An internal promoter underlies the difference in disease severity between N- and C-terminal truncation mutations of Titin in zebrafish.

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.

COPA mutations impair ER-Golgi transport and cause hereditary autoimmune-mediated lung disease and arthritis.

Unbiased genetic studies have uncovered surprising molecular mechanisms in human cellular immunity and autoimmunity. We performed whole-exome sequencing and targeted sequencing in five families with an apparent mendelian syndrome of autoimmunity characterized by high-titer autoantibodies, inflammatory arthritis and interstitial lung disease. We identified four unique deleterious variants in the COPA gene (encoding coatomer subunit α) affecting the same functional domain. Hypothesizing that mutant COPA leads to defective intracellular transport via coat protein complex I (COPI), we show that COPA variants impair binding to proteins targeted for retrograde Golgi-to-ER transport. Additionally, expression of mutant COPA results in ER stress and the upregulation of cytokines priming for a T helper type 17 (TH17) response. Patient-derived CD4(+) T cells also demonstrate significant skewing toward a TH17 phenotype that is implicated in autoimmunity. Our findings uncover an unexpected molecular link between a vesicular transport protein and a syndrome of autoimmunity manifested by lung and joint disease.

Estimating genotype error rates from high-coverage next-generation sequence data.

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.

Effects of the absence of apolipoprotein e on lipoproteins, neurocognitive function, and retinal function.

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. RESULTS: The 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 ß-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.

A polymorphism in TLR2 is associated with arterial thrombosis in a multiethnic population of patients with systemic lupus erythematosus.

OBJECTIVE: Thrombosis is a serious complication of systemic lupus erythematosus (SLE). Studies that have investigated the genetics of thrombosis in SLE are limited. We undertook this study to assess the association of previously implicated candidate genes, particularly Toll-like receptor (TLR) genes, with pathogenesis of thrombosis. METHODS: We genotyped 3,587 SLE patients from 3 multiethnic populations for 77 single-nucleotide polymorphisms (SNPs) in 10 genes, primarily in TLRs 2, 4, 7, and 9, and we also genotyped 64 ancestry-informative markers (AIMs). We first analyzed association with arterial and venous thrombosis in the combined population via logistic regression, adjusting for top principal components of the AIMs and other covariates. We also subjected an associated SNP, rs893629, to meta-analysis (after stratification by ethnicity and study population) to confirm the association and to test for study population or ethnicity effects. RESULTS: In the combined analysis, the SNP rs893629 in the KIAA0922/TLR2 region was significantly associated with arterial thrombosis (logistic P = 6.4 × 10(-5) , false discovery rate P = 0.0044). Two additional SNPs in TLR2 were also suggestive: rs1816702 (logistic P = 0.002) and rs4235232 (logistic P = 0.009). In the meta-analysis by study population, the odds ratio (OR) for arterial thrombosis with rs893629 was 2.44 (95% confidence interval 1.58-3.76), without evidence for heterogeneity (P = 0.78). By ethnicity, the effect was most significant among African Americans (OR 2.42, P = 3.5 × 10(-4) ) and European Americans (OR 3.47, P = 0.024). CONCLUSION: TLR2 gene variation is associated with thrombosis in SLE, particularly among African Americans and European Americans. There was no evidence of association among Hispanics, and results in Asian Americans were limited due to insufficient sample size. These results may help elucidate the pathogenesis of this important clinical manifestation.

Newborn screening for SCID identifies patients with ataxia telangiectasia.

PURPOSE: Severe 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. METHODS: Whole 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. RESULTS: Exome 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). CONCLUSIONS: T 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.