Dominant ELOVL1 mutation causes neurological disorder with ichthyotic keratoderma, spasticity, hypomyelination and dysmorphic features.

BACKGROUND: Ichthyosis and neurological involvement occur in relatively few known Mendelian disorders caused by mutations in genes relevant both for epidermis and neural function. OBJECTIVES: To identify the cause of a similar phenotype of ichthyotic keratoderma, spasticity, mild hypomyelination (on MRI) and dysmorphic features (IKSHD) observed in two unrelated paediatric probands without family history of disease. METHODS: Whole exome sequencing was performed in both patients. The functional effect of prioritised variant in ELOVL1 (very-long-chain fatty acids (VLCFAs) elongase) was analysed by VLCFA profiling by gas chromatography-mass spectrometry in stably transfected HEK2932 cells and in cultured patient's fibroblasts. RESULTS: Probands shared novel heterozygous ELOVL1 p.Ser165Phe mutation (de novo in one family, while in the other family, father could not be tested). In transfected cells p.Ser165Phe: (1) reduced levels of FAs C24:0-C28:0 and C26:1 with the most pronounced effect for C26:0 (P=7.8×10-6 vs HEK293 cells with wild type (wt) construct, no difference vs naïve HEK293) and (2) increased levels of C20:0 and C22:0 (P=6.3×10-7, P=1.2×10-5, for C20:0 and C22:0, respectively, comparison vs HEK293 cells with wt construct; P=2.2×10-7, P=1.9×10-4, respectively, comparison vs naïve HEK293). In skin fibroblasts, there was decrease of C26:1 (P=0.014), C28:0 (P=0.001) and increase of C20:0 (P=0.033) in the patient versus controls. There was a strong correlation (r=0.92, P=0.008) between the FAs profile of patient's fibroblasts and that of p.Ser165Phe transfected HEK293 cells. Serum levels of C20:0-C26:0 FAs were normal, but the C24:0/C22:0 ratio was decreased. CONCLUSION: The ELOVL1 p.Ser165Phe mutation is a likely cause of IKSHD.

Multiple myeloma-associated hDIS3 mutations cause perturbations in cellular RNA metabolism and suggest hDIS3 PIN domain as a potential drug target.

hDIS3 is a mainly nuclear, catalytic subunit of the human exosome complex, containing exonucleolytic (RNB) and endonucleolytic (PIN) active domains. Mutations in hDIS3 have been found in ∼10% of patients with multiple myeloma (MM). Here, we show that these mutations interfere with hDIS3 exonucleolytic activity. Yeast harboring corresponding mutations in DIS3 show growth inhibition and changes in nuclear RNA metabolism typical for exosome dysfunction. Construction of a conditional DIS3 knockout in the chicken DT40 cell line revealed that DIS3 is essential for cell survival, indicating that its function cannot be replaced by other exosome-associated nucleases: hDIS3L and hRRP6. Moreover, HEK293-derived cells, in which depletion of endogenous wild-type hDIS3 was complemented with exogenously expressed MM hDIS3 mutants, proliferate at a slower rate and exhibit aberrant RNA metabolism. Importantly, MM mutations are synthetically lethal with the hDIS3 PIN domain catalytic mutation both in yeast and human cells. Since mutations in PIN domain alone have little effect on cell physiology, our results predict the hDIS3 PIN domain as a potential drug target for MM patients with hDIS3 mutations. It is an interesting example of intramolecular synthetic lethality with putative therapeutic potential in humans.

TENT5-mediated polyadenylation of mRNAs encoding secreted proteins is essential for gametogenesis in mice.

Cytoplasmic polyadenylation plays a vital role in gametogenesis; however, the participating enzymes and substrates in mammals remain unclear. Using knockout and knock-in mouse models, we describe the essential role of four TENT5 poly(A) polymerases in mouse fertility and gametogenesis. TENT5B and TENT5C play crucial yet redundant roles in oogenesis, with the double knockout of both genes leading to oocyte degeneration. Additionally, TENT5B-GFP knock-in females display a gain-of-function infertility effect, with multiple chromosomal aberrations in ovulated oocytes. TENT5C and TENT5D both regulate different stages of spermatogenesis, as shown by the sterility in males following the knockout of either gene. Finally, Tent5a knockout substantially lowers fertility, although the underlying mechanism is not directly related to gametogenesis. Through direct RNA sequencing, we discovered that TENT5s polyadenylate mRNAs encoding endoplasmic reticulum-targeted proteins essential for gametogenesis. Sequence motif analysis and reporter mRNA assays reveal that the presence of an endoplasmic reticulum-leader sequence represents the primary determinant of TENT5-mediated regulation.

Disrupting interaction between miR-132 and Mmp9 3'UTR improves synaptic plasticity and memory in mice.

As microRNAs have emerged to be important regulators of molecular events occurring at the synapses, the new questions about their regulatory effect on the behavior have araised. In the present study, we show for the first time that the dysregulated specific targeting of miR132 to Mmp9 mRNA in the mouse brain results in the increased level of Mmp9 protein, which affects synaptic plasticity and has an effect on memory formation. Our data points at the importance of complex and precise regulation of the Mmp9 level by miR132 in the brain.

Cytoplasmic polyadenylation by TENT5A is required for proper bone formation.

Osteoblasts orchestrate bone formation through the secretion of type I collagen and other constituents of the matrix on which hydroxyapatite crystals mineralize. Here, we show that TENT5A, whose mutations were found in congenital bone disease osteogenesis imperfecta patients, is a cytoplasmic poly(A) polymerase playing a crucial role in regulating bone mineralization. Direct RNA sequencing revealed that TENT5A is induced during osteoblast differentiation and polyadenylates mRNAs encoding Col1α1, Col1α2, and other secreted proteins involved in osteogenesis, increasing their expression. We postulate that TENT5A, possibly together with its paralog TENT5C, is responsible for the wave of cytoplasmic polyadenylation of mRNAs encoding secreted proteins occurring during bone mineralization. Importantly, the Tent5a knockout (KO) mouse line displays bone fragility and skeletal hypomineralization phenotype resulting from quantitative and qualitative collagen defects. Thus, we report a biologically relevant posttranscriptional regulator of collagen production and, more generally, bone formation.

Immunoglobulin expression and the humoral immune response is regulated by the non-canonical poly(A) polymerase TENT5C.

TENT5C is a non-canonical cytoplasmic poly(A) polymerase highly expressed by activated B cells to suppress their proliferation. Here we measure the global distribution of poly(A) tail lengths in responsive B cells using a Nanopore direct RNA-sequencing approach, showing that TENT5C polyadenylates immunoglobulin mRNAs regulating their half-life and consequently steady-state levels. TENT5C is upregulated in differentiating plasma cells by innate signaling. Compared with wild-type, Tent5c-/- mice produce fewer antibodies and have diminished T-cell-independent immune response despite having more CD138high plasma cells as a consequence of accelerated differentiation. B cells from Tent5c-/- mice also have impaired capacity of the secretory pathway, with reduced ER volume and unfolded protein response. Importantly, these functions of TENT5C are dependent on its enzymatic activity as catalytic mutation knock-in mice display the same defect as Tent5c-/-. These findings define the role of the TENT5C enzyme in the humoral immune response.

The non-canonical poly(A) polymerase FAM46C acts as an onco-suppressor in multiple myeloma.

FAM46C is one of the most frequently mutated genes in multiple myeloma. Here, using a combination of in vitro and in vivo approaches, we demonstrate that FAM46C encodes an active non-canonical poly(A) polymerase which enhances mRNA stability and gene expression. Reintroduction of active FAM46C into multiple myeloma cell lines, but not its catalytically-inactive mutant, leads to broad polyadenylation and stabilization of mRNAs strongly enriched with those encoding endoplasmic reticulum-targeted proteins and induces cell death. Moreover, silencing of FAM46C in multiple myeloma cells expressing WT protein enhance cell proliferation. Finally, using a FAM46C-FLAG knock-in mouse strain, we show that the FAM46C protein is strongly induced during activation of primary splenocytes and that B lymphocytes isolated from newly generated FAM46C KO mice proliferate faster than those isolated from their WT littermates. Concluding, our data clearly indicate that FAM46C works as an onco-suppressor, with the specificity for B-lymphocyte lineage from which multiple myeloma originates. FAM46C is one of the most frequently mutated genes in multiple myeloma (MM), but its molecular function remains unknown. Here the authors show that FAM46C is a poly(A) polymerase and that loss of function of FAM46C drives multiple myeloma through the destabilisation of ER response transcripts.

Association of the BRCA1 promoter polymorphism rs11655505 with the risk of familial breast and/or ovarian cancer.

Germline mutations in the BRCA1 tumor suppressor gene predispose affected individuals to breast cancer; however, incomplete cancer penetrance and the presence of phenocopies in BRCA1 families also indicate genetic and environmental modifiers of breast cancer risk. In this study, we have tested the single nucleotide polymorphism rs1655505 of the BRCA1 promoter, as candidate for the modifier of breast cancer risk. The polymorphic variants were genotyped in BRCA1-negative (729), familial breast and/or ovarian cancer cases (FBOC), including cases with a reported maternal history (154), nonfamilal (sporadic) cases (600), hereditary breast/ovarian cases with BRCA1 mutations (190) and population controls (1,590) from Central Poland. An association with the risk of FBOC was observed for the minor (T) allele and (TT) genotype (T: p = 0.006, OR = 1.40, 95% CI = 1.10-1.79; TT: p = 0.001, OR = 2.23, 95% CI = 1.37-3.62) in female cases with a reported maternal history, specifically in women with the onset of disease after 50 years of age (T: p = 0.004, OR = 1.77, 95% CI = 1.20-2.62; TT: p = 0.001, OR = 3.7, 95% CI = 1.62-8.46). The presented evidence suggests a need to conduct larger studies on the association between genetic variations at the BRCA1 promoter and the breast cancer risk, according to maternal/paternal lineage.

Aldolase B mutations and prevalence of hereditary fructose intolerance in a Polish population.

We studied 28 Polish hereditary fructose intolerant (HFI) patients (26 unrelated) by direct sequencing of the ALDOB coding region/splice sites. Eight different mutations were found including two novel ones (each found in two unrelated individuals): c.250delC (frameshift) and c.522 C > G (p.Y174X). The most frequent mutation c.448 G > C (p.A150P, 67% of chromosomes) was screened for in a group of 1049 randomly selected unrelated individuals. Eight (1:131) carriers were found allowing to estimate the HFI prevalence in Poland as 1:31,000.