Familial visceral myopathy (FVM) is a rare heritable and heterogeneous condition due to impaired smooth muscle function. We identified a family segregating 11 individuals with a spectrum of visceral symptoms involving the small intestine, colon, biliary tract, urinary tract and uterus. Whole-exome sequencing revealed a novel heterozygous tandem base substitution c.806_807delinsAA (p.(Gly269Glu)) in ACTG2, encoding smooth muscle actin γ-2, in affected family members. Variants in ACTG2 were recently identified in FVM with intestinal pseudo-obstruction as well as with the congenital megacystics-microcolonintestinal hypoperistalsis syndrome. In our family, eight affected members presented with severe complications from the biliary and/or the urinary tracts in addition to gastrointestinal pseudo-obstructions. Furthermore, all affected mothers had a history of assisted deliveries owing to poor progress during labor and weak uterine contractions. The variable involvement of multiple smooth muscle-dependent organs in our family, including the biliary tract and the uterus, add to the phenotypic spectrum associated with ACTG2 missense variants.

We have redesigned probes for in situ proximity ligation assay (PLA), resulting in more efficient localized detection of target proteins. In situ PLA depends on recognition of target proteins by pairs of antibody-oligonucleotide conjugates (PLA probes), which jointly give rise to DNA circles that template localized rolling circle amplification reactions. The requirement for dual recognition of the target proteins improves selectivity by ignoring any cross-reactivity not shared by the antibodies, and it allows detection of protein-protein interactions and post-translational modifications. We herein describe an improved design of the PLA probes –UnFold probes – where all elements required for formation of circular DNA strands are incorporated in the probes. Premature interactions between the UnFold probes are prevented by including an enzymatic “unfolding” step in the detection reactions. This allows DNA circles to form by pairs of reagents only after excess reagents have been removed. We demonstrate the performance of UnFold probes for detection of protein-protein interactions and post-translational modifications in fixed cells and tissues, revealing considerably more efficient signal generation. We also apply the UnFold probes to detect IL-6 in solution phase after capture on solid supports, demonstrating increased sensitivity over both normal sandwich enzyme-linked immunosorbent assays and conventional PLA assays.

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The invention of the microscope has been fundamental for the understanding of tissue architecture and subcellular structures. With the advancement of higher magnification microscopes came the development of various molecular biology tools such as Förster resonance energy transfer (FRET) and in situ proximity ligation assay (in situ PLA) to monitor protein interactions. Microscopy has become a commonly used method for the investigation of molecular events within the cell, for the identification of key players in signaling networks, and the activation of these pathways. Multiple approaches are available for functional analyses in single cells. They provide information not only on the localization of proteins at a given time point, but also on their expression levels and activity states, allowing us to pinpoint hallmarks of different cellular identities within tissues in health and disease. Clever solutions to increase the sensitivity of molecular tools, the possibilities for multiplexing, as well as image resolution have recently been introduced; however, these methods have their pros and cons. Therefore, one needs to carefully consider the biological question of interest along with the nature of the sample before choosing the most suitable method or combination of methods. Herein, we review a few of the most exciting microscopy-based molecular techniques for proteomic analysis and cover the benefits as well as the disadvantages of their use.

Induced pluripotent stem cells (iPSCs) have brought great promises for disease modeling and cell-based therapies. One concern related to the use of reprogrammed somatic cells is the loss of genomic integrity and chromosome stability, a hallmark for cancer and many other human disorders. We investigated 16 human iPSC lines reprogrammed by nonintegrative Sendai virus (SeV) and another 16 iPSC lines generated by integrative lentivirus for genetic changes. At early passages we detected cytogenetic rearrangements in 44% (7/16) of iPSC lines generated by lentiviral integration whereas the corresponding figure was 6% (1/16) using SeV-based delivery. The rearrangements were numerical and/or structural with chromosomes 5 and 12 as the most frequently involved chromosomes. Three iPSC lines with chromosome 5 aberrations were derived from one and the same donor. We present in this study the aberrant karyotypes including a duplication of chromosome 5q13q33 that restricts a candidate region for growth advantage. Our results suggest that the use of integrative lentivirus confers a higher risk for cytogenetic abnormalities at early passages when compared to SeV-based reprogramming. In combination, our findings expand the knowledge on acquired cytogenetic aberrations in iPSC after reprogramming and during culture.

Induced pluripotent stem cell (iPSC) technology has become an important tool for disease modeling. Insufficient data on the variability among iPSC lines derived from a single somatic parental cell line have in practice led to generation and analysis of several, usually three, iPSC sister lines from each parental cell line. We established iPSC lines from a human fibroblast line (HDF-K1) and used transcriptome sequencing to investigate the variation among three sister lines (iPSC-K1A, B, and C). For comparison, we analyzed the transcriptome of an iPSC line (iPSC-K5B) derived from a different fibroblast line (HDF-K5), a human embryonic stem cell (ESC) line (ESC-HS181), as well as the two parental fibroblast lines. All iPSC lines fulfilled stringent criteria for pluripotency. In an unbiased cluster analysis, all stem cell lines (four iPSCs and one ESC) clustered together as opposed to the parental fibroblasts. The transcriptome profiles of the three iPSC sister lines were indistinguishable from each other, and functional pathway analysis did not reveal any significant hits. In contrast, the expression profiles of the ESC line and the iPSC-K5B line were distinct from that of the sister lines iPSC-K1A, B, and C. Differentiation to embryoid bodies and subsequent analysis of germ layer markers in the five stem cell clones confirmed that the distribution of their expression profiles was retained. Taken together, our observations stress the importance of using iPSCs of different parental origin rather than several sister iPSC lines to distinguish disease-associated mechanisms from genetic background effects in disease modeling.

Pure hair and nail ectodermal dysplasia (PHNED) comprises a heterogeneous group of rare heritable disorders characterized by brittle hair, hypotrichosis, onychodystrophy and micronychia. Autosomal recessive (AR) PHNED has previously been associated with mutations in either KRT85 or HOXC13 on chromosome 12p11.1-q14.3. We investigated a consanguineous Pakistani family with AR PHNED linked to the keratin gene cluster on 12p11.1 but without detectable mutations in KRT85 and HOXC13. Whole exome sequencing of affected individuals revealed homozygosity for a rare c.821T>C variant (p.Phe274Ser) in the KRT74 gene that segregates AR PHNED in the family. The transition alters the highly conserved Phe274 residue in the coil 1B domain required for long-range dimerization of keratins, suggesting that the mutation compromises the stability of intermediate filaments. Immunohistochemical (IHC) analyses confirmed a strong keratin-74 expression in the nail matrix, the nail bed and the hyponychium of mouse distal digits, as well as in normal human hair follicles. Furthermore, hair follicles and epidermis of an affected family member stained negative for Keratin-74 suggesting a loss of function mechanism mediated by the Phe274Ser substitution. Our observations show for the first time that homozygosity for a KRT74 missense variant may be associated with AR PHNED. Heterozygous KRT74 mutations have previously been associated with autosomal dominant woolly hair/hypotrichosis simplex (ADWH). Thus, our findings expand the phenotypic spectrum associated with KRT74 mutations and imply that a subtype of AR PHNED is allelic with ADWH.

Optimization of proximity-dependent initiation of hybridization chain reaction for use at varying concentrations of salt and temperature.