Rare X chromosome abnormalities in systemic lupus erythematosus and Sjögren's syndrome

Objective: Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE) are related by clinical and serologic manifestations as well as genetic risks. Both diseases are more commonly found in women than in men, at a ratio of ~10 to 1. Common X chromosome aneuploidies, 47,XXY and 47,XXX, are enriched among men and women, respectively, in either disease, suggesting a dose effect on the X chromosome. Methods: We examined cohorts of SS and SLE patients by constructing intensity plots of X chromosome single-nucleotide polymorphism alleles, along with determining the karyotype of selected patients. Results: Among ~2,500 women with SLE, we found 3 patients with a triple mosaic, consisting of 45,X/46,XX/47,XXX. Among ~2,100 women with SS, 1 patient had 45,X/46,XX/47,XXX, with a triplication of the distal p arm of the X chromosome in the 47,XXX cells. Neither the triple mosaic nor the partial triplication was found among the controls. In another SS cohort, we found a mother/daughter pair with partial triplication of this same region of the X chromosome. The triple mosaic occurs in ~1 in 25,000–50,000 live female births, while partial triplications are even rarer. Conclusion: Very rare X chromosome abnormalities are present among patients with either SS or SLE and may inform the location of a gene(s) that mediates an X dose effect, as well as critical cell types in which such an effect is operative. © 2017, American College of Rheumatolog

Chemoselective Coupling Preserves the Substrate Integrity of Surface-Immobilized Oligonucleotides for Emulsion PCR-Based Gene Library Construction

Combinatorial bead libraries figure prominently in next-generation sequencing and are also important tools for in vitro evolution. The most common methodology for generating such bead libraries, emulsion PCR (emPCR), enzymatically extends bead-immobilized oligonucleotide PCR primers in emulsion droplets containing a single progenitor library member. Primers are almost always immobilized on beads via noncovalent biotin–streptavidin binding. Here, we describe covalent bead functionalization with primers (∼10⁶ primers/2.8-μm-diameter bead) via either azide–alkyne click chemistry or Michael addition. The primers are viable polymerase substrates (4–7% bead-immobilized enzymatic extension product yield from one thermal cycle). Carbodiimide-activated carboxylic acid beads only react with oligonucleotides under conditions that promote nonspecific interactions (low salt, low pH, no detergent), comparably immobilizing primers on beads, but yielding no detectable enzymatic extension product. Click-functionalized beads perform satisfactorily in emPCR of a site-saturation mutagenesis library, generating monoclonal templated beads (10⁴–10⁵ copies/bead, 1.4-kb amplicons). This simpler, chemical approach to primer immobilization may spur more economical library preparation for high-throughput sequencing and enable more complex surface elaboration for in vitro evolution

DNA-Encoded Solid-Phase Synthesis: Encoding Language Design and Complex Oligomer Library Synthesis

The promise of exploiting combinatorial synthesis for small molecule discovery remains unfulfilled due primarily to the “structure elucidation problem”: the back-end mass spectrometric analysis that significantly restricts one-bead-one-compound (OBOC) library complexity. The very molecular features that confer binding potency and specificity, such as stereochemistry, regiochemistry, and scaffold rigidity, are conspicuously absent from most libraries because isomerism introduces mass redundancy and diverse scaffolds yield uninterpretable MS fragmentation. Here we present DNA-encoded solid-phase synthesis (DESPS), comprising parallel compound synthesis in organic solvent and aqueous enzymatic ligation of unprotected encoding dsDNA oligonucleotides. Computational encoding language design yielded 148 thermodynamically optimized sequences with Hamming string distance ≥ 3 and total read length <100 bases for facile sequencing. Ligation is efficient (70% yield), specific, and directional over 6 encoding positions. A series of isomers served as a testbed for DESPS’s utility in split-and-pool diversification. Single-bead quantitative PCR detected 9 × 10⁴ molecules/bead and sequencing allowed for elucidation of each compound’s synthetic history. We applied DESPS to the combinatorial synthesis of a 75 645-member OBOC library containing scaffold, stereochemical and regiochemical diversity using mixed-scale resin (160-μm quality control beads and 10-μm screening beads). Tandem DNA sequencing/MALDI-TOF MS analysis of 19 quality control beads showed excellent agreement (<1 ppt) between DNA sequence-predicted mass and the observed mass. DESPS synergistically unites the advantages of solid-phase synthesis and DNA encoding, enabling single-bead structural elucidation of complex compounds and synthesis using reactions normally considered incompatible with unprotected DNA. The widespread availability of inexpensive oligonucleotide synthesis, enzymes, DNA sequencing, and PCR make implementation of DESPS straightforward, and may prompt the chemistry community to revisit the synthesis of more complex and diverse libraries

Integrated, Continuous Emulsion Creamer

Automated and reproducible sample handling is a key requirement for high-throughput compound screening and currently demands heavy reliance on expensive robotics in screening centers. Integrated droplet microfluidic screening processors are poised to replace robotic automation by miniaturizing biochemical reactions to the droplet scale. These processors must generate, incubate, and sort droplets for continuous droplet screening, passively handling millions of droplets with complete uniformity, especially during the key step of sample incubation. Here, we disclose an integrated microfluidic emulsion creamer that packs (“creams”) assay droplets by draining away excess oil through microfabricated drain channels. The drained oil coflows with creamed emulsion and then reintroduces the oil to disperse the droplets at the circuit terminus for analysis. Creamed emulsion assay incubation time dispersion was 1.7%, 3-fold less than other reported incubators. The integrated, continuous emulsion creamer (ICEcreamer) was used to miniaturize and optimize measurements of various enzymatic activities (phosphodiesterase, kinase, bacterial translation) under multiple- and single-turnover conditions. Combining the ICEcreamer with current integrated microfluidic DNA-encoded library bead processors eliminates potentially cumbersome instrumentation engineering challenges and is compatible with assays of diverse target class activities commonly investigated in drug discovery

High-throughput identification of DNA-encoded IgG ligands that distinguish active and latent Mycobacterium tuberculosis infections

The circulating antibody repertoire encodes a patient's health status and pathogen exposure history, but identifying antibodies with diagnostic potential usually requires knowledge of the antigen(s). We previously circumvented this problem by screening libraries of bead-displayed small molecules against case and control serum samples to discover "epitope surrogates" (Ligands of IgGs enriched in the case sample). Here, we describe an improved version of this technology that employs DNA-encoded libraries and high-throughput FACS-based screening to discover epitope surrogates that differentiate noninfectious/latent (LTB) patients from infectious/active TB (ATB) patients, which is imperative for proper treatment selection and antibiotic stewardship. Normal control/LTB (10 patients each, NCL) and ATB (10 patients) serum pools were screened against a library (5 × 10 beads, 448 000 unique compounds) using fluorescent antihuman IgG to label hit compound beads for FACS. Deep sequencing decoded all hit structures and each hit's occurrence frequencies. ATB hits were pruned of NCL hits and prioritized for resynthesis based on occurrence and homology. Several structurally homologous families were identified and 16/21 resynthesized representative hits validated as selective ligands of ATB serum IgGs (p < 0.005). The native secreted TB protein Ag85B (though not the E. coli recombinant form) competed with one of the validated ligands for binding to antibodies, suggesting that it mimics a native Ag85B epitope. The use of DNA-encoded libraries and FACS-based screening in epitope surrogate discovery reveals thousands of potential hit structures. Distilling this list down to several consensus chemical structures yielded a diagnostic panel for ATB composed of thermally stable and economically produced small molecule ligands in place of protein antigens

Rare X Chromosome Abnormalities in Systemic Lupus Erythematosus and Sjögren's Syndrome

"Objective: Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE) are related by clinical and serologic manifestations as well as genetic risks. Both diseases are more commonly found in women than in men, at a ratio of ~10 to 1. Common X chromosome aneuploidies, 47,XXY and 47,XXX, are enriched among men and women, respectively, in either disease, suggesting a dose effect on the X chromosome. Methods: We examined cohorts of SS and SLE patients by constructing intensity plots of X chromosome single-nucleotide polymorphism alleles, along with determining the karyotype of selected patients. Results: Among ~2,500 women with SLE, we found 3 patients with a triple mosaic, consisting of 45,X/46,XX/47,XXX. Among ~2,100 women with SS, 1 patient had 45,X/46,XX/47,XXX, with a triplication of the distal p arm of the X chromosome in the 47,XXX cells. Neither the triple mosaic nor the partial triplication was found among the controls. In another SS cohort, we found a mother/daughter pair with partial triplication of this same region of the X chromosome. The triple mosaic occurs in ~1 in 25,000–50,000 live female births, while partial triplications are even rarer. Conclusion: Very rare X chromosome abnormalities are present among patients with either SS or SLE and may inform the location of a gene(s) that mediates an X dose effect, as well as critical cell types in which such an effect is operative. © 2017, American College of Rheumatology

Rare X Chromosome Abnormalities in Systemic Lupus Erythematosus and Sjögren's Syndrome

Objective: Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE) are related by clinical and serologic manifestations as well as genetic risks. Both diseases are more commonly found in women than in men, at a ratio of ~10 to 1. Common X chromosome aneuploidies, 47,XXY and 47,XXX, are enriched among men and women, respectively, in either disease, suggesting a dose effect on the X chromosome. Methods: We examined cohorts of SS and SLE patients by constructing intensity plots of X chromosome single-nucleotide polymorphism alleles, along with determining the karyotype of selected patients. Results: Among ~2,500 women with SLE, we found 3 patients with a triple mosaic, consisting of 45,X/46,XX/47,XXX. Among ~2,100 women with SS, 1 patient had 45,X/46,XX/47,XXX, with a triplication of the distal p arm of the X chromosome in the 47,XXX cells. Neither the triple mosaic nor the partial triplication was found among the controls. In another SS cohort, we found a mother/daughter pair with partial triplication of this same region of the X chromosome. The triple mosaic occurs in ~1 in 25,000–50,000 live female births, while partial triplications are even rarer. Conclusion: Very rare X chromosome abnormalities are present among patients with either SS or SLE and may inform the location of a gene(s) that mediates an X dose effect, as well as critical cell types in which such an effect is operative. © 2017, American College of Rheumatolog

Klinefelter's syndrome (47,XXY) is in excess among men with Sjögren's syndrome

Primary Sjögren's syndrome (pSS) has a strong female bias. We evaluated an X chromosome dose effect by analyzing 47,XXY (Klinefelter's syndrome, 1 in 500 live male births) among subjects with pSS. 47,XXY was determined by examination of fluorescence intensity of single nucleotide polymorphisms from the X and Y chromosomes. Among 136 pSS men there were 4 with 47,XXY. This was significantly different from healthy controls (1 of 1254 had 47,XXY, p = 0.0012 by Fisher's exact test) as well men with rheumatoid arthritis (0 of 363 with 47,XXY), but not different compared to men with systemic lupus erythematosus (SLE) (4 of 136 versus 8 of 306, Fisher's exact test p = NS). These results are consistent with the hypothesis that the number of X chromosomes is critical for the female bias of pSS, a property that may be shared with SLE but not RA. © 2016