Phenotypic heterogeneity in individuals with MECOM variants in 2 families

MECOM encodes the transcriptional regulators, EVI1 and MDS1-EVI1, from two distinct transcription start sites. EVI1 plays important roles in hematopoiesis and stem cell self-renewal. Recently, our group and others revealed that individuals with MECOM variants present diverse hematological and skeletal defects, including radioulnar synostosis (RUS). In the present study, we analyzed two families suspected with MECOM-associated syndrome. In family 1, a MECOM splicing variant (c.2285+1G>A) was identified in an individual with bone marrow failure (TRS4) without RUS and her mother, who had mild leukocytopenia, thrombocytopenia, and bilateral RUS. A copy neutral loss of heterozygosity decreasing the variant allele frequency was observed in the bone marrow of TRS4 and the peripheral blood leukocytes of her mother. However, TRS4 remained transfusion-dependent. In family 2, a MECOM variant (c.2208-4A>G), which was predicted to cause a cryptic acceptor site that results in a 3-base insertion (an insertion of Ser) in the mRNA, was identified in the proband, with bone marrow failure; this variant was also observed in her brother and father, both of whom have skeletal malformations, but no cytopenia. RT-PCR using leukocytes revealed a transcript with a 3-bp insertion in the proband, her brother, and the father, suggesting that the transcript variant with a 3-bp insertion is independent of blood phenotype. Collectively, these results suggest the presence of intrafamilial clinical heterogeneity in both families with MECOM splicing variants. Somatic genetic event may complicate the understanding of clinical variability among family members.

Fanconi Anemia Patients from an Indigenous Community in Mexico Carry a New Founder Pathogenic Variant in FANCG

Fanconi anemia (FA) is a rare genetic disorder caused by pathogenic variants (PV) in at least 22 genes, which cooperate in the FA/BRCA pathway to maintain genome stability. PV in FANCA, FANCC, and FANCG account for most cases (~90%). This study evaluated the chromosomal, molecular, and phenotypic findings of a novel founder FANCG PV, identified in three patients with FA from the Mixe community of Oaxaca, Mexico. All patients presented chromosomal instability and a homozygous PV, FANCG: c.511-3_511-2delCA, identified by next-generation sequencing analysis. Bioinformatics predictions suggest that this deletion disrupts a splice acceptor site promoting the exon 5 skipping. Analysis of Cytoscan 750K arrays for haplotyping and global ancestry supported the Mexican origin and founder effect of the variant, reaffirming the high frequency of founder PV in FANCG. The degree of bone marrow failure and physical findings (described through the acronyms VACTERL-H and PHENOS) were used to depict the phenotype of the patients. Despite having a similar frequency of chromosomal aberrations and genetic constitution, the phenotype showed a wide spectrum of severity. The identification of a founder PV could help for a systematic and accurate genetic screening of patients with FA suspicion in this population.

Retroperitoneal leiomyosarcoma in a female patient with a germline splicing variant RAD51D c.904-2A > T: a case report

Background RAD51D (RAD51 paralog D) is an intermediate cancer susceptibility gene for primary ovarian cancer, including fallopian tube and peritoneal carcinomas and breast cancer. Although gynecological non-epithelial tumors such as uterine sarcomas are associated with genomic instability, including BRCA impairment, there is no clear evidence of the relationship between RAD51D variants and the risk of sarcoma development. Case presentation A Japanese woman in her 50s underwent multiple surgical resections and several regimens of chemotherapy for tumors that originated in the retroperitoneum and recurred in the peritoneum over a clinical course of approximately 4 years. The peritoneal tumor was histologically diagnosed as a leiomyosarcoma and was genetically identified to show a splice variant of RAD51D c.904-2A > T [NM_002878] through tumor profiling performed as a part of cancer precision medicine. The confirmatory genetic test performed after genetic counseling revealed that the RAD51D splicing variant detected in her tumor was of germline origin. In silico analyses supported the possible pathogenicity of the detected splice variant of RAD51D with a predicted attenuation in mRNA transcription and truncated protein production due to frameshifting, which was attributed to a single-nucleotide alteration in the splicing acceptor site at the 3′-end of intron 9 of RAD51D. Considering her unfavorable clinical outcome, which showed a highly aggressive phenotype of leiomyosarcoma with altered RAD51D, this case provided novel evidence for the relationship of a RAD51D splicing variant with malignant tumor development or progression. We report the findings of this rare case with possible involvement of the germline variant of RAD51D c.904-2A > T as a potential predisposing factor for malignant tumors, including leiomyosarcoma. Conclusions We present the findings of a case of leiomyosarcoma in the peritoneum of a female patient with a novel germline splicing variant of RAD51D as potential evidence for the pathogenicity of the variant and its involvement in the risk of sarcoma etiology and/or development. To the best of our knowledge, this is the first case report describing a leiomyosarcoma carrying a germline RAD51D splicing variant and elucidating its pathogenicity on the basis of computational prediction of the impairment of normal transcription and the presumed loss of functional protein production.

Functionally distinct roles for eEF2K in the control of ribosome availability and p-body abundance

Processing bodies (p-bodies) are a prototypical phase-separated RNA-containing granule. Their abundance is highly dynamic and has been linked to translation. Yet, the molecular mechanisms responsible for coordinate control of the two processes are unclear. Here, we uncover key roles for eEF2 kinase (eEF2K) in the control of ribosome availability and p-body abundance. eEF2K acts on a sole known substrate, eEF2, to inhibit translation. We find that the eEF2K agonist nelfinavir abolishes p-bodies in sensory neurons and impairs translation. To probe the latter, we used cryo-electron microscopy. Nelfinavir stabilizes vacant 80S ribosomes. They contain SERBP1 in place of mRNA and eEF2 in the acceptor site. Phosphorylated eEF2 associates with inactive ribosomes that resist splitting in vitro. Collectively, the data suggest that eEF2K defines a population of inactive ribosomes resistant to recycling and protected from degradation. Thus, eEF2K activity is central to both p-body abundance and ribosome availability in sensory neurons.

Genetic manipulation of pyoverdine non-ribosomal peptide synthetases to identify genetic constraints to effective domain recombination

<p>Non-ribosomal peptide synthetases (NRPSs) synthesise small highly diverse peptides with a wide range of activities, such as antibiotics, anticancer drugs, and immunosuppressants. NRPS synthesis often resembles an assembly line, in which each module acts in a linear order to add one monomer to the growing peptide chain. In the basic mechanism of synthesis, an adenylation (A) domain within each module activates a specific monomer. Once activated, the monomer is attached to an immediately downstream thiolation (T) domain via a prosthetic phosphopantheine group, which acts as a flexible arm to pass the substrate between catalytic domains. A condensation (C) domain, upstream to the A-T domains, catalyses peptide bond formation between an acceptor substrate attached to the T domain and a donor substrate attached to the T domain of the upstream module. The peptide remains attached to the T domain of the acceptor substrate, and then acts as the donor substrate for the next C domain. When peptide synthesis reaches the final module, the peptide is released by a thioesterase (TE) domain. The linear mode of synthesis and discrete functional domains within each module gives the potential to generate new products by substituting domains or entire modules with ones that activate alternative substrates. Attempts to create new products using domain and module substitution often result in a loss of activity. The work in this thesis focuses on identifying barriers to effective domain substitution. The NRPS enzyme pvdD, which adds the final residue to the eleven residue non-ribosomal peptide pyoverdine, was developed as a model for domain substitution. The primary benefit for using this model is that pyoverdine creates easily detectible fluorescent products. The first set of experiments focused on testing the limitations of A domain and C-A domain substitutions to alter pyoverdine. Nine A domain and nine C-A domain substitution pvdD variants were constructed and used to complement a P. aeruginosa PAO1 pvdD deletion strain. The A domain substitutions that specified the wild type substrate were highly functional, whereas A domains that specified other substrates resulted in low levels of wild type pyoverdine production. This suggests the acceptor site substrate specificity of the C domain limited the success of A domain substitutions, rather than disruption of the C/A domain junction. In contrast, although C-A domain substitutions in pvdD in some cases synthesised novel pyoverdines, the majority lost function for unknown reasons. The high success rate A domain substitutions (when not limited by the acceptor site specificity of the C domain) suggested the addition of new C domains was a likely cause for loss of function. The second set of experiments investigated whether disrupting the protein interface between C domains and their upstream T domains may cause a loss in function of C-A domain substitutions. However, domain substitutions of T domains were found to have a high rate of success. Therefore, the results thus far confirmed that disrupting interactions of the C domain with A domains or T domains does not have a large affect on enzyme activity. An alternative explanation for the loss in function with C-A domain substitutions is that C domains translocated to a new enzyme are unable to process the new incoming donor peptide chain because of substrate specificity or steric constraints. To develop methods to circumvent limitations caused by the C domain, the final part of this thesis examined acceptor substrate specificity of C domains. Acceptor site substrate specificity was chosen over donor site specificity as it acts on only an amino acid rather than peptide chain. The substrate specificity was narrowed down to a small subsection of the C domain. This was an initial study of C domain substrate specificity, which may guide future development of relaxed specificity C domains.</p>

Genetic manipulation of pyoverdine non-ribosomal peptide synthetases to identify genetic constraints to effective domain recombination

<p>Non-ribosomal peptide synthetases (NRPSs) synthesise small highly diverse peptides with a wide range of activities, such as antibiotics, anticancer drugs, and immunosuppressants. NRPS synthesis often resembles an assembly line, in which each module acts in a linear order to add one monomer to the growing peptide chain. In the basic mechanism of synthesis, an adenylation (A) domain within each module activates a specific monomer. Once activated, the monomer is attached to an immediately downstream thiolation (T) domain via a prosthetic phosphopantheine group, which acts as a flexible arm to pass the substrate between catalytic domains. A condensation (C) domain, upstream to the A-T domains, catalyses peptide bond formation between an acceptor substrate attached to the T domain and a donor substrate attached to the T domain of the upstream module. The peptide remains attached to the T domain of the acceptor substrate, and then acts as the donor substrate for the next C domain. When peptide synthesis reaches the final module, the peptide is released by a thioesterase (TE) domain. The linear mode of synthesis and discrete functional domains within each module gives the potential to generate new products by substituting domains or entire modules with ones that activate alternative substrates. Attempts to create new products using domain and module substitution often result in a loss of activity. The work in this thesis focuses on identifying barriers to effective domain substitution. The NRPS enzyme pvdD, which adds the final residue to the eleven residue non-ribosomal peptide pyoverdine, was developed as a model for domain substitution. The primary benefit for using this model is that pyoverdine creates easily detectible fluorescent products. The first set of experiments focused on testing the limitations of A domain and C-A domain substitutions to alter pyoverdine. Nine A domain and nine C-A domain substitution pvdD variants were constructed and used to complement a P. aeruginosa PAO1 pvdD deletion strain. The A domain substitutions that specified the wild type substrate were highly functional, whereas A domains that specified other substrates resulted in low levels of wild type pyoverdine production. This suggests the acceptor site substrate specificity of the C domain limited the success of A domain substitutions, rather than disruption of the C/A domain junction. In contrast, although C-A domain substitutions in pvdD in some cases synthesised novel pyoverdines, the majority lost function for unknown reasons. The high success rate A domain substitutions (when not limited by the acceptor site specificity of the C domain) suggested the addition of new C domains was a likely cause for loss of function. The second set of experiments investigated whether disrupting the protein interface between C domains and their upstream T domains may cause a loss in function of C-A domain substitutions. However, domain substitutions of T domains were found to have a high rate of success. Therefore, the results thus far confirmed that disrupting interactions of the C domain with A domains or T domains does not have a large affect on enzyme activity. An alternative explanation for the loss in function with C-A domain substitutions is that C domains translocated to a new enzyme are unable to process the new incoming donor peptide chain because of substrate specificity or steric constraints. To develop methods to circumvent limitations caused by the C domain, the final part of this thesis examined acceptor substrate specificity of C domains. Acceptor site substrate specificity was chosen over donor site specificity as it acts on only an amino acid rather than peptide chain. The substrate specificity was narrowed down to a small subsection of the C domain. This was an initial study of C domain substrate specificity, which may guide future development of relaxed specificity C domains.</p>

A deep intronic mutation in AR gene causing androgen insensitivity syndrome: difficulties of diagnostics

Partial androgen resistance syndrome (PAIS) is the most difficult form of disorders/differences of sex development 46,XY (DSD 46,XY) for choosing of patient management. To date, there are no clear biochemical criteria, especially before puberty, that allow differentiating PAIS from other PAIS-like forms of DSD 46, XY, and genetic verification of the partial form of AIS plays an important role. Meanwhile, according to the literature, mutations in the coding region of AR gene have not been identified in more than 50% of patients with suspected AIS. We performed an extensive analysis of the AR gene in a patient with clinical and laboratory signs of AIS and found a deep intron mutation in the AR gene (p. 2450–42G>A). This variant creates an alternative splice acceptor site resulted a disturbance of the AR function. These findings indicate the need for extensive genetic analysis in a cohort of patients with suspected CPA in the absence of mutations in the AR gene using standard methods of genetic diagnosis.

Adenine Base-Editing-Mediated Exon Skipping Induces Gene Knockout in Cultured Pig Cells

Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif (PAM) sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified “all-in-one” ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The “all-in-one” ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3’ end of the intron 5) and splice donor site (GT sequence at the 5’ end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.

A Cytosolic Reductase Pathway is Required for Efficient N-Glycosylation of an STT3B-Dependent Acceptor Site

N-linked glycosylation of proteins entering the secretory pathway is an essential modification required for protein stability and function. Previously, it has been shown that there is a temporal relationship between protein folding and glycosylation, which influences the occupancy of specific glycosylation sites. Here we use an in vitro translation system that reproduces the initial stages of secretory protein translocation, folding and glycosylation under defined redox conditions. We found that the efficiency of glycosylation of hemopexin was dependent upon a robust NADPH-dependent cytosolic reductive pathway, which could also be mimicked by the addition of a membrane impermeable reducing agent. The identified hypoglycosylated acceptor site is adjacent to a cysteine involved in a short range disulfide, which has been shown to be dependent on the STT3B-containing oligosaccharyl transferase. We also show that efficient glycosylation at this site is influenced by the cytosolic reductive pathway acting on both STT3A and STT3B-dependent glycosylation. Our results provide further insight into the important role of the ER redox conditions in glycosylation site occupancy and demonstrate a link between redox conditions in the cytosol and glycosylation efficiency.