Combined measurements of mRNA and protein expression in single cells enable in-depth analysis of cellular states. We present SPARC, an approach that combines single-cell RNA-sequencing with proximity extension essays to simultaneously measure global mRNA and 89 intracellular proteins in individual cells. We show that mRNA expression fails to accurately reflect protein abundance at the time of measurement, although the direction of changes is in agreement during neuronal differentiation. Moreover, protein levels of transcription factors better predict their downstream effects than do their corresponding transcripts. Finally, we highlight that protein expression variation is overall lower than mRNA variation, but relative protein variation does not reflect the mRNA level. Our results demonstrate that mRNA and protein measurements in single cells provide different and complementary information regarding cell states. SPARC presents a state-of-the-art co-profiling method that overcomes current limitations in throughput and protein localization, including removing the need for cell fixation.

Amplification of DNA is required as a mandatory step during library preparation in most targeted sequencing protocols. This can be a critical limitation when targeting regions that are highly repetitive or with extreme guanine‐cytosine (GC) content, including repeat expansions associated with human disease. Here we used an amplification‐free protocol for targeted enrichment utilizing the CRISPR/Cas9 system (No‐Amp Targeted sequencing) in combination with Single Molecule, Real‐Time (SMRT) sequencing for studying repeat elements in the huntingtin (HTT) gene, where an expanded CAG repeat is causative for Huntington disease. We also developed a robust data analysis pipeline for repeat element analysis that is independent of alignment of reads to a reference genome. The method was applied to eleven diagnostic blood samples, and for all 22 alleles the resulting CAG repeat count agreed with previous results based on fragment analysis. The amplification‐free protocol also allowed for studying somatic variability of repeat elements in our samples, without the interference of PCR stutter. In summary, with No‐Amp Targeted sequencing in combination with our analysis pipeline, we could accurately study repeat elements that are difficult to investigate using PCR‐based methods. This article is protected by copyright. All rights reserved

Welander distal myopathy (WDM) is an adult onset autosomal dominant disorder characterized by distal limb weakness, which progresses slowly from the fifth decade. All WDM patients are of Swedish or Finnish descent and share a rare chromosome 2p13 haplotype. We restricted the WDM-associated haplotype followed by whole exome sequencing. Within the conserved haplotype, we identified a single heterozygous mutation c.1150G>A (p.E384K) in T-cell intracellular antigen-1 (TIA1) in all WDM patients investigated (n = 43). The TIA1 protein regulates splicing, and translation through direct interaction with mRNA and the p.E384K mutation is located in the C-terminal Q-rich domain that interacts with the U1-C splicing factor. TIA1 has been shown to prevent skipping of SMN2 exon 7, and we show that WDM patients have increased levels of spliced SMN2 in skeletal muscle cells when compared with controls. Immunostaining of WDM muscle biopsies showed accumulation of TIA1 and stress granulae proteins adjacent to intracellular inclusions, a typical finding in WDM. The combined findings strongly suggest that the TIA1 mutation causes perturbed RNA splicing and cellular stress resulting in WDM. The selection against the mutation is likely to be negligible and the age of the TIA1 founder mutation was calculated to approximately 1,050 years, which coincides with the epoch of early seafaring across the Baltic Sea.

There are 3 major sweat-producing glands present in skin; eccrine, apocrine, and apoeccrine glands. Due to the high rate of secretion, eccrine sweating is a vital regulator of body temperature in response to thermal stress in humans; therefore, an inability to sweat (anhidrosis) results in heat intolerance that may cause impaired consciousness and death. Here, we have reported 5 members of a consanguineous family with generalized, isolated anhidrosis, but morphologically normal eccrine sweat glands. Whole-genome analysis identified the presence of a homozygous missense mutation in ITPR2, which encodes the type 2 inositol 1,4,5-trisphosphate receptor (InsP₃R2), that was present in all affected family members. We determined that the mutation is localized within the pore forming region of InsP₃R2 and abrogates Ca²⁺ release from the endoplasmic reticulum, which suggests that intracellular Ca²⁺ release by InsP₃R2 in clear cells of the sweat glands is important for eccrine sweat production. Itpr2^–/– mice exhibited a marked reduction in sweat secretion, and evaluation of sweat glands from Itpr2^–/– animals revealed a decrease in Ca²⁺ response compared with controls. Together, our data indicate that loss of InsP₃R2-mediated Ca²⁺ release causes isolated anhidrosis in humans and suggest that specific InsP₃R inhibitors have the potential to reduce sweat production in hyperhidrosis.