Advances in genomics have allowed unbiased genetic studies of human disease with unexpected insights into the molecular mechanisms of cellular immunity and autoimmunity1. We performed whole exome sequencing (WES) and targeted sequencing in patients with an apparent Mendelian syndrome of autoimmune disease characterized by high-titer autoantibodies, inflammatory arthritis and interstitial lung disease (ILD). In five families, we identified four unique deleterious variants in the Coatomer subunit alpha (COPA) gene all located within the same functional domain. We hypothesized that mutant COPA leads to a defect in intracellular transport mediated by coat protein complex I (COPI)2–4. We show that COPA variants impair binding of proteins targeted for retrograde Golgi to ER transport and demonstrate that expression of mutant COPA leads to ER stress and the upregulation of Th17 priming cytokines. Consistent with this pattern of cytokine expression, patients demonstrated a significant skewing of CD4+ T cells toward a T helper 17 (Th17) phenotype, an effector T cell population implicated in autoimmunity5,6. Our findings uncover an unexpected molecular link between a vesicular transport protein and a syndrome of autoimmunity manifested by lung and joint disease. These findings provide a unique opportunity to understand how alterations in cellular homeostasis caused by a defect in the intracellular trafficking pathway leads to the generation of human autoimmune disease.

Hyperglycemia-linked oxidative stress and/or consequent endoplasmic reticulum stress are the causative factors of pathogenesis of diabetic retinopathy. Dietary bioactive components which mitigate oxidative stress may serve as potential chemopreventative agents to prevent or slow down the disease progression. Wolfberry is a traditional Asian fruit consumed for years to prevent aging eye diseases in Asian countries. Here we report that dietary wolfberry ameliorated mouse retinal abnormality at the early stage of type 2 diabetes in db/db mice. Male mice at 6 weeks of age were fed the control diet with or without 1 % (kCal) wolfberry for 8 weeks. Dietary wolfberry restored the thickness of the whole retina, in particular the inner nuclear layer and photoreceptor layer, and the integrity of retinal pigment epithelia (RPE), and the ganglion cell number in db/db mice. Western blotting of whole retinal cell lysates revealed that addition of wolfberry lowered expression of endoplasmic reticulum (ER) stress biomarkers BiP, PERK, ATF6, and caspase-12; and restored AMPK, thioredoxin, Mn SOD, and FOXO3α activities. To determine if our observations were due to the high contents of zeaxanthin and lutein in wolfberry additional studies using these carotenoids were conducted. Using the human adult diploid RPE cell line ARPE-19 we demonstrated that both zeaxanthin and lutein could mimic wolfberry preventive effect on activation of AMPK, thioredoxin, Mn SOD, FOXO3α activities, normalize cellular reactive oxygen species, and attenuate ER stress in ARPE-19 cells exposed to a high glucose challenge. The zeaxanthin preventive effect was abolished by siRNA knockdown of AMPKα. These results suggested that AMPK activation appeared to play a key role in upregulated expression of thioredoxin and Mn SOD, and mitigation of cellular oxidative stress and/or ER stress by wolfberry and zeaxanthin and/or lutein. Taken together, dietary wolfberry on retinal protection in diabetic mice is, at least partially, due to zeaxanthin and/or lutein.

Exome and whole-genome sequencing studies are becoming increasingly common, but little is known about the accuracy of the genotype calls made by the commonly used platforms. Here we use replicate high-coverage sequencing of blood and saliva DNA samples from four European-American individuals to estimate lower bounds on the error rates of Complete Genomics and Illumina HiSeq whole-genome and whole-exome sequencing. Error rates for nonreference genotype calls range from 0.1% to 0.6%, depending on the platform and the depth of coverage. Additionally, we found (1) no difference in the error profiles or rates between blood and saliva samples; (2) Complete Genomics sequences had substantially higher error rates than Illumina sequences had; (3) error rates were higher (up to 6%) for rare or unique variants; (4) error rates generally declined with genotype quality (GQ) score, but in a nonlinear fashion for the Illumina data, likely due to loss of specificity of GQ scores greater than 60; and (5) error rates increased with increasing depth of coverage for the Illumina data. These findings, especially (3)- (5), suggest that caution should be taken in interpreting the results of next-generation sequencing-based association studies, and even more so in clinical application of this technology in the absence of validation by other more robust sequencing or genotyping methods.

Truncating mutations in the giant sarcomeric protein Titin result in dilated cardiomyopathy and skeletal myopathy. The most severely affected dilated cardiomyopathy patients harbor Titin truncations in the C-terminal two-thirds of the protein, suggesting that mutation position might influence disease mechanism. Using CRISPR/Cas9 technology, we generated six zebrafish lines with Titin truncations in the N-terminal and C-terminal regions. Although all exons were constitutive, C-terminal mutations caused severe myopathy whereas N-terminal mutations demonstrated mild phenotypes. Surprisingly, neither mutation type acted as a dominant negative. Instead, we found a conserved internal promoter at the precise position where divergence in disease severity occurs, with the resulting protein product partially rescuing N-terminal truncations. In addition to its clinical implications, our work may shed light on a long-standing mystery regarding the architecture of the sarcomere.DOI: http://dx.doi.org/10.7554/eLife.09406.001

Summary We recently showed in a single centre trial that low‐dose secondary prophylaxis in severe/moderate haemophilia patients with arthropathy is feasible and beneficial. However, this regimen has not been validated in a multicentre setting and what obstacles are there to prophylaxis remain unclear. (i) Benefit study: to confirm the benefits of similar prophylaxis protocol in severe/moderate haemophilia A (HA) in a multicentre setting in China. (ii) Follow‐up obstacle study: to investigate obstacles in compliance to prophylaxis treatment. (i) Benefit study: severe/moderate HA children with arthropathy from 15 centres were enrolled to undergo an 8‐week on‐demand treatment, followed by 6 to 12‐week low‐dose secondary prophylaxis. Outcomes compared in the two periods include joint and severe bleeding, daily activities and factor consumption. (ii) Obstacle study: questionnaires to investigators to collect data on patient and centre factors contributing to inability to comply with prophylaxis. We enrolled 191 patients from 15 centres. Sixty‐six (34.6%) from three centres completed the prophylaxis protocol, and they had significantly decreased bleeding (78.8% haemarthrosis and 68.9% severe bleedings) and improved daily activities with no increase in factor consumption over that in the on‐demand therapy period. The remaining 125 patients from 12 centres were not compliant to the prophylaxis protocol; questionnaire data indicated that the major obstacles were inability of patients/parents to accept (41.7%) or to adhere (33.3%) to the prophylaxis protocol, mostly because of failure to understand the benefits and to accept the frequent injections. Non‐availability of a centre comprehensive care team was another important determinant. Short‐term low‐dose secondary prophylactic therapy is beneficial without increasing factors consumption for severe/moderate HA with arthropathy in a multi‐centre setting in China. Obstacles to overcome must include improvement in comprehensive care and in education to patient/parents and healthcare personnel.

IMPORTANCE The identification of a patient with a rare form of severe dysbetalipoproteinemia allowed the study of the consequences of total absence of apolipoprotein E (apoE).OBJECTIVES To discover the molecular basis of this rare disorder and to determine the effects of complete absence of apoE on neurocognitive and visual function and on lipoprotein metabolism.DESIGN, SETTING, AND PARTICIPANTS Whole-exome sequencing was performed on the patient's DNA. He underwent detailed neurological and visual function testing and lipoprotein analysis. Lipoprotein analysis was also performed in the Cardiovascular Research Institute, University of California, San Francisco, on blood samples from the proband's mother, wife, 2 daughters, and normolipidemic control participants.MAIN OUTCOME MEASURES Whole-exome sequencing, lipoprotein analysis, and neurocognitive function. RESULTSThe patient was homozygous for an ablative APOE frameshift mutation (c.291del, p.E97fs). No other mutations likely to contribute to the phenotype were discovered, with the possible exception of two, in ABCC2 (p.I670T) and LIPC (p.G137R). Despite complete absence of apoE, he had normal vision, exhibited normal cognitive, neurological, and retinal function, had normal findings on brain magnetic resonance imaging, and had normal cerebrospinal fluid levels of β-amyloid and tau proteins. He had no significant symptoms of cardiovascular disease except a suggestion of myocardial ischemia on treadmill testing and mild atherosclerosis noted on carotid ultrasonography. He had exceptionally high cholesterol content (760 mg/dL; to convert to millimoles per liter, multiply by 0.0259) and a high cholesterol to triglycerides ratio (1.52) in very low-density lipoproteins with elevated levels of small-diameter high-density lipoproteins, including high levels of prebeta-1 high-density lipoprotein. Intermediate-density lipoproteins, low-density lipoproteins, and very low-density lipoproteins contained elevated apoA-I and apoA-IV levels. The patient's apoC-III and apoC-IV levels were decreased in very low-density lipoproteins. Electron microscopy revealed large lamellar particles having electron-opaque cores attached to electron-lucent zones in intermediate-density and low-density lipoproteins. Low-density lipoprotein particle diameters were distributed bimodally.CONCLUSIONS AND RELEVANCE Despite a profound effect on lipoprotein metabolism, detailed neurocognitive and retinal studies failed to demonstrate any defects. This suggests that functions of apoE in the brain and eye are not essential or that redundant mechanisms exist whereby its role can be fulfilled. Targeted knockdown of apoE in the central nervous system might be a therapeutic modality in neurodegenerative disorders.

PurposeSevere combined immunodeficiency (SCID) is characterized by failure of T lymphocyte development and absent or very low T cell receptor excision circles (TRECs), DNA byproducts of T cell maturation. Newborn screening for TRECs to identify SCID is now performed in several states using PCR of DNA from universally collected dried blood spots (DBS). In addition to infants with typical SCID, TREC screening identifies infants with T lymphocytopenia who appear healthy and in whom a SCID diagnosis cannot be confirmed. Deep sequencing was employed to find causes of T lymphocytopenia in such infants.MethodsWhole exome sequencing and analysis were performed in infants and their parents. Upon finding deleterious mutations in the ataxia telangiectasia mutated (ATM) gene, we confirmed the diagnosis of ataxia telangiectasia (AT) in two infants and then tested archival newborn DBS of additional AT patients for TREC copy number.ResultsExome sequencing and analysis led to 2 unsuspected gene diagnoses of AT. Of 13 older AT patients for whom newborn DBS had been stored, 7 samples tested positive for SCID under the criteria of California’s newborn screening program. AT children with low neonatal TRECs had low CD4 T cell counts subsequently detected (R = 0.64).ConclusionsT lymphocytopenia in newborns can be a feature of AT, as revealed by TREC screening and exome sequencing. Although there is no current cure for the progressive neurological impairment of AT, early detection permits avoidance of infectious complications, while providing information for families regarding reproductive recurrence risks and increased cancer risks in patients and carriers.

Reading disability (RD) or dyslexia is a common neurogenetic disorder. Two genes, KIAA0319 and DCDC2, have been identified by association studies of the DYX2 locus on 6p21.3. We previously identified a 2445 bp deletion, and a compound STR within the deleted region (BV677278), in intron 2 of DCDC2. The deletion and several alleles of the STR are strongly associated with RD (P = 0.00002). In this study we investigated whether BV677278 is a regulatory region for DCDC2 by electrophoretic mobility shift and luciferase reporter assays. We show that oligonucleotide probes from the STR bind nuclear protein from human brain, and that alleles of the STR have a range of DCDC2-specific enhancer activities. Five alleles displayed strong enhancer activity and increased gene expression, while allele 1 showed no enhancer activity. These studies suggest that the association of BV677278 with RD reflects a role as a modifier of DCDC2 expression.