Chlamydomonas FAP70 is a component of the previously uncharacterized ciliary central apparatus projection C2a.

Cilia are essential organelles required for cell signaling and motility. Nearly all motile cilia have a '9+2' axoneme composed of nine outer doublet microtubules plus two central microtubules; the central microtubules together with their projections are termed the central apparatus (CA). In Chlamydomonas reinhardtii, a model organism for studying cilia, 30 proteins are known CA components, and ∼36 more are predicted to be CA proteins. Among the candidate CA proteins is the highly conserved FAP70 (CFAP70 in humans), which also has been reported to be associated with the doublet microtubules. Here, we determined by super-resolution structured illumination microscopy that FAP70 is located exclusively in the CA, and show by cryo-electron microscopy that its N-terminus is located at the base of the C2a projection of the CA. We also found that fap70-1 mutant axonemes lack most of the C2a projection. Mass spectrometry revealed that fap70-1 axonemes lack not only FAP70 but two other conserved candidate CA proteins, FAP65 (CFAP65 in humans) and FAP147 (MYCBPAP in humans). Finally, FAP65 and FAP147 co-immunoprecipitated with HA-tagged FAP70. Taken together, these data identify FAP70, FAP65 and FAP147 as the first defining components of the C2a projection.

Tetrahydrobiopterin deficiency in the pathogenesis of Fabry disease.

Fabry disease is caused by deficient activity of α-galactosidase A and subsequent accumulation of glycosphingolipids (mainly globotriaosylceramide, Gb3), leading to multisystem organ dysfunction. Oxidative stress and nitric oxide synthase (NOS) uncoupling are thought to contribute to Fabry cardiovascular diseases. We hypothesized that decreased tetrahydrobiopterin (BH4) plays a role in the pathogenesis of Fabry disease. We found that BH4 was decreased in the heart and kidney but not in the liver and aorta of Fabry mice. BH4 was also decreased in the plasma of female Fabry patients, which was not corrected by enzyme replacement therapy (ERT). Gb3 levels were inversely correlated with BH4 levels in animal tissues and cultured patient cells. To investigate the role of BH4 deficiency in disease phenotypes, 12-month-old Fabry mice were treated with gene transfer-mediated ERT or substrate reduction therapy (SRT) for 6 months. In the Fabry mice receiving SRT but not ERT, BH4 deficiency was restored, concomitant with ameliorated cardiac and renal hypertrophy. Additionally, glutathione levels were decreased in Fabry mouse tissues in a sex-dependent manner. Renal BH4 levels were closely correlated with glutathione levels and inversely correlated with cardiac and kidney weight. In conclusion, this study showed that BH4 deficiency occurs in Fabry disease and may contribute to the pathogenesis of the disease through oxidative stress associated with a reduced antioxidant capacity of cells and NOS uncoupling. This study also suggested dissimilar efficacy of ERT and SRT in correcting pre-existing pathologies in Fabry disease.

Low frequency of Fabry disease in patients with common heart disease.

PURPOSE: To test the hypothesis that undiagnosed patients with Fabry disease exist among patients affected by common heart disease. METHODS: Globotriaosylceramide in random whole urine using tandem mass spectroscopy, α-galactosidase A activity in dried blood spots, and next-generation sequencing of pooled or individual genomic DNA samples supplemented by Sanger sequencing. RESULTS: We tested 2,256 consecutive patients: 852 women (median age 65 years (19-95)) and 1,404 men (median age 65 years (21-92)). The primary diagnoses were coronary artery disease (n = 994), arrhythmia (n = 607), cardiomyopathy (n = 138), and valvular disease (n = 568). Urinary globotriaosylceramide was elevated in 15% of patients and 15 males had low α-galactosidase A activity. GLA variants found included R118C (n = 2), D83N, and D313Y (n = 7); IVS6-22 C>T, IVS4-16 A>G, IVS2+990C>A, 5'UTR-10 C>T (n = 4), IVS1-581 C>T, IVS1-1238 G>A, 5'UTR-30 G>A, IVS2+590C>T, IVS0-12 G>A, IVS4+68A>G, IVS0-10 C>T, IVS2-81-77delCAGCC, IVS2-77delC. Although the pathogenicity of several of these missense mutations and complex intronic haplotypes has been controversial, none of the patients screened in this study were diagnosed definitively with Fabry disease. CONCLUSION: This population of patients with common heart disease did not contain a substantial number of patients with undiagnosed Fabry disease. GLA gene sequencing is superior to urinary globotriaosylceramide or α-galactosidase A activity in the screening for Fabry disease.

Whole exome sequencing in patients with white matter abnormalities.

Here we report whole exome sequencing (WES) on a cohort of 71 patients with persistently unresolved white matter abnormalities with a suspected diagnosis of leukodystrophy or genetic leukoencephalopathy. WES analyses were performed on trio, or greater, family groups. Diagnostic pathogenic variants were identified in 35% (25 of 71) of patients. Potentially pathogenic variants were identified in clinically relevant genes in a further 7% (5 of 71) of cases, giving a total yield of clinical diagnoses in 42% of individuals. These findings provide evidence that WES can substantially decrease the number of unresolved white matter cases. Ann Neurol 2016;79:1031-1037.

Molecular basis for globotriaosylceramide regulation and enzyme uptake in immortalized aortic endothelial cells from Fabry mice.

Fabry disease is caused by deficient activity of α-galactosidase A and subsequent intracellular accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3). Vascular endothelial cells may play important roles in disease pathogenesis, and are one of the main target cell types in therapeutic interventions. In this study, we generated immortalized aortic endothelial cell lines from a mouse model of Fabry disease. These cells retained endothelial cell-specific markers and functions. Gb3 expression level in one of these clones (referred to as FMEC2) was highly susceptible to culture media, and appeared to be regulated by glucosylceramide synthase. Results also showed that Gb3 could be upregulated by hydrocortisone. FMEC2 express the mannose 6-phosphate receptor and sortilin but not the mannose receptor. Uptake studies suggested that sortilin plays a role in the binding and internalization of mammalian cell-produced α-galactosidase A. Moss-aGal (a plant-made enzyme) was endocytosed by FMEC2 via a receptor other than the aforementioned receptors. In conclusion, this study suggests that glucosylceramide synthase and hydrocortisone may play important roles in modulating Gb3 levels in Fabry mouse aortic endothelial cells, and that endocytosis of recombinant α-galactosidase A involves a combination of multiple receptors depending on the properties of the enzyme.

A clinical approach to the diagnosis of patients with leukodystrophies and genetic leukoencephelopathies.

Leukodystrophies (LD) and genetic leukoencephalopathies (gLE) are disorders that result in white matter abnormalities in the central nervous system (CNS). Magnetic resonance (MR) imaging (MRI) has dramatically improved and systematized the diagnosis of LDs and gLEs, and in combination with specific clinical features, such as Addison's disease in Adrenoleukodystrophy or hypodontia in Pol-III related or 4H leukodystrophy, can often resolve a case with a minimum of testing. The diagnostic odyssey for the majority LD and gLE patients, however, remains extensive--many patients will wait nearly a decade for a definitive diagnosis and at least half will remain unresolved. The combination of MRI, careful clinical evaluation and next generation genetic sequencing holds promise for both expediting the diagnostic process and dramatically reducing the number of unresolved cases. Here we present a workflow detailing the Global Leukodystrophy Initiative (GLIA) consensus recommendations for an approach to clinical diagnosis, including salient clinical features suggesting a specific diagnosis, neuroimaging features and molecular genetic testing. We also discuss recommendations on the use of broad-spectrum next-generation sequencing in instances of ambiguous MRI or clinical findings. We conclude with a proposal for systematic trials of genome-wide agnostic testing as a first line diagnostic in LDs and gLEs given the increasing number of genes associated with these disorders.

BAYSIC: a Bayesian method for combining sets of genome variants with improved specificity and sensitivity.

BACKGROUND: Accurate genomic variant detection is an essential step in gleaning medically useful information from genome data. However, low concordance among variant-calling methods reduces confidence in the clinical validity of whole genome and exome sequence data, and confounds downstream analysis for applications in genome medicine.Here we describe BAYSIC (BAYeSian Integrated Caller), which combines SNP variant calls produced by different methods (e.g. GATK, FreeBayes, Atlas, SamTools, etc.) into a more accurate set of variant calls. BAYSIC differs from majority voting, consensus or other ad hoc intersection-based schemes for combining sets of genome variant calls. Unlike other classification methods, the underlying BAYSIC model does not require training using a "gold standard" of true positives. Rather, with each new dataset, BAYSIC performs an unsupervised, fully Bayesian latent class analysis to estimate false positive and false negative error rates for each input method. The user specifies a posterior probability threshold according to the user's tolerance for false positive and false negative errors; lowering the posterior probability threshold allows the user to trade specificity for sensitivity while raising the threshold increases specificity in exchange for sensitivity. RESULTS: We assessed the performance of BAYSIC in comparison to other variant detection methods using ten low coverage (~5X) samples from The 1000 Genomes Project, a tumor/normal exome pair (40X), and exome sequences (40X) from positive control samples previously identified to contain clinically relevant SNPs. We demonstrated BAYSIC's superior variant-calling accuracy, both for somatic mutation detection and germline variant detection. CONCLUSIONS: BAYSIC provides a method for combining sets of SNP variant calls produced by different variant calling programs. The integrated set of SNP variant calls produced by BAYSIC improves the sensitivity and specificity of the variant calls used as input. In addition to combining sets of germline variants, BAYSIC can also be used to combine sets of somatic mutations detected in the context of tumor/normal sequencing experiments.

Free sialic acid storage disease without sialuria.

We performed high-resolution in vitro proton nuclear magnetic resonance spectroscopy on cerebrospinal fluid and urine samples of 44 patients with leukodystrophies of unknown cause. Free sialic acid concentration was increased in cerebrospinal fluid of two siblings with mental retardation and mild hypomyelination. By contrast, urinary excretion of free sialic acid in urine was normal on repeated testing by two independent methods. Both patients were homozygous for the K136E mutation in SLC17A5, the gene responsible for the free sialic acid storage diseases. Our findings demonstrate that mutations in the SLC17A5 gene have to be considered in patients with hypomyelination, even in the absence of sialuria.

The unity and diversity of the ciliary central apparatus.

Nearly all motile cilia and flagella (terms here used interchangeably) have a '9+2' axoneme containing nine outer doublet microtubules and two central microtubules. The central pair of microtubules plus associated projections, termed the central apparatus (CA), is involved in the control of flagellar motility and is essential for the normal movement of '9+2' cilia. Research using the green alga Chlamydomonas reinhardtii, an important model system for studying cilia, has provided most of our knowledge of the protein composition of the CA, and recent work using this organism has expanded the number of known and candidate CA proteins nearly threefold. Here we take advantage of this enhanced proteome to examine the genomes of a wide range of eukaryotic organisms, representing all of the major phylogenetic groups, to identify predicted orthologues of the C. reinhardtii CA proteins and explore how widely the proteins are conserved and whether there are patterns to this conservation. We also discuss in detail two contrasting groups of CA proteins-the ASH-domain proteins, which are broadly conserved, and the PAS proteins, which are restricted primarily to the volvocalean algae. This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.

TIM, a targeted insertional mutagenesis method utilizing CRISPR/Cas9 in Chlamydomonas reinhardtii.

Generation and subsequent analysis of mutants is critical to understanding the functions of genes and proteins. Here we describe TIM, an efficient, cost-effective, CRISPR-based targeted insertional mutagenesis method for the model organism Chlamydomonas reinhardtii. TIM utilizes delivery into the cell of a Cas9-guide RNA (gRNA) ribonucleoprotein (RNP) together with exogenous double-stranded (donor) DNA. The donor DNA contains gene-specific homology arms and an integral antibiotic-resistance gene that inserts at the double-stranded break generated by Cas9. After optimizing multiple parameters of this method, we were able to generate mutants for six out of six different genes in two different cell-walled strains with mutation efficiencies ranging from 40% to 95%. Furthermore, these high efficiencies allowed simultaneous targeting of two separate genes in a single experiment. TIM is flexible with regard to many parameters and can be carried out using either electroporation or the glass-bead method for delivery of the RNP and donor DNA. TIM achieves a far higher mutation rate than any previously reported for CRISPR-based methods in C. reinhardtii and promises to be effective for many, if not all, non-essential nuclear genes.

Functionally pathogenic EARS2 variants in vitro may not manifest a phenotype in vivo.

OBJECTIVE: To investigate the genetic etiology of a patient diagnosed with leukoencephalopathy, brain calcifications, and cysts (LCC). METHODS: Whole-exome sequencing was performed on a patient with LCC and his unaffected family members. The variants were subject to in silico and in vitro functional testing to determine pathogenicity. RESULTS: Whole-exome sequencing uncovered compound heterozygous mutations in EARS2, c.328G>A (p.G110S), and c.1045G>A (p.E349K). This gene has previously been implicated in the autosomal recessive leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL). The p.G110S mutation has been found in multiple patients with LTBL. In silico analysis supported pathogenicity in the second variant. In vitro functional testing showed a significant mitochondrial dysfunction demonstrated by an ∼11% decrease in the oxygen consumption rate and ∼43% decrease in the maximum respiratory rate in the patient's skin fibroblasts compared with the control. EARS2 protein levels were reduced to 30% of normal controls in the patient's fibroblasts. These deficiencies were corrected by the expression of the wild-type EARS2 protein. However, a further unrelated genetic investigation of our patient revealed the presence of biallelic variants in a small nucleolar RNA (SNORD118) responsible for LCC. CONCLUSIONS: Here, we report seemingly pathogenic EARS2 mutations in a single patient with LCC with no biochemical or neuroimaging presentations of LTBL. This patient illustrates that variants with demonstrated impact on protein function should not necessarily be considered clinically relevant. CLINICALTRIALSGOV IDENTIFIER: NCT00001671.

ANGPTL8 reverses established adriamycin cardiomyopathy by stimulating adult cardiac progenitor cells.

Established adriamycin cardiomyopathy is a lethal disease. When congestive heart failure develops, mortality is approximately 50% in a year. It has been known that ANGPTLs has various functions in lipid metabolism, inflammation, cancer cell invasion, hematopoietic stem activity and diabetes. We hypothesized that ANGPTL8 is capable of maintaining heart function by stimulating adult cardiac progenitor cells to initiate myocardial regeneration. We employed UTMD to deliver piggybac transposon plasmids with the human ANGPTL8 gene to the liver of rats with adriamycin cardiomyopathy. After ANGPTL8 gene liver delivery, overexpression of transgenic human ANGPTL8 was found in rat liver cells and blood. UTMD- ANGPTL8 gene therapy restored LV mass, fractional shortening index, and LV posterior wall diameter to nearly normal. Our results also showed that ANGPTL8 reversed established ADM cardiomyopathy. This was associated with activation of ISL-1 positive cardiac progenitor cells in the epicardium. A time-course experiment shown that ISL-1 cardiac progenitor cells proliferated and formed a niche in the epicardial layer and then migrated into sub-epicardium. The observed myocardial regeneration accompanying reversal of adriamycin cardiomyopathy was associated with upregulation of PirB expression on the cell membrane of cardiac muscle cells or progenitor cells stimulated by ANGPTL8.