Ulnar-mammary syndrome (UMS) is a rare, autosomal dominant disorder characterized by anomalies affecting the limbs, apocrine glands, dentition, and genital development. This syndrome is caused by haploinsufficiency in the T-Box3 gene (TBX3), with considerable variability in the clinical phenotype being observed even within families. We describe a one-year-old female with unilateral, postaxial polydactyly, and bilateral fifth fingernail duplication. Next-generation sequencing revealed a novel, likely pathogenic, variant predicted to affect the canonical splice site in intron 3 of the TBX3 gene (c.804 + 1G > A, IVS3 + 1G > A). This variant was inherited from the proband’s father who was also diagnosed with UMS with the additional clinical finding of congenital, sagittal craniosynostosis. Subsequent whole genome analysis in the proband’s father detected a variant in the EFNA4 gene (c.178C > T, p.His60Tyr), which has only been reported to be associated with sagittal craniosynostosis in one patient prior to this report but reported in other cranial suture synostosis. The findings in this family extend the genotypic spectrum of UMS, as well as the phenotypic spectrum of EFNA4-related craniosynostosis.

Background: Loose anagen hair syndrome is a recently described genetic form of non-scarring alopecia that occurs in children and is due to poorly anchored hair shafts during the anagen phase. It can occur alone or in association with hair pathology or complex systemic phenotypes. Methods: We report a mother and daughter with loose anagen hair syndrome that is associated with wooly hair, although it shows variable expressivity. We studied the family using genomic sequencing and identified an intronic variant in their KRT71 that segregates in an autosomal dominant pattern and is suspected to affect splicing in the tail domain of this hair follicle keratin. We studied this variant with a minigene experimental approach. Results: We provide experimental evidence that the identified intronic variant affects splicing in the tail domain, which is critical to the biomechanical properties of the keratin intermediate filaments. We demonstrate that it affects splicing by adding 12 bases to the mature transcript and consequently four amino acids to the peptide. Conclusion: We suspect that this variant is responsible for the poorly anchored and finely curled hair in the mother and daughter, which leads to a proposed diagnosis of autosomal dominant wooly hair, as well as loose anagen hair syndrome. We thus expand the variant spectrum of KRT71 and its associated phenotypes to include both disorders.

The study of cytoskeletal proteins has been limited by some of their intrinsic properties, such as fixed intracellular localization and insolubility, which limit the implementation of many protein–protein interaction assays. Far Western blotting is a powerful biochemical technique used to detect and quantify protein–protein interactions on a membrane, addressing limitations in cytoskeletal research. This method combines traditional Western blotting with protein overlay assays to identify and characterize binding partners in complex samples. Here, we present a detailed protocol for the use of far Western blotting to probe keratin–keratin interactions, including instructions for protein sample preparation, electrophoretic separation, membrane transfer, protein labeling, buffer dilutions, and detection steps. We further extend the use of far Western blotting to compare relative binding and enhance assay sensitivity by incorporating a Western blot as a loading control. This approach enables the analysis of samples that are too dilute for reliable membrane staining. Key considerations for enhancing specificity and sensitivity are discussed, along with troubleshooting tips. This approach offers a versatile tool for studying protein interaction networks, providing valuable insights into cellular signaling and molecular mechanisms, and can be conducted in any laboratory capable of performing a Western blot.