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Glycosphingolipid Changes in Plasma in Parkinson's Disease Independent of Glucosylceramide Levels.
BACKGROUND: Alteration in glycosphingolipids (GSLs) in Parkinson's disease (PD) still needs to be determined. OBJECTIVES: We evaluated if PD subjects show abnormal GSLs levels compared to healthy controls (HC) and if GSLs correlate with clinical features. METHODS: We analyzed GSLs and glucosylceramide (GlcCer) in plasma using two normal-phase high-performance liquid chromatography assays; clinico-demographic data were extracted. RESULTS: Eighty PD subjects and 25 HCs were analyzed. Levels of GlcCer, GD1b, Gb4, GalNAcGA1, and b-series were higher in PD patients than in HCs; total GSLs, GT1b, GM1a, GM3, GM2, and a-series levels were lower in PD patients than in HCs. Changes in GSLs were present in PD subjects, with GlcCer levels similar to those in HCs. The results were similar after excluding certain GBA1 mutation carriers. Movement Disorder Society Unified Parkinson's Disease Rating Scale, Part III, correlated with Gb4 and Montreal Cognitive Assessment with GD1b levels. CONCLUSIONS: Multiple GSL abnormalities in plasma were detected in patients with and without GlcCer changes, indicating a broader shift in lipid homeostasis. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society.
A novel, highly sensitive and specific biomarker for Niemann-Pick type C1 disease.
BACKGROUND: Lysosomal storage disorders (LSDs), are a heterogeneous group of rare disorders caused by defects in genes encoding for proteins involved in the lysosomal degradation of macromolecules. They occur at a frequency of about 1 in 5,000 live births, though recent neonatal screening suggests a higher incidence. New treatment options for LSDs demand a rapid, early diagnosis of LSDs if maximal clinical benefit is to be achieved. METHODS: Here, we describe a novel, highly specific and sensitive biomarker for Niemann-Pick Type C disease type 1 (NPC1), lyso-sphingomyelin-509. We cross-validate this biomarker with cholestane-3β,5α,6β-triol and relative lysosomal volume. The primary cohort for establishment of the biomarker contained 135 NPC1 patients, 66 NPC1 carriers, 241 patients with other LSDs and 46 healthy controls. RESULTS: With a sensitivity of 100.0% and specificity of 91.0% a cut-off of 1.4 ng/ml was established. Comparison with cholestane-3β,5α,6β-triol and relative acidic compartment volume measurements were carried out with a subset of 125 subjects. Both cholestane-3β,5α,6β-triol and lyso-Sphingomyelin-509 were sufficient in establishing the diagnosis of NPC1 and correlated with disease severity. CONCLUSION: In summary, we have established a new biomarker for the diagnosis of NPC1, and further studies will be conducted to assess correlation to disease progress and monitoring treatment.
Measuring relative lysosomal volume for monitoring lysosomal storage diseases.
Biomarkers are important tools in medicine, which can be used for monitoring disease progression and response to therapy. One of the main problems in rare lysosomal storage diseases is that there are over 70 different diseases, all with different biochemical storage profiles. Developing biochemical biomarkers therefore requires an individual assay per disease/subgroup of diseases. An alternative approach is to develop an assay that is independent of the specific macromolecules stored. This chapter discusses an assay that may serve as a universal biomarker for these diseases and measures the expansion of the late endosomal/lysosomal system. We have developed an assay that takes advantage of a commercially available late endosomal/lysosomal probe, LysoTracker, which becomes trapped in the acidic compartment of cells and emits a fluorescent signal that can be detected using flow cytometry. In this chapter, we detail the methodology behind this assay and discuss the factors that need to be considered when establishing this assay in clinical and research settings.
Treatment with miglustat reverses the lipid-trafficking defect in Niemann-Pick disease type C.
Niemann-Pick disease type C (NP-C) is a hereditary neurovisceral lipid storage disorder. Although traditionally considered a primary cholesterol storage disorder, a variety of glycolipids accumulate in NP-C cells, which resemble those from glycosphingolipidosis patients. Substrate reduction therapy (SRT) with miglustat, an inhibitor of glycosphingolipid biosynthesis, is a novel therapy for the glycosphingolipidoses. We report the use of SRT in a patient with NP-C. We show that depletion of glycosphingolipids by miglustat treatment reduces pathological lipid storage, improves endosomal uptake and normalises lipid trafficking in peripheral blood B lymphocytes. The demonstration that treatment with miglustat, which has no direct effect on cholesterol metabolism, corrects the abnormal lipid trafficking seen in B lymphocytes in NP-C indicates that glycosphingolipid accumulation is the primary pathogenetic event in NP-C. These observations support the use of SRT in patients with this devastating neurodegenerative disease.
Invariant natural killer T cells are not affected by lysosomal storage in patients with Niemann-Pick disease type C.
Invariant natural killer T (iNKT) cells are a specialised subset of T cells that are restricted to the MHC class I like molecule, CD1d. The ligands for iNKT cells are lipids, with the canonical superagonist being α-galactosylceramide, a non-mammalian glycosphingolipid. Trafficking of CD1d through the lysosome is required for the development of murine iNKT cells. Niemann-Pick type C (NPC) disease is a lysosomal storage disorder caused by dysfunction in either of two lysosomal proteins, NPC1 or NPC2, resulting in the storage of multiple lipids, including glycosphingolipids. In the NPC1 mouse model, iNKT cells are virtually undetectable, which is likely due to the inability of CD1d to be loaded with the selecting ligand due to defective lysosomal function and/or CD1d trafficking. However, in this study we have found that in NPC1 patients iNKT cells are present at normal frequencies, with no phenotypic or functional differences. In addi-tion, antigen-presenting cells derived from NPC1 patients are functionally competent to present several different CD1d/iNKT-cell ligands. This further supports the hypothesis that there are different trafficking requirements for the development of murine and human iNKT cells, and a functional lysosomal/late-endosomal compartment is not required for human iNKT-cell development.
A sensitive and specific LC-MS/MS method for rapid diagnosis of Niemann-Pick C1 disease from human plasma.
Niemann-Pick type C1 (NPC1) disease is a rare, progressively fatal neurodegenerative disease for which there are no FDA-approved therapies. A major barrier to developing new therapies for this disorder has been the lack of a sensitive and noninvasive diagnostic test. Recently, we demonstrated that two cholesterol oxidation products, specifically cholestane-3β,5α,6β-triol (3β,5α,6β-triol) and 7-ketocholesterol (7-KC), were markedly increased in the plasma of human NPC1 subjects, suggesting a role for these oxysterols in diagnosis of NPC1 disease and evaluation of therapeutics in clinical trials. In the present study, we describe the development of a sensitive and specific LC-MS/MS method for quantifying 3β,5α,6β-triol and 7-KC human plasma after derivatization with N,N-dimethylglycine. We show that dimethylglycine derivatization successfully enhanced the ionization and fragmentation of 3β,5α,6β-triol and 7-KC for mass spectrometric detection of the oxysterol species in human plasma. The oxysterol dimethylglycinates were resolved with high sensitivity and selectivity, and enabled accurate quantification of 3β,5α,6β-triol and 7-KC concentrations in human plasma. The LC-MS/MS assay was able to discriminate with high sensitivity and specificity between control and NPC1 subjects, and offers for the first time a noninvasive, rapid, and highly sensitive method for diagnosis of NPC1 disease.
Glycosphingolipid storage leads to the enhanced degradation of the B cell receptor in Sandhoff disease mice.
Glycosphingolipid storage diseases are a group of inherited metabolic diseases in which glycosphingolipids accumulate due to their impaired lysosomal breakdown. Splenic B cells isolated from NPC1, Sandhoff, GM1-gangliosidosis and Fabry disease mouse models showed large (20- to 30-fold) increases in disease specific glycosphingolipids and up to a 4-fold increase in cholesterol. The magnitude of glycosphingolipid storage was in the order NPC1 > Sandhoff approximately GM1 gangliosidosis > Fabry. Except for Fabry disease, glycosphingolipid storage led to an increase in the lysosomal compartment and altered glycosphingolipid trafficking. In order to investigate the consequences of storage on B cell function, the levels of surface expression of B cell IgM receptor and its associated components were quantitated in Sandhoff B cells, since they are all raft-associated on activation. Both the B cell receptor, CD21 and CD19 had decreased cell surface expression. In contrast, CD40 and MHC II, surface receptors that do not associate with lipid rafts, were unchanged. Using a pulse chase biotinylation procedure, surface B cell receptors on a Sandhoff lymphoblast cell line were found to have a significantly decreased half-life. Increased co-localization of fluorescently conjugated cholera toxin and lysosomes was also observed in Sandhoff B cells. Glycosphingolipid storage leads to the enhanced formation of lysosomal lipid rafts, altered endocytic trafficking and increased degradation of the B cell receptor.
Relative acidic compartment volume as a lysosomal storage disorder-associated biomarker.
Lysosomal storage disorders (LSDs) occur at a frequency of 1 in every 5,000 live births and are a common cause of pediatric neurodegenerative disease. The relatively small number of patients with LSDs and lack of validated biomarkers are substantial challenges for clinical trial design. Here, we evaluated the use of a commercially available fluorescent probe, Lysotracker, that can be used to measure the relative acidic compartment volume of circulating B cells as a potentially universal biomarker for LSDs. We validated this metric in a mouse model of the LSD Niemann-Pick type C1 disease (NPC1) and in a prospective 5-year international study of NPC patients. Pediatric NPC subjects had elevated acidic compartment volume that correlated with age-adjusted clinical severity and was reduced in response to therapy with miglustat, a European Medicines Agency–approved drug that has been shown to reduce NPC1-associated neuropathology. Measurement of relative acidic compartment volume was also useful for monitoring therapeutic responses of an NPC2 patient after bone marrow transplantation. Furthermore, this metric identified a potential adverse event in NPC1 patients receiving i.v. cyclodextrin therapy. Our data indicate that relative acidic compartment volume may be a useful biomarker to aid diagnosis, clinical monitoring, and evaluation of therapeutic responses in patients with lysosomal disorders.
Analysis of fluorescently labeled glycosphingolipid-derived oligosaccharides following ceramide glycanase digestion and anthranilic acid labeling.
Interest in cellular glycosphingolipid (GSL) function has necessitated the development of a rapid and sensitive method to both analyze and characterize the full complement of structures present in various cells and tissues. An optimized method to characterize oligosaccharides released from glycosphingolipids following ceramide glycanase digestion has been developed. The procedure uses the fluorescent compound anthranilic acid (2-aminobenzoic acid; 2-AA) to label oligosaccharides prior to analysis using normal-phase high-performance liquid chromatography. The labeling procedure is rapid, selective, and easy to perform and is based on the published method of Anumula and Dhume [Glycobiology 8 (1998) 685], originally used to analyze N-linked oligosaccharides. It is less time consuming than a previously published 2-aminobenzamide labeling method [Anal. Biochem. 298 (2001) 207] for analyzing GSL-derived oligosaccharides, as the fluorescent labeling is performed on the enzyme reaction mixture. The purification of 2-AA-labeled products has been improved to ensure recovery of oligosaccharides containing one to four monosaccharide units, which was not previously possible using the Anumula and Dhume post-derivatization purification procedure. This new approach may also be used to analyze both N- and O-linked oligosaccharides.
Urinary excretion and metabolism of miglustat and valproate in patients with Niemann-Pick type C1 disease: One- and two-dimensional solution-state (1)H NMR studies.
Niemann-Pick type C1 (NP-C1) disease is a neurodegenerative lysosomal storage disease for which the only approved therapy is miglustat (MGS). In this study we explored the applications and value of both one- and two-dimensional high-resolution NMR analysis strategies to the detection and quantification of MGS and its potential metabolites in urine samples collected from NP-C1 disease patients (n=47), and also applied these techniques to the analysis of the anticonvulsant drug valproate and one of its major metabolites in ca. 30% of these samples (i.e. from those who were also receiving this agent for the control of epileptic seizures). A combination of high-resolution 1D and 2D TOCSY/NOESY techniques confirmed the identity of MGS in the urinary (1)H NMR profiles of NP-C1 patients treated with this agent (n=25), and its quantification was readily achievable via electronic integration of selected 1D resonance intensities. However, this analysis provided little or no evidence for its metabolism in vivo, observations consistent with those acquired in corresponding experiments performed involving an in vitro microsomal system. Contrastingly, the major valproate metabolite 1-O-valproyl-β-glucuronide was readily detectable and quantifiable in 14/47 of the urine samples investigated, despite some resonance overlap problems (identification of this agent was confirmed by experiments involving equilibration of these samples with β-glucuronidase, a process liberating free valproate). In order to facilitate and validate the detection of MGS in urine specimens, full assignments of the (1)H NMR spectra of MGS in both buffered aqueous (pH 7.10) and deuterated methanol solvent systems were also made. The pharmacological and bioanalytical significance of data acquired are discussed, with special reference to the advantages offered by high-resolution NMR analysis.
Altered distribution and function of natural killer cells in murine and human Niemann-Pick disease type C1.
Niemann-Pick type C (NPC) is a neurodegenerative lysosomal storage disorder caused by defects in the lysosomal proteins NPC1 or NPC2. NPC cells are characterized by reduced lysosomal calcium levels and impaired sphingosine transport from lysosomes. Natural killer (NK) cells kill virally infected/transformed cells via degranulation of lysosome-related organelles. Their trafficking from lymphoid tissues into the circulation is dependent on sphingosine-1-phosphate (S1P) gradients, sensed by S1P receptor 5 (S1P5). We hypothesized that NK-cell function and trafficking could be affected in NPC disease due to the combined effects of the lysosomal calcium defect and sphingosine storage. In an NPC1 mouse model, we found the frequency of NK cells was altered and phenocopied S1P5-deficient mice, consistent with defects in S1P levels. NK cells from NPC1 mice also had a defect in cytotoxicity due to a failure in degranulation of cytotoxic granules, which was associated with reduced lysosomal calcium levels. Affected NPC1 patients and NPC1 heterozygote carriers had reduced NK-cell numbers in their blood and showed similar phenotypic and developmental changes to those observed in the NPC1 mouse. These findings highlight the effects of lysosomal storage on the peripheral immune system.
A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency.
Superparamagnetic iron oxide nanoparticles (SPIONs) have mainly been used as cellular carriers for genes and therapeutic products, while their use in subcellular organelle isolation remains underexploited. We engineered SPIONs targeting distinct subcellular compartments. Dimercaptosuccinic acid-coated SPIONs are internalized and accumulate in late endosomes/lysosomes, while aminolipid-SPIONs reside at the plasma membrane. These features allowed us to establish standardized magnetic isolation procedures for these membrane compartments with a yield and purity permitting proteomic and lipidomic profiling. We validated our approach by comparing the biomolecular compositions of lysosomes and plasma membranes isolated from wild-type and Niemann-Pick disease type C1 (NPC1) deficient cells. While the accumulation of cholesterol and glycosphingolipids is seen as a primary hallmark of NPC1 deficiency, our lipidomics analysis revealed the buildup of several species of glycerophospholipids and other storage lipids in selectively late endosomes/lysosomes of NPC1-KO cells. While the plasma membrane proteome remained largely invariable, we observed pronounced alterations in several proteins linked to autophagy and lysosomal catabolism reflecting vesicular transport obstruction and defective lysosomal turnover resulting from NPC1 deficiency. Thus the use of SPIONs provides a major advancement in fingerprinting subcellular compartments, with an increased potential to identify disease-related alterations in their biomolecular compositions.
NMR analysis reveals significant differences in the plasma metabolic profiles of Niemann Pick C1 patients, heterozygous carriers, and healthy controls.
Niemann-Pick type C1 (NPC1) disease is a rare autosomal recessive, neurodegenerative lysosomal storage disorder, which presents with a range of clinical phenotypes and hence diagnosis remains a challenge. In view of these difficulties, the search for a novel, NPC1-specific biomarker (or set of biomarkers) is a topic of much interest. Here we employed high-resolution 1H nuclear magnetic resonance spectroscopy coupled with advanced multivariate analysis techniques in order to explore and seek differences between blood plasma samples acquired from NPC1 (untreated and miglustat treated), heterozygote, and healthy control subjects. Using this approach, we were able to identify NPC1 disease with 91% accuracy confirming that there are significant differences in the NMR plasma metabolic profiles of NPC1 patients when compared to healthy controls. The discrimination between NPC1 (both miglustat treated and untreated) and healthy controls was dominated by lipoprotein triacylglycerol 1H NMR resonances and isoleucine. Heterozygote plasma samples displayed also increases in the intensities of selected lipoprotein triacylglycerol 1H NMR signals over those of healthy controls. The metabolites identified could represent useful biomarkers in the future and provide valuable insight in to the underlying pathology of NPC1 disease.
Machine learning-based prediction of anxiety disorders using blood metabolite and social trait data from the UK Biobank
Anxiety disorders are the most prevalent type of mental health disorders and are characterised by excessive fear and worry. Despite affecting one in four individuals within their lifetime, there remains a gap in our understanding regarding the underlying pathophysiology of anxiety disorders, which limits the development of novel treatment options. Exploring blood-based biomarkers of anxiety disorder offers the potential to predict the risk of clinically significant anxiety in the general population, increase our understanding of anxiety pathophysiology, and to reveal options for preventative treatment. Here, using psychosocial variables in combination with blood and urine biomarkers, reported in the UK Biobank, we sought to predict future anxiety onset. Machine learning accurately predicted (ROC AUC: ∼0.83) ICD-10-coded anxiety diagnoses up to 5 years (mean 3.5 years) after blood sampling, against lifetime anxiety-free controls. Analysis of the blood biochemistry measures indicated that anxious individuals were more anaemic and exhibited higher levels of markers of systemic inflammation than controls. However, blood biomarkers alone were not predictive of resilience or susceptibility to anxiety disorders in a subset of individuals rigorously matched for a wide range of psychosocial covariates (ROC AUC: ∼0.50). Overall, we demonstrate that the integration of biological and psychosocial risk factors is an effective tool to screen for and predict anxiety disorder onset in the general population.
Small-molecule dissolution of stress granules by redox modulation benefits ALS models.
Neurodegenerative diseases, such as amyotrophic lateral sclerosis, are often associated with mutations in stress granule proteins. Aberrant stress granule condensate formation is associated with disease, making it a potential target for pharmacological intervention. Here, we identified lipoamide, a small molecule that specifically prevents cytoplasmic condensation of stress granule proteins. Thermal proteome profiling showed that lipoamide stabilizes intrinsically disordered domain-containing proteins, including SRSF1 and SFPQ, which are stress granule proteins necessary for lipoamide activity. SFPQ has redox-state-specific condensate dissolving behavior, which is modulated by the redox-active lipoamide dithiolane ring. In animals, lipoamide ameliorates aging-associated aggregation of a stress granule reporter protein, improves neuronal morphology and recovers motor defects caused by amyotrophic lateral sclerosis-associated FUS and TDP-43 mutants. Thus, lipoamide is a well-tolerated small-molecule modulator of stress granule condensation, and dissection of its molecular mechanism identified a cellular pathway for redox regulation of stress granule formation.
Integrating TSPO-PET imaging with metabolomics for enhanced prognostic accuracy in multiple sclerosis.
BACKGROUND: Predicting disease progression in multiple sclerosis (MS) remains challenging. PET imaging with 18 kDa translocator protein (TSPO) radioligands can detect microglial and astrocyte activation beyond MRI-visible lesions, which has been shown to be highly predictive of disease progression. We previously demonstrated that nuclear magnetic resonance (NMR)-based metabolomics could accurately distinguish between relapsing-remitting (RRMS) and secondary progressive MS (SPMS). This study investigates whether combining TSPO imaging with metabolomics enhances predictive accuracy in a similar setting. METHODS: Blood samples were collected from 87 MS patients undergoing PET imaging with the TSPO-binding radioligand 11C-PK11195 in Finland. Patient disability was assessed using the expanded disability status scale (EDSS) at baseline and 1 year later. Serum metabolomics was performed to identify biomarkers associated with TSPO binding and disease progression. RESULTS: Greater TSPO availability in the normal-appearing white matter and perilesional regions correlated with higher EDSS. Serum metabolites glutamate (p=0.02), glutamine (p=0.006), and glucose (p=0.008), detected by NMR, effectively distinguished future progressors. These three metabolites alone predicted progression with the same accuracy as TSPO-PET imaging (AUC 0.78; p=0.0001), validated in an independent cohort. Combining serum metabolite data with PET imaging significantly improved predictive power, achieving an AUC of 0.98 (p<0.0001). CONCLUSION: Measuring three specific serum metabolites is as effective as TSPO imaging in predicting MS progression. However, integrating TSPO imaging with serum metabolite analysis substantially enhances predictive accuracy. Given the simplicity and affordability of NMR analysis, this approach could lead to more personalised, accessible treatment strategies and serve as a valuable tool for clinical trial stratification.
Early plasma ceramide and sphingomyelin levels reflect APOE genotype but not familial Alzheimer's disease gene mutations in female 5xFAD mice, with brain-region specific sphingolipid alterations.
Pathophysiological changes associated with Alzheimer's disease (AD) begin decades before dementia onset, with age and APOE ε4 genotype as major risk factors [1-4]. Primary risk factors for developing AD include aging and number of copies of the apolipoprotein E (APOE) ε4 allele. Altered sphingolipid metabolism is increasingly implicated in early AD. However, the relationship between early plasma and brain sphingolipid changes-particularly in the context of APOE genotype-remains poorly defined. In this study, we analyzed plasma and brain sphingolipid profiles in transgenic AD mice carrying human APOE3 or APOE4 variants, with or without familial AD mutations (E3FAD and E4FAD). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we assessed 110 sphingolipid species across four major classes (ceramides (Cers), hexosylceramides (HexCers), lactosylceramides (LacCers), and sphingomyelins (SMs)) at 2, 4, and 6 months in plasma and at 6 months in brain tissue in the cortex, hippocampus, striatum, and cerebellum. Our results demonstrate that early plasma sphingolipid alterations are largely driven by APOE genotype rather than AD pathology. Specifically, APOE4 carriers showed significant increases in SM species and reductions in Cer species compared to APOE3 carriers, independent of age or AD genotype. Brain lipid profiles showed minimal changes across genotypes after region correction. However, combined p-value analyses revealed APOE- and EFAD-dependent differences in the composition of primarily cortical sphingolipids. ROC analyses demonstrated high discriminative power of plasma sphingolipids for APOE, but not for AD genotype. These findings suggest that early plasma lipid profiles in female 5xFAD mice are more strongly influenced by APOE genotype than by overt AD pathology, potentially reflecting systemic pathways linked to APOE4-associated AD risk, while early disease-associated changes in the brain appear to be subtle and region-specific. These results underscore the importance of accounting for APOE genotype in early-stage AD lipidomic studies and in the interpretation of peripheral lipid biomarkers.
Increased TMEM106B levels lead to lysosomal dysfunction which affects synaptic signaling and neuronal health
Background: Genetic variation in Transmembrane protein 106B (TMEM106B) is known to influence the risk and presentation in several neurodegenerative diseases and modifies healthy aging. While evidence from human studies suggests that the risk allele is associated with higher levels of TMEM106B, the contribution of elevated levels of TMEM106B to neurodegeneration and aging has not been assessed and it remains unclear how TMEM106B modulates disease risk. Methods: To study the effect of increased TMEM106B levels, we generated Cre-inducible transgenic mice expressing human wild-type TMEM106B. We evaluated lysosomal and neuronal health using in vitro and in vivo assays including transmission electron microscopy, immunostainings, behavioral testing, electrophysiology, and bulk RNA sequencing. Results: We created the first transgenic mouse model that successfully overexpresses TMEM106B, with a 4- to 8-fold increase in TMEM106B protein levels in heterozygous (hTMEM106B(+)) and homozygous (hTMEM106B(++)) animals, respectively. We showed that the increase in TMEM106B protein levels induced lysosomal dysfunction and age-related downregulation of genes associated with neuronal plasticity, learning, and memory. Increased TMEM106B levels led to altered synaptic signaling in 12-month-old animals which further exhibited an anxiety-like phenotype. Finally, we observed mild neuronal loss in the hippocampus of 21-month-old animals. Conclusion: Characterization of the first transgenic mouse model that overexpresses TMEM106B suggests that higher levels of TMEM106B negatively impacts brain health by modifying brain aging and impairing the resilience of the brain to the pathomechanisms of neurodegenerative disorders. This novel model will be a valuable tool to study the involvement and contribution of increased TMEM106B levels to aging and will be essential to study the many age-related diseases in which TMEM106B was genetically shown to be a disease- and risk-modifier.