele p.(Ala615Thr) was observed heterozygously or homozygously in 8 and 5 individuals respectively (7 and 4 families). Two siblings in one family, while identical for the HGSNAT locus, but discordant for retinal disease, suggest the influence of trans-acting genetic or environmental modifying factors.