A Novel Splicing Variant of COL2A1 in a Fetus with Achondrogenesis Type II: Interpretation of Pathogenicity of In-Frame Deletions.

Achondrogenesis type II (ACG2) is a lethal skeletal dysplasia caused by dominant pathogenic variants in COL2A1. Most of the variants found in patients with ACG2 affect the glycine residue included in the Gly-X-Y tripeptide repeat that characterizes the type II collagen helix. In this study, we reported a case of a novel splicing variant of COL2A1 in a fetus with ACG2. An NGS analysis of fetal DNA revealed a heterozygous variant c.1267-2_1269del located in intron 20/exon 21. The variant occurred de novo since it was not detected in DNA from the blood samples of parents. We generated an appropriate minigene construct to study the effect of the variant detected. The minigene expression resulted in the synthesis of a COL2A1 messenger RNA lacking exon 21, which generated a predicted in-frame deleted protein. Usually, in-frame deletion variants of COL2A1 cause a phenotype such as Kniest dysplasia, which is milder than ACG2. Therefore, we propose that the size and position of an in-frame deletion in COL2A1 may be relevant in determining the phenotype of skeletal dysplasia.

Functional characterization of p.Pro409His variant in HNF1A, a hypomorphic mutation involved in pancreatic ß-cell dysfunction.

AIMS: HNF1A is a gene coding for the transcription factor HNF1-α, mutated in some forms of MODY and type 2 diabetes mellitus characterized by a strong genetic component. The penetrance of HNF1A variants differs considerably; thus, to assess the genetic risk of diabetes in carrier subjects of a HNF1A mutant allele, a functional characterization of mutant forms is of paramount importance. METHODS: The HNF1A gene was sequenced in two patients with partly discordant diabetic phenotype, carrying the p.Pro409His variant. To evaluate the pathogenicity of the variant, we measured the transactivation power of the corresponding P408H HNF1-α mutant mouse form on HNF1-α target promoters. RESULTS: We found a lower but detectable activity of transactivation of the mutant form compared with the wild-type form and we excluded mechanisms of protein degradation or nuclear mislocalization. CONCLUSIONS: The HNF1A mutation p.Pro409His can be considered a mild variant that confers a moderate risk of type 2 diabetes mellitus in heterozygous carriers.

Recombinant TAT-BMI-1 fusion protein induces ex vivo expansion of human umbilical cord blood-derived hematopoietic stem cells.

Transplantation of hematopoietic stem cells (HSCs) is a well-established therapeutic approach for numerous disorders. HSCs are typically derived from bone marrow or peripheral blood after cytokine-induced mobilization. Umbilical cord blood (CB) represents an appealing alternative HSC source, but the small amounts of the individual CB units have limited its applications. The availability of strategies for safe ex vivo expansion of CB-derived HSCs (CB-HSCs) may allow to extend the use of these cells in adult patients and to avoid the risk of insufficient engraftment or delayed hematopoietic recovery.Here we describe a system for the ex vivo expansion of CB-HSCs based on their transient exposure to a recombinant TAT-BMI-1 chimeric protein. BMI-1 belongs to the Polycomb family of epigenetic modifiers and is recognized as a central regulator of HSC self-renewal. Recombinant TAT-BMI-1 produced in bacteria was able to enter the target cells via the HIV TAT-derived protein transduction peptide covalently attached to BMI-1, and conserved its biological activity. Treatment of CB-CD34+ cells for 3 days with repeated addition of 10 nM purified TAT-BMI-1 significantly enhanced total cell expansion as well as that of primitive hematopoietic progenitors in culture. Importantly, TAT-BMI-1-treated CB-CD34+ cells displayed a consistently higher rate of multi-lineage long-term repopulating activity in primary and secondary xenotransplants in immunocompromised mice. Thus, recombinant TAT-BMI-1 may represent a novel, effective reagent for ex vivo expansion of CB-HSC for therapeutic purposes.