Kaposi sarcoma (KS), caused by Herpesvirus-8 (HHV-8; KSHV), shows sporadic, endemic, and epidemic forms. While familial clustering of KS was previously recorded, the molecular basis of hereditary predilection to KS remains largely unknown. We demonstrate through genetic studies that a dominantly inherited missense mutation in BPTF segregates with a phenotype of classical KS in multiple immunocompetent individuals in two families. Using an rKSHV.219-infected CRISPR/cas9-model, we show that BPTFI2012T mutant cells exhibit higher latent-to-lytic ratio, decreased virion production, increased LANA staining, and latent phenotype in viral transcriptomics. RNA-sequencing demonstrated that KSHV infection dysregulated oncogenic-like response and P53 pathways, MAPK cascade, and blood vessel development pathways, consistent with KS. BPTFI2012T also enriched pathways of viral genome regulation and replication, immune response, and chemotaxis, including downregulation of IFI16, SHFL HLAs, TGFB1, and HSPA5, all previously associated with KSHV infection and tumorigenesis. Many of the differentially expressed genes are regulated by Rel-NF-kappa B, which regulates immune processes, cell survival, and proliferation and is pivotal to oncogenesis. We thus demonstrate BPTF mutation-mediated monogenic hereditary predilection of KSHV virus-induced oncogenesis, and suggest BPTF as a drug target.

BackgroundOtosclerosis is a common cause of adult-onset progressive hearing loss, affecting 0.3%–0.4% of the population. It results from dysregulation of bone homeostasis in the otic capsule, most commonly leading to fixation of the stapes bone, impairing sound conduction through the middle ear. Otosclerosis has a well-known genetic predisposition including familial cases with apparent autosomal dominant mode of inheritance. While linkage analysis and genome-wide association studies suggested an association with several genomic loci and with genes encoding structural proteins involved in bone formation or metabolism, the molecular genetic pathophysiology of human otosclerosis is yet mostly unknown.MethodsWhole-exome sequencing, linkage analysis, generation of CRISPR mutant mice, hearing tests and micro-CT.ResultsThrough genetic studies of kindred with seven individuals affected by apparent autosomal dominant otosclerosis, we identified a disease-causing variant inSMARCA4, encoding a key component of the PBAF chromatin remodelling complex. We generated CRISPR-Cas9 transgenic mice carrying the human mutation in the mouseSMARCA4orthologue. MutantSmarca4+/E1548Kmice exhibited marked hearing impairment demonstrated through acoustic startle response and auditory brainstem response tests. Isolated ossicles of the auditory bullae of mutant mice exhibited a highly irregular structure of the incus bone, and their in situ micro-CT studies demonstrated the anomalous structure of the incus bone, causing disruption in the ossicular chain.ConclusionWe demonstrate that otosclerosis can be caused by a variant inSMARCA4, with a similar phenotype of hearing impairment and abnormal bone formation in the auditory bullae in transgenic mice carrying the human mutation in the mouseSMARCA4orthologue.

Ehlers–Danlos syndromes (EDS) are a group of connective tissue disorders caused by mutations in collagen and collagen-interacting genes. We delineate a novel form of EDS with vascular features through clinical and histopathological phenotyping and genetic studies of a three-generation pedigree, displaying an apparently autosomal dominant phenotype of joint hypermobility and frequent joint dislocations, atrophic scarring, prolonged bleeding time and age-related aortic dilatation and rupture. Coagulation tests as well as platelet counts and function were normal. Reticular dermis displayed highly disorganized collagen fibers and transmission electron microscopy (TEM) revealed abnormally shaped fibroblasts and endothelial cells, with high amount and irregular shape of extracellular matrix (ECM) substance, especially near blood vessels. Genetic analysis unraveled a heterozygous mutation in THBS2 (NM_003247.5:c.2686T>C, p.Cys896Arg). We generated CRISPR/Cas9 knock-in (KI) mice, bearing the heterozygous human mutation in the mouse ortholog. The KI mice demonstrated phenotypic traits correlating with those observed in the human subjects, as evidenced by morphologic, histologic, and TEM analyses, in conjunction with bleeding time assays. Our findings delineate a novel form of human EDS with classical-like elements combined with vascular features, caused by a heterozygous THBS2 missense mutation. We further demonstrate a similar phenotype in heterozygous THBS2Cys896Arg KI mice, in line with previous studies in Thbs2 homozygous null-mutant mice. Notably, THBS2 encodes Thrombospondin-2, a secreted homotrimeric matricellular protein that directly binds the ECM-shaping Matrix Metalloproteinase 2 (MMP2), mediating its clearance. THBS2 loss-of-function attenuates MMP2 clearance, enhancing MMP2-mediated proteoglycan cleavage, causing ECM abnormalities similar to those seen in the human and mouse disease we describe.

BACKGROUND:Sex-specific predilection in neurological diseases caused by mutations in autosomal genes is a phenomenon whose molecular basis is poorly understood. We studied females of consanguineous Bedouin kindred presenting with severe global developmental delay and epilepsy. METHODS:Linkage analysis, whole exome sequencing, generation of CRISPR/cas9 knock-in mice, mouse behaviour and molecular studies RESULTS: Linkage analysis and whole exome sequencing studies of the affected kindred delineated a ~5 Mbp disease-associated chromosome 2q35 locus, containing a novel homozygous frameshift truncating mutation in ZNF142, in line with recent studies depicting similar ZNF142 putative loss-of-function human phenotypes with female preponderance. We generated knock-in mice with a truncating mutation adjacent to the human mutation in the mouse ortholog. Behaviour studies of homozygous Zfp142R1508* mice showed significant phenotype only in mutant females, with learning and memory deficits, hyperactivity and aberrant loss of fear of open spaces. Bone marrow and spleen of homozygous Zfp142R1508* mice showed depletion of lymphoid and haematopoietic cells, mostly in females. RT-PCR showed lower expression of Zpf142 in brain compartments of female versus male wild-type mice. RNA-seq studies of hippocampus, hypothalamus, cortex and cerebellum of female wild-type versus homozygous Zfp142R1508* mice demonstrated differentially expressed genes. Notably, expression of Taok1 in the cortex and of Mllt6 in the hippocampus was downregulated in homozygous Zfp142R1508* mice. Taok1 mutations have been associated with aberrant neurodevelopment and behaviour. Mllt6 expression is regulated by sex hormones and Mllt6 null-mutant mice present with haematopoietic, immune system and female-specific behaviour phenotypes. CONCLUSION:ZNF142 mutation downregulates Mllt6 and Taok1, causing a neurodevelopmental phenotype in humans and mice with female preponderance.

Abstract Pathogenic variants underlying Mendelian diseases often disrupt the normal physiology of a few tissues and organs. However, variant effect prediction tools that aim to identify pathogenic variants are typically oblivious to tissue contexts. Here we report a machine-learning framework, denoted “Tissue Risk Assessment of Causality by Expression for variants” (TRACEvar, https://netbio.bgu.ac.il/TRACEvar/ ), that offers two advancements. First, TRACEvar predicts pathogenic variants that disrupt the normal physiology of specific tissues. This was achieved by creating 14 tissue-specific models that were trained on over 14,000 variants and combined 84 attributes of genetic variants with 495 attributes derived from tissue omics. TRACEvar outperformed 10 well-established and tissue-oblivious variant effect prediction tools. Second, the resulting models are interpretable, thereby illuminating variants’ mode of action. Application of TRACEvar to variants of 52 rare-disease patients highlighted pathogenicity mechanisms and relevant disease processes. Lastly, the interpretation of all tissue models revealed that top-ranking determinants of pathogenicity included attributes of disease-affected tissues, particularly cellular process activities. Collectively, these results show that tissue contexts and interpretable machine-learning models can greatly enhance the etiology of rare diseases.

With the increasing importance of genomic data in understanding genetic diseases, there is an essential need for efficient and user-friendly tools that simplify variant analysis. Although multiple tools exist, many present barriers such as steep learning curves, limited reference genome compatibility, or costs. We developed VARista, a free web-based tool, to address these challenges and provide a streamlined solution for researchers, particularly those focusing on rare monogenic diseases. VARista offers a user-centric interface that eliminates much of the technical complexity typically associated with variant analysis. The tool directly supports VCF files generated using reference genomes hg19, hg38, and the emerging T2T, with seamless remapping capabilities between them. Features such as gene summaries and links, tissue and cell-specific gene expression data for both adults and fetuses, as well as automated PCR design and integration with tools such as SpliceAI and AlphaMissense, enable users to focus on the biology and the case itself. As we demonstrate, VARista proved effective in narrowing down potential disease-causing variants, prioritizing them effectively, and providing meaningful biological context, facilitating rapid decision-making. VARista stands out as a freely available and comprehensive tool that consolidates various aspects of variant analysis into a single platform that embraces the forefront of genomic advancements. Its design inherently supports a shift in focus from technicalities to critical thinking, thereby promoting better-informed decisions in genetic disease research. Given its unique capabilities and user-centric design, VARista has the potential to become an essential asset for the genomic research community. https://VARista.link.

The biallelic variants of the POP1 gene are associated with the anauxetic dysplasia (AAD OMIM 607095), a rare skeletal dysplasia, characterized by prenatal rhizomelic shortening of limbs and generalized joint hypermobility. Affected individuals usually have normal neurodevelopmental milestones. Here we present three cases from the same family with likely pathogenic homozygous POP1 variant and a completely novel phenotype: a girl with global developmental delay and autism, microcephaly, peculiar dysmorphic features and multiple congenital anomalies. Two subsequent pregnancies were terminated due to multiple congenital malformations. Fetal DNA samples revealed the same homozygous variant in the POP1 gene. Expression of the RMRP was reduced in the proband compared with control and slightly reduced in both heterozygous parents, carriers for this variant. To our knowledge, this is the first report of this new phenotype, associated with a novel likely pathogenic variant in POP1. Our findings expand the phenotypic spectrum of POP1-related disorders.

Background Developmental dysplasia of the hip (DDH), formerly termed congenital dislocation of the hip, is the most common congenital disease of the musculoskeletal system in newborns. While familial predilection to DDH has been well documented, the molecular genetics/pathways of this common disorder are poorly understood. Methods Linkage analysis and whole exome sequencing; real-time PCR studies of skin fibroblasts. Results Consanguineous Bedouin kindred presented with DDH with apparent autosomal recessive heredity. Linkage analysis and whole exome sequencing delineated a single 3.2 Mbp disease-associated chromosome 1 locus (maximal multipoint Logarithm of the Odds score 2.3), containing a single homozygous variant with a relevant expression pattern: addition of threonine in TRIM33 (NM_015906.4); c.1648_1650dup. TRIM33 encodes a protein that acts both in the TGF-β and the BMP pathways; however, it has been mostly studied regarding its function in the TGF-β pathway. Since BMPs are known to act in bone formation, we focused on the BMP pathway, in which TRIM33 functions as a transcription factor, both an activator and repressor. Skin fibroblasts of two affected girls and a heterozygous TRIM33 variant carrier were assayed through reverse-transcription PCR for expression of genes known to be downstream of TRIM33 in the BMP pathway: fibroblasts of affected individuals showed significantly reduced expression of DLX5 , significantly increased expression of BGLAP , increased expression of ALPL and no change in expression of RUNX2 or of TRIM33 itself. Conclusions DDH can be caused by a biallelic variant in TRIM33 , affecting the BMP pathway.

Hemolytic-uremic syndrome (HUS), mostly secondary to infectious diseases, is a common cause of acute kidney injury in children. It is characterized by progressive acute kidney failure due to severe thrombotic microangiopathy, associated with nonimmune, Coombs-negative hemolytic anemia and thrombocytopenia. HUS is caused mostly by Shiga toxin-producing E. Coli, and to a lesser extent by Streptococcus pneumonia. In Streptococcus pneumonia HUS (pHUS), bacterial neuraminidase A exposes masked O-glycan sugar residues on erythrocytes, known as the T antigen, triggering a complement cascade causing thrombotic microangiopathy. Atypical HUS (aHUS) is a life-threatening genetic form of the disease, whose molecular mechanism is only partly understood. Through genetic studies, we demonstrate a novel X-linked form of aHUS that is caused by a de-novo missense mutation in C1GALT1C1:c.266 C > T,p.(T89I), encoding a T-synthase chaperone essential for the proper formation and incorporation of the T antigen on erythrocytes. We demonstrate the presence of exposed T antigen on the surface of mutant erythrocytes, causing aHUS in a mechanism similar to that suggested in pHUS. Our findings suggest that both aHUS caused by mutated C1GALT1C1 and pHUS are mediated by the lectin-complement-pathway, not comprehensively studied in aHUS. We thus delineate a shared molecular basis of aHUS and pHUS, highlighting possible therapeutic opportunities.

How do aberrations in widely expressed genes lead to tissue‐selective hereditary diseases? Previous attempts to answer this question were limited to testing a few candidate mechanisms. To answer this question at a larger scale, we developed “Tissue Risk Assessment of Causality by Expression” (TRACE), a machine learning approach to predict genes that underlie tissue‐selective diseases and selectivity‐related features. TRACE utilized 4,744 biologically interpretable tissue‐specific gene features that were inferred from heterogeneous omics datasets. Application of TRACE to 1,031 disease genes uncovered known and novel selectivity‐related features, the most common of which was previously overlooked. Next, we created a catalog of tissue‐associated risks for 18,927 protein‐coding genes ( https://netbio.bgu.ac.il/trace/ ). As proof‐of‐concept, we prioritized candidate disease genes identified in 48 rare‐disease patients. TRACE ranked the verified disease gene among the patient's candidate genes significantly better than gene prioritization methods that rank by gene constraint or tissue expression. Thus, tissue selectivity combined with machine learning enhances genetic and clinical understanding of hereditary diseases. An interpretable machine‐learning framework predicts disease genes for tissue‐selective hereditary diseases. The framework highlights known and novel tissue‐selectivity features and enhances genetic diagnosis of rare‐disease patients. An interpretable machine‐learning framework predicts disease genes for tissue‐selective hereditary diseases. The framework highlights known and novel tissue‐selectivity features and enhances genetic diagnosis of rare‐disease patients.

Knudson's "two hit" hypothesis, mostly associated with cancer, relates to a primary heterozygous germline mutation complemented by a somatic mutation in the second allele. When the somatic "second hit" is a deletion mutation, the heterozygosity due to the first hit is lost ("loss of heterozygosity"). As the rate of germline mutations is almost two orders of magnitude lower than that of somatic mutations, de-novo germline mutations causing autosomal recessive diseases in carriers of inherited heterozygous mutations are not common. We delineate a case of high myopia presenting at infancy with mild diminution of retinal responses. Exome sequencing identified a paternally inherited apparently homozygous missense mutation in RBP3. Chromosomal microarrays delineated a de-novo germline heterozygous deletion encompassing RBP3, verified through revision of WES data. Thus, we demonstrate an inherited RBP3 missense mutation complemented by a de-novo germline RBP3 deletion, causing loss of heterozygosity of the inherited mutation. We describe a novel RBP3 missense mutation, report the first isolated RBP3 deletion, and demonstrate infantile high myopia as an initial presentation of RBP3 disease. Notably, we highlight de-novo germline deletion mutations causing "loss of heterozygosity" of inherited heterozygous mutations, culminating in autosomal recessive diseases, and discuss the scarce literature.

Hyperinsulinism/hyperammonemia (HI/HA) syndrome has been known to be caused by dominant gain-of-function mutations in GLUD1, encoding the mitochondrial enzyme glutamate dehydrogenase. Pathogenic GLUD1 mutations enhance enzymatic activity by reducing its sensitivity to allosteric inhibition by GTP. Two recent independent studies showed that a similar HI/HA phenotype can be caused by biallelic mutations in SLC25A36, encoding pyrimidine nucleotide carrier 2 (PNC2), a mitochondrial nucleotide carrier that transports pyrimidine and guanine nucleotides across the inner mitochondrial membrane: one study reported a single case caused by a homozygous truncating mutation in SLC25A36 resulting in lack of expression of SLC25A36 in patients' fibroblasts. A second study described two siblings with a splice site mutation in SLC25A36, causing reduction of mitochondrial GTP content, putatively leading to hyperactivation of glutamate dehydrogenase. In an independent study, through combined linkage analysis and exome sequencing, we demonstrate in four individuals of two Bedouin Israeli related families the same disease-causing SLC25A36 (NM_018155.3) c.284 + 3A > T homozygous splice-site mutation found in the two siblings. We demonstrate that the mutation, while causing skipping of exon 3, does not abrogate expression of mRNA and protein of the mutant SLC25A36 in patients' blood and fibroblasts. Affected individuals had hyperinsulinism, hyperammonemia, borderline low birth weight, tonic-clonic seizures commencing around 6 months of age, yet normal intellect and no significant other morbidities. Chronic constipation, hypothyroidism, and developmental delay previously described in a single patient were not found. We thus verify that biallelic SLC25A36 mutations indeed cause HI/HA syndrome and clearly delineate the disease phenotype.

Genomic sequences residing within introns of few genes have been shown to act as enhancers affecting expression of neighboring genes. We studied an autosomal recessive phenotypic continuum of microphthalmia, anophthalmia and ocular coloboma, with no apparent coding-region disease-causing mutation. Homozygosity mapping of several affected Jewish Iranian families, combined with whole genome sequence analysis, identified a 0.5 Mb disease-associated chromosome 2q35 locus (maximal LOD score 6.8) harboring an intronic founder variant in NHEJ1 , not predicted to affect NHEJ1. The human NHEJ1 intronic variant lies within a known specifically limb-development enhancer of a neighboring gene, Indian hedgehog (Ihh), known to be involved in eye development in mice and chickens. Through mouse and chicken molecular development studies, we demonstrated that this variant is within an Ihh enhancer that drives gene expression in the developing eye and that the identified variant affects this eye-specific enhancer activity. We thus delineate an Ihh enhancer active in mammalian eye development whose variant causes human microphthalmia, anophthalmia and ocular coloboma. The findings highlight disease causation by an intronic variant affecting the expression of a neighboring gene, delineating molecular pathways of eye development.

Myopathy is the main adverse effect of the widely prescribed statin drug class. Statins exert their beneficial effect by inhibiting HMG CoA-reductase, the rate-controlling enzyme of the mevalonate pathway. The mechanism of statin myopathy is yet to be resolved, and its treatment is insufficient. Through homozygosity mapping and whole exome sequencing, followed by functional analysis using confocal microscopy and biochemical and biophysical methods, we demonstrate that a distinct form of human limb girdle muscular disease is caused by a pathogenic homozygous loss-of-function missense mutation in HMG CoA reductase (HMGCR), encoding HMG CoA-reductase. We biochemically synthesized and purified mevalonolactone, never administered to human patients before, and establish the safety of its oral administration in mice. We then show that its oral administration is effective in treating a human patient with no significant adverse effects. Furthermore, we demonstrate that oral mevalonolactone resolved statin-induced myopathy in mice. We conclude that HMGCR mutation causes a late-onset severe progressive muscular disease, which shows similar features to statin-induced myopathy. Our findings indicate that mevalonolactone is effective both in the treatment of hereditary HMGCR myopathy and in a murine model of statin myopathy. Further large clinical trials are in place to enable the clinical use of mevalonolactone both in the rare orphan disease and in the more common statin myopathy.

Primary microcephaly and Seckel syndrome are rare genetically and clinically heterogenous brain development disorders. Several exonic/splicing mutations are reported for these disorders to date, but ∼40% of all cases remain unexplained. We aimed to uncover the genetic correlate(s) in a family of multiple siblings with microcephaly. A novel homozygous intronic variant (NC_000013.10:g.25459823T>C) in CENPJ (13q12) segregating with all four affected male siblings was identified by exome sequencing and validated by targeted linkage approach (logarithm of the odds score 1.8 at θ 0.0). RT-PCR of CENPJ in affected siblings using their EBV derived cell lines showed aberrant transcripts suggestive of exon skipping confirmed by Sanger sequencing. Significantly reduced wild type transcript/protein in the affected siblings having the splice variant indicates a leaky gene expression of pathological relevance. Based on known CENPJ function, assessing for mitotic alterations revealed defect in centrosome duplication causing mono/multicentrosome(s) at prophase, delayed metaphase, and unequal chromosomal segregation in patient cells. Clinical features witnessed in this study expand the spectrum of CENPJ-associated primary microcephaly and Seckel syndrome. Furthermore, besides the importance of regulatory variants in classical monogenic disorders these findings provide new insights into splice site biology with possible implications for ASO-based therapies.