Functional characterization of 2,832 JAG1 variants supports reclassification for Alagille syndrome and improves guidance for clinical variant interpretation.

Pathogenic variants in the JAG1 gene are a primary cause of the multi-system disorder Alagille syndrome. Although variant detection rates are high for this disease, there is uncertainty associated with the classification of missense variants that leads to reduced diagnostic yield. Consequently, up to 85% of reported JAG1 missense variants have uncertain or conflicting classifications. We generated a library of 2,832 JAG1 nucleotide variants within exons 1-7, a region with a high number of reported missense variants, and designed a high-throughput assay to measure JAG1 membrane expression, a requirement for normal function. After calibration using a set of 175 known or predicted pathogenic and benign variants included within the variant library, 486 variants were characterized as functionally abnormal (n = 277 abnormal and n = 209 likely abnormal), of which 439 (90.3%) were missense. We identified divergent membrane expression occurring at specific residues, indicating that loss of the wild-type residue itself does not drive pathogenicity, a finding supported by structural modeling data and with broad implications for clinical variant classification both for Alagille syndrome and globally across other disease genes. Of 144 uncertain variants reported in patients undergoing clinical or research testing, 27 had functionally abnormal membrane expression, and inclusion of our data resulted in the reclassification of 26 to likely pathogenic. Functional evidence augments the classification of genomic variants, reducing uncertainty and improving diagnostics. Inclusion of this repository of functional evidence during JAG1 variant reclassification will significantly affect resolution of variant pathogenicity, making a critical impact on the molecular diagnosis of Alagille syndrome.

Germ Granule Evolution Provides Mechanistic Insight into Drosophila Germline Development.

The copackaging of mRNAs into biomolecular condensates called germ granules is a conserved strategy to posttranscriptionally regulate germline mRNAs. In Drosophila melanogaster, mRNAs accumulate in germ granules by forming homotypic clusters, aggregates containing multiple transcripts from the same gene. Nucleated by Oskar (Osk), homotypic clusters are generated through a stochastic seeding and self-recruitment process that requires the 3' untranslated region (UTR) of germ granule mRNAs. Interestingly, the 3' UTR belonging to germ granule mRNAs, such as nanos (nos), have considerable sequence variations among Drosophila species and we hypothesized that this diversity influences homotypic clustering. To test our hypothesis, we investigated the homotypic clustering of nos and polar granule component (pgc) in four Drosophila species and concluded that clustering is a conserved process used to enrich germ granule mRNAs. However, we discovered germ granule phenotypes that included significant changes in the abundance of transcripts present in species' homotypic clusters, which also reflected diversity in the number of coalesced primordial germ cells within their embryonic gonads. By integrating biological data with computational modeling, we found that multiple mechanisms underlie naturally occurring germ granule diversity, including changes in nos, pgc, osk levels and/or homotypic clustering efficacy. Furthermore, we demonstrated how the nos 3' UTR from different species influences nos clustering, causing granules to have ∼70% less nos and increasing the presence of defective primordial germ cells. Our results highlight the impact that evolution has on germ granules, which should provide broader insight into processes that modify compositions and activities of other classes of biomolecular condensate.

Evolutionary changes in germ granule mRNA content are driven by multiple mechanisms in Drosophila.

The co-packaging of mRNAs into biomolecular condensates called germ granules is a conserved strategy to post-transcriptionally regulate mRNAs that function in germline development and maintenance. In D. melanogaster, mRNAs accumulate in germ granules by forming homotypic clusters, aggregates that contain multiple transcripts from a specific gene. Nucleated by Oskar (Osk), homotypic clusters in D. melanogaster are generated through a stochastic seeding and self-recruitment process that requires the 3' UTR of germ granule mRNAs. Interestingly, the 3' UTR belonging to germ granule mRNAs, such as nanos (nos), have considerable sequence variations among Drosophila species. Thus, we hypothesized that evolutionary changes in the 3' UTR influences germ granule development. To test our hypothesis, we investigated the homotypic clustering of nos and polar granule component (pgc) in four Drosophila species and concluded that homotypic clustering is a conserved developmental process used to enrich germ granule mRNAs. Additionally, we discovered that the number of transcripts found in nos and/or pgc clusters could vary significantly among species. By integrating biological data with computational modeling, we determined that multiple mechanisms underlie naturally occurring germ granule diversity, including changes in nos, pgc, osk levels, and/or homotypic clustering efficacy. Finally, we found that the nos 3' UTR from different species can alter the efficacy of nos homotypic clustering, resulting in germ granules with reduced nos accumulation. Our findings highlight the impact that evolution has on the development of germ granules and may provide insight into processes that modify the content of other classes of biomolecular condensates.

Nebulous without white: annotated long-read genome assembly and CRISPR/Cas9 genome engineering in Drosophila nebulosa.

The diversity among Drosophila species presents an opportunity to study the molecular mechanisms underlying the evolution of biological phenomena. A challenge to investigating these species is that, unlike the plethora of molecular and genetics tools available for D. melanogaster research, many other species do not have sequenced genomes; a requirement for employing these tools. Selecting transgenic flies through white (w) complementation has been commonly practiced in numerous Drosophila species. While tolerated, the disruption of w is associated with impaired vision, among other effects in D. melanogaster. The D. nebulosa fly has a unique mating behavior which requires vision, and is thus unable to successfully mate in dark conditions. Here, we hypothesized that the disruption of w will impede mating success. As a first step, using PacBio long-read sequencing, we assembled a high-quality annotated genome of D. nebulosa. Using these data, we employed CRISPR/Cas9 to successfully disrupt the w gene. As expected, D. nebulosa males null for w did not court females, unlike several other mutant strains of Drosophila species whose w gene has been disrupted. In the absence of mating, no females became homozygous null for w. We conclude that gene disruption via CRISPR/Cas9 genome engineering is a successful tool in D. nebulosa, and that the w gene is necessary for mating. Thus, an alternative selectable marker unrelated to vision is desirable.