High-Throughput Splicing Assays Identify Known and Novel WT1 Exon 9 Variants in Nephrotic Syndrome.

Introduction: Frasier syndrome (FS) is a rare Mendelian form of nephrotic syndrome (NS) caused by variants which disrupt the proper splicing of WT1. This key transcription factor gene is alternatively spliced at exon 9 to produce 2 isoforms ("KTS+" and "KTS-"), which are normally expressed in the kidney at a ∼2:1 (KTS+:KTS-) ratio. FS results from variants that reduce this ratio by disrupting the splice donor of the KTS+ isoform. FS is extremely rare, and it is unclear whether any variants beyond the 8 already known could cause FS. Methods: To prospectively identify other splicing-disruptive variants, we leveraged a massively parallel splicing assay. We tested every possible single nucleotide variant (n = 519) in and around WT1 exon 9 for effects upon exon inclusion and KTS+/- ratio. Results: Splice disruptive variants (SDVs) made up 11% of the tested point variants overall and were tightly concentrated near the canonical acceptor and the KTS+/- alternate donors. Our map successfully identified all 8 known FS or focal segmental glomerulosclerosis (FSGS) variants and 16 additional novel variants which were comparably disruptive to these known pathogenic variants. We also identified 19 variants that, conversely, increased the KTS+/KTS- ratio, of which 2 are observed in unrelated individuals with 46,XX ovotesticular disorder of sex development (46,XX OTDSD). Conclusion: This splicing effect map can serve as functional evidence to guide the clinical interpretation of newly observed variants in and around WT1 exon 9.

Aberrant activation of TCL1A promotes stem cell expansion in clonal haematopoiesis.

Mutations in a diverse set of driver genes increase the fitness of haematopoietic stem cells (HSCs), leading to clonal haematopoiesis1. These lesions are precursors for blood cancers2-6, but the basis of their fitness advantage remains largely unknown, partly owing to a paucity of large cohorts in which the clonal expansion rate has been assessed by longitudinal sampling. Here, to circumvent this limitation, we developed a method to infer the expansion rate from data from a single time point. We applied this method to 5,071 people with clonal haematopoiesis. A genome-wide association study revealed that a common inherited polymorphism in the TCL1A promoter was associated with a slower expansion rate in clonal haematopoiesis overall, but the effect varied by driver gene. Those carrying this protective allele exhibited markedly reduced growth rates or prevalence of clones with driver mutations in TET2, ASXL1, SF3B1 and SRSF2, but this effect was not seen in clones with driver mutations in DNMT3A. TCL1A was not expressed in normal or DNMT3A-mutated HSCs, but the introduction of mutations in TET2 or ASXL1 led to the expression of TCL1A protein and the expansion of HSCs in vitro. The protective allele restricted TCL1A expression and expansion of mutant HSCs, as did experimental knockdown of TCL1A expression. Forced expression of TCL1A promoted the expansion of human HSCs in vitro and mouse HSCs in vivo. Our results indicate that the fitness advantage of several commonly mutated driver genes in clonal haematopoiesis may be mediated by TCL1A activation.

GCAF(TMEM251) regulates lysosome biogenesis by activating the mannose-6-phosphate pathway.

The mannose-6-phosphate (M6P) biosynthetic pathway for lysosome biogenesis has been studied for decades and is considered a well-understood topic. However, whether this pathway is regulated remains an open question. In a genome-wide CRISPR/Cas9 knockout screen, we discover TMEM251 as the first regulator of the M6P modification. Deleting TMEM251 causes mistargeting of most lysosomal enzymes due to their loss of M6P modification and accumulation of numerous undigested materials. We further demonstrate that TMEM251 localizes to the Golgi and is required for the cleavage and activity of GNPT, the enzyme that catalyzes M6P modification. In zebrafish, TMEM251 deletion leads to severe developmental defects including heart edema and skeletal dysplasia, which phenocopies Mucolipidosis Type II. Our discovery provides a mechanism for the newly discovered human disease caused by TMEM251 mutations. We name TMEM251 as GNPTAB cleavage and activity factor (GCAF) and its related disease as Mucolipidosis Type V.

Longitudinal profiling of clonal hematopoiesis provides insight into clonal dynamics.

BACKGROUND: Clonal hematopoiesis of indeterminate potential (CHIP), the age-related expansion of mutant hematopoietic stem cells, confers risk for multiple diseases of aging including hematologic cancer and cardiovascular disease. Whole-exome or genome sequencing can detect CHIP, but due to those assays' high cost, most population studies have been cross-sectional, sequencing only a single timepoint per individual. RESULTS: We developed and validated a cost-effective single molecule molecular inversion probe sequencing (smMIPS) assay for detecting CHIP, targeting the 11 most frequently mutated genes in CHIP along with 4 recurrent mutational hotspots. We sequenced 548 multi-timepoint samples collected from 182 participants in the Women's Health Initiative cohort, across a median span of 16 years. We detected 178 driver mutations reaching variant allele frequency ≥ 2% in at least one timepoint, many of which were detectable well below this threshold at earlier timepoints. The majority of clonal mutations (52.1%) expanded over time (with a median doubling period of 7.43 years), with the others remaining static or decreasing in size in the absence of any cytotoxic therapy. CONCLUSIONS: Targeted smMIPS sequencing can sensitively measure clonal dynamics in CHIP. Mutations that reached the conventional threshold for CHIP (2% frequency) tended to continue growing, indicating that after CHIP is acquired, it is generally not lost. The ability to cost-effectively profile CHIP longitudinally will enable future studies to investigate why some CHIP clones expand, and how their dynamics relate to health outcomes at a biobank scale.

Massively parallel functional testing of MSH2 missense variants conferring Lynch syndrome risk.

The lack of functional evidence for the majority of missense variants limits their clinical interpretability and poses a key barrier to the broad utility of carrier screening. In Lynch syndrome (LS), one of the most highly prevalent cancer syndromes, nearly 90% of clinically observed missense variants are deemed "variants of uncertain significance" (VUS). To systematically resolve their functional status, we performed a massively parallel screen in human cells to identify loss-of-function missense variants in the key DNA mismatch repair factor MSH2. The resulting functional effect map is substantially complete, covering 94% of the 17,746 possible variants, and is highly concordant (96%) with existing functional data and expert clinicians' interpretations. The large majority (89%) of missense variants were functionally neutral, perhaps unexpectedly in light of its evolutionary conservation. These data provide ready-to-use functional evidence to resolve the ∼1,300 extant missense VUSs in MSH2 and may facilitate the prospective classification of newly discovered variants in the clinic.

Inherited causes of clonal haematopoiesis in 97,691 whole genomes.

Age is the dominant risk factor for most chronic human diseases, but the mechanisms through which ageing confers this risk are largely unknown1. The age-related acquisition of somatic mutations that lead to clonal expansion in regenerating haematopoietic stem cell populations has recently been associated with both haematological cancer2-4 and coronary heart disease5-this phenomenon is termed clonal haematopoiesis of indeterminate potential (CHIP)6. Simultaneous analyses of germline and somatic whole-genome sequences provide the opportunity to identify root causes of CHIP. Here we analyse high-coverage whole-genome sequences from 97,691 participants of diverse ancestries in the National Heart, Lung, and Blood Institute Trans-omics for Precision Medicine (TOPMed) programme, and identify 4,229 individuals with CHIP. We identify associations with blood cell, lipid and inflammatory traits that are specific to different CHIP driver genes. Association of a genome-wide set of germline genetic variants enabled the identification of three genetic loci associated with CHIP status, including one locus at TET2 that was specific to individuals of African ancestry. In silico-informed in vitro evaluation of the TET2 germline locus enabled the identification of a causal variant that disrupts a TET2 distal enhancer, resulting in increased self-renewal of haematopoietic stem cells. Overall, we observe that germline genetic variation shapes haematopoietic stem cell function, leading to CHIP through mechanisms that are specific to clonal haematopoiesis as well as shared mechanisms that lead to somatic mutations across tissues.

Loss of Neurokinin-1 Receptor Alters Ocular Surface Homeostasis and Promotes an Early Development of Herpes Stromal Keratitis.

Substance P neuropeptide and its receptor, neurokinin-1 receptor (NK1R), are reported to present on the ocular surface. In this study, mice lacking functional NK1R exhibited an excessive desquamation of apical corneal epithelial cells in association with an increased epithelial cell proliferation and increased epithelial cell density, but decreased epithelial cell size. The lack of NK1R also resulted in decreased density of corneal nerves, corneal epithelial dendritic cells (DCs), and a reduced volume of basal tears. Interestingly, massive accumulation of CD11c+CD11b+ conventional DCs was noted in the bulbar conjunctiva and near the limbal area of corneas from NK1R-/- mice. After ocular HSV-1 infection, the number of conventional DCs and neutrophils infiltrating the infected corneas was significantly higher in NK1R-/- than C57BL/6J mice. This was associated with an increased viral load in infected corneas of NK1R-/- mice. As a result, the number of IFN-γ-secreting virus-specific CD4 T cells in the draining lymph nodes of NK1R-/- mice was much higher than in infected C57BL/6J mice. An increased number of CD4 T cells and mature neutrophils (CD11b+Ly6ghigh) in the inflamed corneas of NK1R-/- mice was associated with an early development of severe herpes stromal keratitis. Collectively, our results show that the altered corneal biology of uninfected NK1R-/- mice along with an enhanced immunological response after ocular HSV-1 infection causes an early development of herpes stromal keratitis in NK1R-/- mice.

Histone deacetylases and phosphorylated polymerase II C-terminal domain recruit Spt6 for cotranscriptional histone reassembly.

Spt6 is a multifunctional histone chaperone involved in the maintenance of chromatin structure during elongation by RNA polymerase II (Pol II). Spt6 has a tandem SH2 (tSH2) domain within its C terminus that recognizes Pol II C-terminal domain (CTD) peptides phosphorylated on Ser2, Ser5, or Try1 in vitro. Deleting the tSH2 domain, however, only has a partial effect on Spt6 occupancy in vivo, suggesting that more complex mechanisms are involved in the Spt6 recruitment. Our results show that the Ser2 kinases Bur1 and Ctk1, but not the Ser5 kinase Kin28, cooperate in recruiting Spt6, genome-wide. Interestingly, the Ser2 kinases promote the association of Spt6 in early transcribed regions and not toward the 3' ends of genes, where phosphorylated Ser2 reaches its maximum level. In addition, our results uncover an unexpected role for histone deacetylases (Rpd3 and Hos2) in promoting Spt6 interaction with elongating Pol II. Finally, our data suggest that phosphorylation of the Pol II CTD on Tyr1 promotes the association of Spt6 with the 3' ends of transcribed genes, independently of Ser2 phosphorylation. Collectively, our results show that a complex network of interactions, involving the Spt6 tSH2 domain, CTD phosphorylation, and histone deacetylases, coordinate the recruitment of Spt6 to transcribed genes in vivo.