Choline transporter-like 1 deficiency causes a new type of childhood-onset neurodegeneration.

Cerebral choline metabolism is crucial for normal brain function, and its homoeostasis depends on carrier-mediated transport. Here, we report on four individuals from three families with neurodegenerative disease and homozygous frameshift mutations (Asp517Metfs*19, Ser126Metfs*8, and Lys90Metfs*18) in the SLC44A1 gene encoding choline transporter-like protein 1. Clinical features included progressive ataxia, tremor, cognitive decline, dysphagia, optic atrophy, dysarthria, as well as urinary and bowel incontinence. Brain MRI demonstrated cerebellar atrophy and leukoencephalopathy. Moreover, low signal intensity in globus pallidus with hyperintensive streaking and low signal intensity in substantia nigra were seen in two individuals. The Asp517Metfs*19 and Ser126Metfs*8 fibroblasts were structurally and functionally indistinguishable. The most prominent ultrastructural changes of the mutant fibroblasts were reduced presence of free ribosomes, the appearance of elongated endoplasmic reticulum and strikingly increased number of mitochondria and small vesicles. When chronically treated with choline, those characteristics disappeared and mutant ultrastructure resembled healthy control cells. Functional analysis revealed diminished choline transport yet the membrane phosphatidylcholine content remained unchanged. As part of the mechanism to preserve choline and phosphatidylcholine, choline transporter deficiency was implicated in impaired membrane homeostasis of other phospholipids. Choline treatments could restore the membrane lipids, repair cellular organelles and protect mutant cells from acute iron overload. In conclusion, we describe a novel childhood-onset neurometabolic disease caused by choline transporter deficiency with autosomal recessive inheritance.

Choline transport links macrophage phospholipid metabolism and inflammation.

Choline is an essential nutrient that is required for synthesis of the main eukaryote phospholipid, phosphatidylcholine. Macrophages are innate immune cells that survey and respond to danger and damage signals. Although it is well-known that energy metabolism can dictate macrophage function, little is known as to the importance of choline homeostasis in macrophage biology. We hypothesized that the uptake and metabolism of choline are important for macrophage inflammation. Polarization of primary bone marrow macrophages with lipopolysaccharide (LPS) resulted in an increased rate of choline uptake and higher levels of PC synthesis. This was attributed to a substantial increase in the transcript and protein expression of the choline transporter-like protein-1 (CTL1) in polarized cells. We next sought to determine the importance of choline uptake and CTL1 for macrophage immune responsiveness. Chronic pharmacological or CTL1 antibody-mediated inhibition of choline uptake resulted in altered cytokine secretion in response to LPS, which was associated with increased levels of diacylglycerol and activation of protein kinase C. These experiments establish a previously unappreciated link between choline phospholipid metabolism and macrophage immune responsiveness, highlighting a critical and regulatory role for macrophage choline uptake via the CTL1 transporter.

Papillary glioneuronal tumor (PGNT) exhibits a characteristic methylation profile and fusions involving PRKCA.

Papillary glioneuronal tumor (PGNT) is a WHO-defined brain tumor entity that poses a major diagnostic challenge. Recently, SLC44A1-PRKCA fusions have been described in PGNT. We subjected 28 brain tumors from different institutions histologically diagnosed as PGNT to molecular and morphological analysis. Array-based methylation analysis revealed that 17/28 tumors exhibited methylation profiles typical for other tumor entities, mostly dysembryoplastic neuroepithelial tumor and hemispheric pilocytic astrocytoma. Conversely, 11/28 tumors exhibited a unique profile, thus constituting a distinct methylation class PGNT. By screening the extended Heidelberg cohort containing over 25,000 CNS tumors, we identified three additional tumors belonging to this methylation cluster but originally histologically diagnosed otherwise. RNA sequencing for the detection of SLC44A1-PRKCA fusions could be performed on 19 of the tumors, 10 of them belonging to the methylation class PGNT. In two additional cases, SLC44A1-PRKCA fusions were confirmed by FISH. We detected fusions involving PRKCA in all cases of this methylation class with material available for analyses: the canonical SLC44A1-PRKCA fusion was observed in 11/12 tumors, while the remaining case exhibited a NOTCH1-PRKCA fusion. Neither of the fusions was found in the tumors belonging to other methylation classes. Our results point towards a high misclassification rate of the morphological diagnosis PGNT and clearly demonstrate the necessity of molecular analyses. PRKCA fusions are highly diagnostic for PGNT, and detection by RNA sequencing enables the identification of rare fusion partners. Methylation analysis recognizes a unique methylation class PGNT irrespective of the nature of the PRKCA fusion.

Mechanism of choline deficiency and membrane alteration in postural orthostatic tachycardia syndrome primary skin fibroblasts.

Fibroblasts from a patient with postural orthostatic tachycardia syndrome (POTS), who presented with low plasma choline and betaine, were studied to determine the metabolic characteristics of the choline deficiency. Choline is required for the synthesis of the phospholipid phosphatidylcholine (PC) and for betaine, an important osmoregulator. Here, choline transport, lipid homeostasis, and mitochondria function were analyzed in skin fibroblasts from POTS and compared with control cells. The choline transporter-like protein 1/solute carrier 44A1 (CTL1/SLC44A1) and mRNA expression were 2-3 times lower in POTS fibroblasts, and choline uptake was reduced 60% (P < 0.05). Disturbances of membrane homeostasis were observed by reduced ratios between PC:phosphatidylethanolamine and sphingomyelin:cholesterol, as well as by modified phospholipid fatty acid composition. Choline deficiency also impaired mitochondria function, which was observed by a reduction in oxygen consumption, mitochondrial potential, and glycolytic activity. When POTS cells were treated with choline, transporter was up-regulated, and uptake of choline increased, offering an option for patient treatment. The characteristics of the POTS fibroblasts described here represent a first model of choline and CTL1/SLC44A1 deficiency, in which choline transport, membrane homeostasis, and mitochondrial function are impaired.

Effective Treatment of Lung Adenocarcinoma With a Novel SLC44A1-BRAF Fusion Using Pembrolizumab Followed by Trametinib: A Case Report.

The serine-threonine protein kinase B-RAF (BRAF) fusions are rarely observed in non-small cell lung cancer (NSCLC) accounting for less than 1%, and therapeutic evidence for molecular-targeted drugs is lacking, unlike for BRAF V600E mutation by RAF and MEK inhibitors. A 75-year-old female patient with no smoking history and mild renal dysfunction developed recurrent lung adenocarcinoma and was initially treated with pembrolizumab immunotherapy followed by chemotherapy using docetaxel showing a certain efficacy but the disease finally progressed. Comprehensive genome profiling showed a novel SLC44A1-BRAF fusion and the tumor progression was controlled with the MEK inhibitor trametinib. Because of the rarity of NSCLC with BRAF fusion, the description of this case would be helpful for the treatment strategy for such tumors.

Polymorphisms in the choline transporter SLC44A1 are associated with reduced cognitive performance in normotypic but not prenatal alcohol-exposed children.

BACKGROUND: Choline is essential for healthy cognitive development. Single nucleotide polymorphisms (SNPs; rs3199966(G), rs2771040(G)) within the choline transporter SLC44A1 increase risk for choline deficiency. In a choline intervention trial of children who experienced prenatal alcohol exposure (PAE), these alleles are associated with improved cognition. OBJECTIVE: This study aimed to determine if SNPs within SLC44A1 are differentially associated with cognition in children with PAE compared with normotypic controls (genotype × exposure). A secondary objective tested for an association of these SNPs and cognition in controls (genotype-only). DESIGN: This is a secondary analysis of data from the Collaborative Initiative on Fetal Alcohol Spectrum Disorders. Participants (163 normotypic controls, 162 PAE) underwent psychological assessments and were genotyped within SLC44A1. Choline status was not assessed. Association analysis between genotype × exposure was performed using an additive genetic model and linear regression to identify the allelic effect. The primary outcome was the interaction between SLC44A1 genotype × exposure status with respect to cognition. The secondary outcome was the cognitive-genotype association in normotypic controls. RESULTS: Genotype × exposure analysis identified 7 SNPs in SLC44A1, including rs3199966(G) and rs2771040(G), and in strong linkage (D' ≥ 0.87), that were associated (adjusted P ≤ 0.05) with reduced performance in measures of general cognition, nonverbal and quantitative reasoning, memory, and executive function (ß, 1.92-3.91). In controls, carriers of rs3199966(GT or GG) had worsened cognitive performance than rs3199966(TT) carriers (ß, 0.46-0.83; P < 0.0001), whereas cognitive performance did not differ by rs3199966 genotype in those with PAE. CONCLUSIONS: Two functional alleles that increase vulnerability to choline deficiency, rs3199966(G) (Ser644Ala) and rs2771040(G) (3' untranslated region), are associated with worsened cognition in otherwise normotypic children. These alleles were previously associated with greater cognitive improvement in children with PAE who received supplemental choline. The findings endorse that choline benefits cognitive development in normotypic children and those with PAE.

Polymorphisms in SLC44A1 are associated with cognitive improvement in children diagnosed with fetal alcohol spectrum disorder: an exploratory study of oral choline supplementation.

BACKGROUND: The essential nutrient choline provides one-carbon units for metabolite synthesis and epigenetic regulation in tissues including brain. Dietary choline intake is often inadequate, and higher intakes are associated with improved cognitive function. OBJECTIVE: Choline supplements confer cognitive improvement for those diagnosed with fetal alcohol spectrum disorder (FASD), a common set of neurodevelopmental impairments; however, the effect sizes have been modest. In this retrospective analysis, we report that genetic polymorphisms affecting choline utilization are associated with cognitive improvement following choline intervention. METHODS: Fifty-two children from the upper midwestern United States and diagnosed with FASD, ages 2-5 y, were randomly assigned to receive choline (500 mg/d; n = 26) or placebo (n = 26) for 9 mo, and were genotyped for 384 choline-related single nucleotide polymorphisms (SNPs). Memory and cognition were assessed at enrollment, study terminus, and at 4-y follow-up for a subset. RESULTS: When stratified by intervention (choline vs. placebo), 14-16 SNPs within the cellular choline transporter gene solute carrier family 44 member 1 (SLC44A1) were significantly associated with performance in an elicited imitation sequential memory task, wherein the effect alleles were associated with the greatest pre-/postintervention improvement. Of these, rs3199966 is a structural variant (S644A) and rs2771040 is a single-nucleotide variant within the 3' untranslated region of the plasma membrane isoform. An additive genetic model best explained the genotype associations. Lesser associations were observed for cognitive outcome and polymorphisms in flavin monooxygenase-3 (FMO3), methylenetetrahydrofolate dehydrogenase-1 (MTHFD1), fatty acid desaturase-2 (FADS2), and adiponectin receptor 1 (ADIPOR1). CONCLUSIONS: These SLC44A1 variants were previously associated with greater vulnerability to choline deficiency. Our data potentially support the use of choline supplements to improve cognitive function in individuals diagnosed with FASD who carry these effect alleles. Although these findings require replication in both retrospective and prospective confirmatory trials, they emphasize the need to incorporate similar genetic analyses of choline-related polymorphisms in other FASD-choline trials, and to test for similar associations within the general FASD population. This trial was registered at www.clinicaltrials.gov as NCT01149538.

In Vitro Hepatitis C Virus Infection and Hepatic Choline Metabolism.

Choline is an essential nutrient required for normal neuronal and muscular development, as well as homeostatic regulation of hepatic metabolism. In the liver, choline is incorporated into the main eukaryotic phospholipid, phosphatidylcholine (PC), and can enter one-carbon metabolism via mitochondrial oxidation. Hepatitis C virus (HCV) is a hepatotropic positive-strand RNA virus that similar to other positive-strand RNA viruses and can impact phospholipid metabolism. In the current study we sought to interrogate if HCV modulates markers of choline metabolism following in vitro infection, while subsequently assessing if the inhibition of choline uptake and metabolism upon concurrent HCV infection alters viral replication and infectivity. Additionally, we assessed whether these parameters were consistent between cells cultured in fetal bovine serum (FBS) or human serum (HS), conditions known to differentially affect in vitro HCV infection. We observed that choline transport in FBS- and HS-cultured Huh7.5 cells is facilitated by the intermediate affinity transporter, choline transporter-like family (CTL). HCV infection in FBS, but not HS-cultured cells diminished CTL1 transcript and protein expression at 24 h post-infection, which was associated with lower choline uptake and lower incorporation of choline into PC. No changes in other transporters were observed and at 96 h post-infection, all differences were normalized. Reciprocally, limiting the availability of choline for PC synthesis by use of a choline uptake inhibitor resulted in increased HCV replication at this early stage (24 h post-infection) in both FBS- and HS-cultured cells. Finally, in chronic infection (96 h post-infection), inhibiting choline uptake and metabolism significantly impaired the production of infectious virions. These results suggest that in addition to a known role of choline kinase, the transport of choline, potentially via CTL1, might also represent an important and regulated process during HCV infection.