International Porphyria Molecular Diagnostic Collaborative: an evidence-based database of verified pathogenic and benign variants for the porphyrias.

With the advent of precision and genomic medicine, a critical issue is whether a disease gene variant is pathogenic or benign. Such is the case for the three autosomal dominant acute hepatic porphyrias (AHPs), including acute intermittent porphyria, hereditary coproporphyria, and variegate porphyria, each resulting from the half-normal enzymatic activities of hydroxymethylbilane synthase, coproporphyrinogen oxidase, and protoporphyrinogen oxidase, respectively. To date, there is no public database that documents the likely pathogenicity of variants causing the porphyrias, and more specifically, the AHPs with biochemically and clinically verified information. Therefore, an international collaborative with the European Porphyria Network and the National Institutes of Health/National Center for Advancing Translational Sciences/National Institute of Diabetes and Digestive and Kidney Diseases (NIH/NCATS/NIDDK)-sponsored Porphyrias Consortium of porphyria diagnostic experts is establishing an online database that will collate biochemical and clinical evidence verifying the pathogenicity of the published and newly identified variants in the AHP-causing genes. The overall goal of the International Porphyria Molecular Diagnostic Collaborative is to determine the pathogenic and benign variants for all eight porphyrias. Here we describe the overall objectives and the initial efforts to validate pathogenic and benign variants in the respective heme biosynthetic genes causing the AHPs.

Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction.

Single nucleotide variants (SNVs) are prevalent genetic factors shaping individual trait profiles and disease susceptibility. The recent development and optimizations of base editors, rubber and pencil genome editing tools now promise to enable direct functional assessment of SNVs in model organisms. However, the lack of bioinformatic tools aiding target prediction limits the application of base editing in vivo. Here, we provide a framework for adenine and cytosine base editing in medaka (Oryzias latipes) and zebrafish (Danio rerio), ideal for scalable validation studies. We developed an online base editing tool ACEofBASEs (a careful evaluation of base-edits), to facilitate decision-making by streamlining sgRNA design and performing off-target evaluation. We used state-of-the-art adenine (ABE) and cytosine base editors (CBE) in medaka and zebrafish to edit eye pigmentation genes and transgenic GFP function with high efficiencies. Base editing in the genes encoding troponin T and the potassium channel ERG faithfully recreated known cardiac phenotypes. Deep-sequencing of alleles revealed the abundance of intended edits in comparison to low levels of insertion or deletion (indel) events for ABE8e and evoBE4max. We finally validated missense mutations in novel candidate genes of congenital heart disease (CHD) dapk3, ube2b, usp44, and ptpn11 in F0 and F1 for a subset of these target genes with genotype-phenotype correlation. This base editing framework applies to a wide range of SNV-susceptible traits accessible in fish, facilitating straight-forward candidate validation and prioritization for detailed mechanistic downstream studies.

DNA contains sequences of four different molecules known as bases that represent our genetic code. In a mutation called a single nucleotide variant (or SNV for short), a single base in the sequence is swapped for another base. This can lead the individual carrying this SNV to produce a slightly different version of a protein to that found in other people. This slightly different protein may not work properly, or may perform a different task. In recent years, researchers have identified thousands of SNVs in humans linked with congenital heart diseases, but the roles of many of these SNVs remain unclear. Tools known as base editors allow researchers to efficiently modify single bases in DNA. Base editors use molecules known as short guide RNAs (or sgRNAs for short) to direct enzymes to specific positions in the DNA to swap, delete or insert a base. The sgRNAs need to be carefully designed to target the correct bases, however, which is a time consuming process. Furthermore, base editors were developed in cells grown in laboratories and so far only a few studies have demonstrated how they could be used in living animals. To overcome these limitations, Cornean, Gierten, Welz et al. developed a framework for base editing in two species of fish that are often used as models in research, namely medaka and zebrafish. The framework uses existing base editors that swap individual target bases and a new online tool ­ referred to as ACEofBASEs ­ to help design the required sgRNAs. The team were able to use the framework to characterize the medaka equivalents of four SNVs that have been previously associated with congenital heart disease in humans. The new framework developed here will help researchers to investigate the roles of SNVs in fish and other animals and validate human disease candidates. This approach could also be used to study the various ways that cells modify proteins by changing the specific bases involved in such modifications.

Long-read nanopore sequencing enables accurate confirmation of a recurrent PMS2 insertion-deletion variant located in a region of complex genomic architecture.

Targeted next generation sequencing (NGS) is the predominant methodology for the molecular genetic diagnosis of inherited conditions. In many laboratories, NGS-identified variants are routinely validated using a different method, to minimize the risk of a false-positive diagnosis. This can be particularly important when pathogenic variants are located in complex genomic regions. In this situation, new long-read sequencing technologies have potential advantages over existing alternatives. However, practical examples of their utility for diagnostic purposes remain scant. Here, we report the use of nanopore sequencing to validate a PMS2 mutation refractory to conventional methods. In a patient who presented with colorectal cancer and loss of PMS2 immunostaining, short-read NGS of Lynch syndrome-associated genes identified the recurrent PMS2 insertion-deletion variant, c.736_741delinsTGTGTGTGAAG (p.Pro246Cysfs*3). Confirmation of this variant using bidirectional Sanger sequencing was impeded by an upstream intron 6 poly(T) tract. Using a locus-specific amplicon template, we undertook nanopore long-read sequencing in order to assess the diagnostic accuracy of this platform. Pairwise comparison between a curated benchmark allele (derived from short-read NGS and unidirectional Sanger sequencing) and the consensus nanopore dataset revealed 100% sequence identity. Our experience provides insight into the robustness and ease of deployment of "third-generation" sequencing for accurate characterisation of pathogenic variants.

A polyclonal allelic expression assay for detecting regulatory effects of transcript variants.

We present an assay to experimentally test the regulatory effects of genetic variants within transcripts using CRISPR/Cas9 followed by targeted sequencing. We applied the assay to 32 premature stop-gained variants across the genome and in two Mendelian disease genes, 33 putative causal variants of eQTLs, and 62 control variants in HEK293T cells, replicating a subset of variants in HeLa cells. We detected significant effects in the expected direction (in 60% of variants), demonstrating the ability of the assay to capture regulatory effects of eQTL variants and nonsense-mediated decay triggered by premature stop-gained variants. The results suggest a utility for validating transcript-level effects of genetic variants.

Application of CRISPR Tools for Variant Interpretation and Disease Modeling in Inherited Retinal Dystrophies.

Inherited retinal dystrophies are an assorted group of rare diseases that collectively account for the major cause of visual impairment of genetic origin worldwide. Besides clinically, these vision loss disorders present a high genetic and allelic heterogeneity. To date, over 250 genes have been associated to retinal dystrophies with reported causative variants of every nature (nonsense, missense, frameshift, splice-site, large rearrangements, and so forth). Except for a fistful of mutations, most of them are private and affect one or few families, making it a challenge to ratify the newly identified candidate genes or the pathogenicity of dubious variants in disease-associated loci. A recurrent option involves altering the gene in in vitro or in vivo systems to contrast the resulting phenotype and molecular imprint. To validate specific mutations, the process must rely on simulating the precise genetic change, which, until recently, proved to be a difficult endeavor. The rise of the CRISPR/Cas9 technology and its adaptation for genetic engineering now offers a resourceful suite of tools to alleviate the process of functional studies. Here we review the implementation of these RNA-programmable Cas9 nucleases in culture-based and animal models to elucidate the role of novel genes and variants in retinal dystrophies.

Unmet Needs in Dystonia: Genetics and Molecular Biology-How Many Dystonias?

Genetic findings of the past years have provided ample evidence for a substantial etiologic heterogeneity of dystonic syndromes. While an increasing number of genes are being identified for Mendelian forms of isolated and combined dystonias using classical genetic mapping and whole-exome sequencing techniques, their precise role in the molecular pathogenesis is still largely unknown. Also, the role of genetic risk factors in the etiology of sporadic dystonias is still enigmatic. Only the systematic ascertainment and precise clinical characterization of very large cohorts with dystonia, combined with systematic genetic studies, will be able to unravel the complex network of factors that determine disease risk and phenotypic expression.