Short-read sequencing has enabled the de novo assembly of several individual human genomes, but with inherent limitations in characterizing repeat elements. Here we sequence a Chinese individual HX1 by single-molecule real-time (SMRT) long-read sequencing, construct a physical map by NanoChannel arrays and generate a de novo assembly of 2.93 Gb (contig N50: 8.3 Mb, scaffold N50: 22.0 Mb, including 39.3 Mb N-bases), together with 206 Mb of alternative haplotypes. The assembly fully or partially fills 274 (28.4%) N-gaps in the reference genome GRCh38. Comparison to GRCh38 reveals 12.8 Mb of HX1-specific sequences, including 4.1 Mb that are not present in previously reported Asian genomes. Furthermore, long-read sequencing of the transcriptome reveals novel spliced genes that are not annotated in GENCODE and are missed by short-read RNA-Seq. Our results imply that improved characterization of genome functional variation may require the use of a range of genomic technologies on diverse human populations.

The complement system consists of effector proteins, regulators, and receptors that participate in host defense against pathogens. Activation of the complement system, via the classical pathway (CP), has long been recognized in immune complex-mediated tissue injury, most notably systemic lupus erythematosus (SLE). Paradoxically, a complete deficiency of an early component of the CP, as evidenced by homozygous genetic deficiencies reported in human, are strongly associated with the risk of developing SLE or a lupus-like disease. Similarly, isotype deficiency attributable to a gene copy-number (GCN) variation and/or the presence of autoantibodies directed against a CP component or a regulatory protein that result in an acquired deficiency are relatively common in SLE patients. Applying accurate assay methodologies with rigorous data validations, low GCNs of total C4, and heterozygous and homozygous deficiencies of C4A have been shown as medium to large effect size risk factors, while high copy numbers of total C4 or C4A as prevalent protective factors, of European and East-Asian SLE. Here, we summarize the current knowledge related to genetic deficiency and insufficiency, and acquired protein deficiencies for C1q, C1r, C1s, C4A/C4B, and C2 in disease pathogenesis and prognosis of SLE, and, briefly, for other systemic autoimmune diseases. As the complement system is increasingly found to be associated with autoimmune diseases and immune-mediated diseases, it has become an attractive therapeutic target. We highlight the recent developments and offer a balanced perspective concerning future investigations and therapeutic applications with a focus on early components of the CP in human systemic autoimmune diseases.

Glutathione reductase (Gsr)3 catalyzes the reduction of glutathione disulfide to glutathione, which plays an important role in the bactericidal function of phagocytes. Since Gsr has been implicated in the oxidative burst in human neutrophils and is abundantly expressed in the lymphoid system, we hypothesized that Gsr-deficient mice would exhibit marked defects during the immune response against bacterial challenge. We report here that Gsr-null mice exhibited enhanced susceptibility to Escherichia coli challenge, indicated by dramatically increased bacterial burden, cytokine storm, striking histological abnormalities, and substantially elevated mortality. Additionally, Gsr-null mice exhibited elevated sensitivity to Staphylococcus aureus. Examination of the bactericidal functions of the neutrophils from Gsr-deficient mice in vitro revealed impaired phagocytosis and defective bacterial killing activities. Although Gsr catalyzes the regeneration of glutathione, a major cellular antioxidant, Gsr-deficient neutrophils paradoxically produced far less reactive oxygen species upon activation both ex vivo and in vivo. Unlike wildtype neutrophils that exhibited a sustained oxidative burst upon stimulation with phorbol ester and fMLP, Gsr-deficient neutrophils displayed a very transient oxidative burst that abruptly ceased shortly after stimulation. Likewise, Gsr-deficient neutrophils also exhibited an attenuated oxidative burst upon encountering E. coli. Biochemical analysis revealed that the hexose monophosphate shunt was compromised in Gsr-deficient neutrophils. Moreover, Gsr-deficient neutrophils displayed a marked impairment in the formation of neutrophil extracellular traps, a bactericidal mechanism which operates after neutrophil death. Thus, Gsr-mediated redox regulation is crucial for bacterial clearance during host defense against massive bacterial challenge.

Objectives Human complement C4 is sophisticatedly complex with multiple layers of diversity. This study aims to elucidate the CNVs of C4A and C4B in disease risk of SLE, and compare the basis of race-specific C4A-deficiency in East-Asians (EA) and Europeans. Patients and Methods Our EA study-population included 999 SLE patients and 1,347 healthy subjects. Variations in gene copy-numbers (GCNs) for total C4, C4A, C4B, long and short genes were determined and validated rigorously by independent genotyping technologies. Genomic regions with C4B96 were investigated to determine the basis of the most basic C4B protein that is concurrent with C4A-deficiency. Results In EA, strong protective effects of high GCNs for total C4 and C4A against SLE were notable; low and medium GCNs for total C4 and C4A, and the absence of short genes were risk factors of SLE. Homozygous C4A-deficiency was infrequent but had an odds-ratio (OR) of 12.4 (p=0.0015). Patients who experienced very-low serum complement were associated with low GCNs of total C4 (OR=3.27, p=7.0×10−7) and C4B (OR=2.55, p=2.5×10−5). Patients with low complement had high frequencies of anti-dsDNA (OR=4.96, p=9.7×10−17), hemolytic anemia (OR=3.89, p=3.6×10−10) and renal disease (OR=2.18, p=8.5×10−6). The monomodular-short haplotype with C4A-deficiency and in linkage-disequilibrium with HLA-DRB1*0301 prevalent in European was scarce in EA. Instead, most EA-subjects with C4A-deficiency shared a recombinant haplotype with bimodular-LS encoding C4B1 and C4B96, which was linked to HLA-DRB1*1501. DNA sequencing revealed the E920K polymorphism for C4B96. Conclusion C4 CNVs and C4A-deficiency are important in the risk and manifestations of East-Asian and European SLE.

Objective Complement-mediated vasculopathy of muscle and skin are clinical features of juvenile dermatomyositis (JDM). We assess gene copy-number variations (CNVs) for complement C4 and its isotypes, C4A and C4B, in genetic risks and pathogenesis of JDM. Methods The study population included 105 JDM patients and 500 healthy European Americans. Gene copy-numbers (GCNs) for total C4, C4A, C4B and HLA-DRB1 genotypes were determined by Southern blots and PCRs. Processed activation product C4d bound to erythrocytes (E-C4d) was measured by flow cytometry. Global gene-expression microarrays were performed in 19 JDM and 7 controls using PAXgene-blood RNA. Differential expression levels for selected genes were validated by qPCR. Results Significantly lower GCNs and differences in distribution of GCN groups for total C4 and C4A were observed between JDM and controls. Lower GCN of C4A in JDM remained among HLA DR3-positive subjects (p=0.015). Homozygous or heterozygous C4A-deficiency was present in 40.0% of JDM compared to 18.2% of controls [odds ratio (OR)=3.00 (1.87–4.79), p=8.2x10−6]. JDM had higher levels of E-C4d than controls (p=0.004). In JDM, C4A-deficient subjects had higher levels of E-C4d (p=0.0003) and higher frequency of elevated levels of multiple serum muscle enzymes at diagnosis (p=0.004). Microarray profiling of blood RNA revealed upregulation of type I Interferon-stimulated genes and lower abundance of transcripts for T-cell and chemokine function genes in JDM, but this was less prominent among C4A-deficient or DR3-positive patients. Conclusions Complement C4A-deficiency appears to be an important factor for the genetic risk and pathogenesis of JDM, particularly in patients with a DR3-positive background.

BackgroundThe course of JDM has improved substantially over the last 70 years with early and aggressive treatments. Yet it remains difficult to detect disease flares as symptoms may be mild; signs of rash and muscle weakness vary widely and are often equivocal; laboratory tests of muscle enzyme levels are often normal; electromyography and muscle biopsy are invasive. Alternative tools are needed to help decide if more aggressive treatment is needed. Our objective is to determine the effectiveness of muscle Magnetic Resonance Imaging (MRI) in detecting JDM flares, and how an MRI affects physician’s decision-making regarding treatment.MethodsThis study was approved by the Institutional Review Board of Nationwide Children’s Hospital. JDM patients were consulted between 1/2005 and 6/2015. MRIs were performed on both lower extremities without contrast sequentially: axial T1, axial T2 fat saturation, axial and coronal inversion recovery, and axial diffusion weighted. The physician decision that a JDM patient was in a flare was considered the gold standard. MRI results were compared with physician’s decisions on whether a relapse had occurred, and if there was a concordance between the assessment methods.ResultsForty-five JDM patients were studied. Eighty percent had weakness at diagnosis, 100% typical rash, and 73% typical nail-fold capillary changes. At diagnosis, muscle enzymes were compatible with JDM generally (CK 52%, LDH 62%, aldolase 72%, AST 54% abnormal). EMG was abnormal in 3/8, muscle biopsy typical of JDM in 10/11, and MRI abnormal demonstrating myositis in 31/40. Thirteen patients had a repeat MRI for possible flares with differing indications. Three repeat MRI’s were abnormal, demonstrating myositis. There was moderate agreement about flares between MRI findings and physician’s treatment decisions (kappa = 0.59). In each abnormal MRI case the physician decided to increase treatment (100% probability for flares). MRI was negative for myositis in 10 patients, by which 7/10 the physicians chose to continue or to taper the medications (70% probability for non-flares).ConclusionA muscle MRI would facilitate objective assessments of JDM flares. When an MRI shows myositis, physicians tend to treat 100% of the time. When an MRI shows no myositis, physicians continued the same medications or tapered medications 70% of the time. Further studies would help confirm the utility and cost-effectiveness of MRI to determine JDM flares.Electronic supplementary materialThe online version of this article (doi:10.1186/s12969-017-0154-4) contains supplementary material, which is available to authorized users.

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Human complement C4 is complex with multiple layers of diversity. This study aims to elucidate the copy-number variations (CNVs) of C4A and C4B in disease risk of SLE, and compare the basis of race-specific C4A-deficiency in East-Asians (EA) and Europeans. Our study-populations included (a) 999 EA-SLE patients and 1,347 healthy subjects; and (b) 232 European SLE patients and 500 healthy subjects. Variations in gene copy-numbers (GCNs) for total C4, C4A, C4B, long and short genes were determined and validated by independent genotyping technologies. Genomic regions with C4B96 were investigated to determine the basis of the most basic C4B protein that is concurrent with C4A-deficiency. In EA, strong protective effects for high GCNs of total C4 and C4A against SLE were notable; low and medium GCNs of total C4 and C4A, and the absence of short genes were risk factors for SLE. Homozygous C4A-deficiency was infrequent but had an odds-ratio (OR) of 12.4 (p=0.0015) in SLE. Low serum complement levels were strongly associated with low GCNs of total C4 (OR=3.27, p=7.0×10−7) and C4B (OR=2.55, p=2.5×10−5). Patients with low complement had high frequencies of anti-dsDNA (OR=4.96, p=9.7×10−17), hemolytic anemia (OR=3.89, p=3.6×10−10) and renal disease (OR=2.18, p=8.5×10−6). The monomodular-short haplotype with C4A-deficiency and in linkage-disequilibrium with HLA-DRB1*0301 prevalent in Europeans was scarce in EA. Instead, most EA-subjects with C4A-deficiency shared a recombinant haplotype with bimodular-LS encoding C4B1 and C4B96, which was linked to HLA-DRB1*1501. DNA-sequencing revealed the E920K polymorphism for C4B96. In conclusion, C4 CNVs and C4A-deficiency are important in the risk and manifestations of East-Asian and European SLE.