(1) Background: Classic Ehlers-Danlos syndrome (cEDS) is a heritable connective tissue disorder characterized by joint hypermobility and skin hyperextensibility with atrophic scarring. Many cEDS individuals carry variants in either the COL5A1 or COL5A2 genes. Mosaicism is relatively common in heritable connective tissue disorders but is rare in EDS. In cEDS, a single example of presumed gonosomal mosaicism for a COL5A1 variant has been published to date. (2) Methods: An 8-year-old girl with cEDS was analyzed by next-generation sequencing (NGS). Segregation was performed by Sanger sequencing in her unaffected parents. In the father, the mosaicism of the variant was further analyzed by targeted NGS and droplet digital PCR (ddPCR) in the blood and by Sanger sequencing in other tissues. (3) Results: The NGS analysis revealed the novel germline heterozygous COL5A1 c.1369G>T, p.(Glu457*) variant in the proband. Sanger chromatogram of the father’s blood specimen suggested the presence of a low-level mosaicism for the COL5A1 variant, which was confirmed by NGS and estimated to be 4.8% by ddPCR. The mosaicism was also confirmed by Sanger sequencing in the father’s saliva, hair bulbs and nails. (4) Conclusions: We described the second case of cEDS caused by paternal gonosomal mosaicism in COL5A1. Parental mosaicism could be an issue in cEDS and, therefore, considered for appropriate genetic counseling.

This study aimed to describe a multisystemic disorder featuring cardiovascular, facial, musculoskeletal, and cutaneous anomalies caused by heterozygous loss-of-function variants in TAB2. Methods: Affected individuals were analyzed by next-generation technologies and genomic array. The presumed loss-of-function effect of identified variants was assessed by luciferase assay in cells transiently expressing TAB2 deleterious alleles. In available patients' fibroblasts, variant pathogenicity was further explored by immunoblot and osteoblast differentiation assays. The transcriptomic profile of fibroblasts was investigated by RNA sequencing. Results: A total of 11 individuals from 8 families were heterozygotes for a novel TAB2 variant. In total, 7 variants were predicted to be null alleles and 1 was a missense change. An additional subject was heterozygous for a 52 kb microdeletion involving TAB2 exons 1 to 3. Luciferase assay indicated a decreased transcriptional activation mediated by NF-κB signaling for all point variants. Immunoblot analysis showed a reduction of TAK1 phosphorylation while osteoblast differentiation was impaired. Transcriptomic analysis identified deregulation of multiple pleiotropic pathways, such as TGFβ-, Ras-MAPK-, and Wnt-signaling networks. Conclusion: Our data defined a novel disorder associated with loss-of-function or, more rarely, hypomorphic alleles in a restricted linker region of TAB2. The pleiotropic manifestations in this disorder partly recapitulate the 6q25.1 (TAB2) microdeletion syndrome and deserve the definition of cardio-facial-cutaneous-articular syndrome.

BACKGROUND: Loss-of-function variants in MID1 are the most common cause of Opitz G/BBB syndrome (OS). The interpretation of intronic variants affecting the splicing is a rising issue in OS. METHODS: Exon sequencing of a 2-year-old boy with OS showed that he was a carrier of the de novo c.1286-10G>T variant in MID1. In silico predictions and minigene assays explored the effect of the variant on splicing. The minigene approach was also applied to two previously identified MID1 c.864+1G>T and c.1285+1G>T variants. RESULTS: Minigene assay demonstrated that the c.1286-10G>T variant generated the inclusion of eight nucleotides that predicted generation of a frameshift. The c.864+1G>T and c.1285+1G>T variants resulted in an in-frame deletion predicted to generate a shorter MID1 protein. In hemizygous males, this allowed reclassification of all the identified variants from "of unknown significance" to "likely pathogenic." CONCLUSIONS: Minigene assay supports functional effects from MID1 intronic variants. This paves the way to the introduction of similar second-tier investigations in the molecular diagnostics workflow of OS. IMPACT:• Causative intronic variants in MID1 are rarely investigated in Opitz syndrome.• MID1 is not expressed in blood and mRNA studies are hardly accessible in routine diagnostics.• Minigene assay is an alternative for assessing the effect of intronic variants on splicing.• This is the first study characterizing the molecular consequences of three MID1 variants for diagnostic purposes and demonstrating the efficacy of minigene assays in supporting their clinical interpretation. •Review of the criteria according to the American College of Medical Genetics reassessed all variants as likely pathogenic.