POS0352 ALLELIC VARIANTS IN THE ABCG2 GENE CAN LEAD TO HYPERURICEMIA AND GOUT

Background:The level of uric acid is largely determined by the functions of urate transporters, which are located in the kidney and intestine. The ABCG2 protein is the major excretor of uric acid and its dysfunction may lead to the development of hyperuricemia and gout.Objectives:The aim of our study was to detect the occurrence and frequency of allelic variants in the ABCG2 gene that can lead to impaired function of the ABCG2 protein and to the development of hyperuricemia and gout.Methods:We examined allelic variants of ABCG2 using PCR amplification and Sanger sequencing of all coding regions and exon-intron boundaries in 359 patients with primary hyperuricemia and gout.Results:We found a rare in-frame deletion p.K360del and 15 missense variants, two of which were common (p.V12M, p.Q141K) and 13 were very rare (p.M71V, p.G74D, p.M131I, p.R147W, p.T153M, p.I242T, p.R236X, p.F373C, p.T421A, p.T434M, p.S476P, p.S572R, p.D620N). The p.R236X variant leads to a premature stop codon. The p.V12M variant probably has a protective effect against gout (minor allele frequency – MAF – in our cohort = 0,025 / MAF in the European population = 0,061), while the p.Q141K variant increases the risk of gout (MAF in our cohort = 0,213 / MAF in the European population = 0,094) (1). As for the rare variants, the p.R147W, p.T153M, p.F373C, p.T434M, p.S476P and p.S572R according to functional analyzes reduce the function of the ABCG2 protein (2). Based on in silico prediction, the impact on reduced function is expected for variants p.M71V, p.G74D, p.M131I, p.R147W, p.I242T, p.F373C, p.T434M, p.S476P and p.S572R.Conclusion:Our data suggest that the common variant p.Q141K and most of the rare variants in the ABCG2 gene affect the function of the ABCG2 urate transporter and are a genetic risk factor for hyperuricemia and gout.References:[1]Stiburkova B, et al. Functional non-synonymous variants of ABCG2 and gout risk. Rheumatology (Oxford). 2017 Nov 1; 56(11):1982-1992.[2]Toyoda Y, et al. Functional characterization of clinically-relevant rare variants in ABCG2 identified in a gout and hyperuricemia cohort. Cells. 2019 Apr 18;8(4).Acknowledgements:This study was supported by the project for conceptual development of research organization 00023728 (Institute of Rheumatology) and RVO VFN64165.Disclosure of Interests:None declared

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POS0354 DETECTION OF ABCG2 VARIANTS IN ENCODING OF URATE TRANSPORTERS ASSOCIATED WITH THE HYPERURICEMIA IN HAEMODIALYSIS PATIENTS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.2084 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 407.2-408

Author(s):

H. Ciferska ◽

K. Pavelcova ◽

J. Vachek ◽

B. Stiburkova

Keyword(s):

Uric Acid ◽

Goodness Of Fit ◽

Rare Variants ◽

Functional Characterization ◽

Hemodialysis Patients ◽

European Population ◽

Reference Sequence ◽

Rank Test ◽

Goodness Of Fit Test ◽

Protective Allele

Background:Hyperuricemia is associated with gout, hypertension, cardiovascular diseases and renal disease. The presence of chronic kidney disease (CKD) is associated with low excretion of the uric acid as the homeostasis in maintaining of serum levels of uric acid is impaired. Progression of CKD is connected to hyperuricemia and lowering levels of the uric acid is one of the most important goals in clinical treatment. Dysfunctional variants of ATP-binding cassette transporter subfamily G member 2 (ABCG2), a major urate transporter in the kidney and intestine, are the major causes of hyperuricemia and gout.A recent studies found that ABCG2 is also a major transporter of uremic toxins and ABCG2 dysfunctional variants are risk genetics factors for mortality in hemodialysis patients.Objectives:The aim of the present study was to investigate the frequencies of ABCG2 variants in cohort of hemodialysis patients.Methods:In this retrospective one centre study 167 patients (age=79,8±11,03;female=74) with end-stage CKD (pre-dialysis n=86; dialysis n=79) were collected. Peak urate levels were 456,3±113,6 μmol/l in pre-dialysis and 572,93±114,56 μmol/l in dialysis. ABCG2 coding regions were analyzed from genomic DNA, as we described previously (1). The reference sequence was defined as version ENST00000237612.7, the reference protein sequence was defined as Q9UNQ0. The chi-square goodness-of-fit test was used to compare minor allele frequencies (MAF), and the log-rank test was used to compare empirical distribution functions.Results:In the CKD cohort, 15 intronic and seven non-synonymous allelic exonic variants were detected: two common (rs2231137/p.V12M; rs2231142/p.Q141K), five ultra rare and/or rare (rs142634180/p.R45Q, rs759726272/p.M131I, rs140207606/p.R236X, rs138606116/p.G354R, rs138892154/p.A607V), and one novel (p.E344D). Common variant p.V12M, previously reported as protective allele, was detected in 12 heterozygous patients with. The p.Q141K variant, detected in the homozygous stage in three patients and in 31 patients as heterozygous variant. Heterozygous rare variants p.R45Q, p. G354R, p.A607V, and novel variant p.E344D were detected in one heterozygous patient each; heterozygous p.M131I and p.R236X in two patients.Conclusion:In this study, significantly higher frequency of dysfunctional ABCG2 variants, common and rare, in comparison with common European population, were identified. On the other hand, the frequency of probably protective allele variant p.V12M was significantly lower in CKD cohort (MAF in our cohort = 0,036/MAF in the European population = 0,061). Further analysis of ABCG2 association with CKD events via ABCG2 inflammation role is necessary. In conclusions, our finding of one novel, five rare and two common non-synonymous ABCG2 allelic variants in a sample of 167 CKD patients suggests that the ABCG2 variants should be considered a risk factor for CKD.References:[1]Toyoda Y, et al. Functional Characterization of Clinically-Relevant Rare Variants in ABCG2 Identified in a Gout and Hyperuricemia Cohort. Cells. 2019;18;8(4).Acknowledgements:This study was supported by the grant from the Czech Republic Ministry of Health RVO 00023728.Disclosure of Interests:None declared

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THU0404 INFLUENCE OF URATE TRANSPORTOSOME FOR HYPERURICEMIA AND GOUT

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.5629 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 439.1-440

Author(s):

B. Stiburkova ◽

K. Pavelcova ◽

J. Bohata ◽

M. Pavlikova ◽

T. Takada ◽

...

Keyword(s):

Uric Acid ◽

Protein Function ◽

Serum Uric Acid Level ◽

Gouty Arthritis ◽

Functional Characterization ◽

Allelic Variants ◽

The Czech Republic ◽

Clinically Significant ◽

Clinical Potential ◽

Abcg2 Gene

Background:Gouty arthritis, caused by a persistent increase in serum uric acid level, can be caused by underexcretion of uric acid by uric transporters; however, the effects of allelic variants of urate transportosome are yet to be fully determined.Objectives:In this study we investigated the effects of 10 genes of urate transportosome in a cohort of patients with primary hyperuricemia and gout.Methods:The cohort consisted of 114 hyperuricemic individuals; 207 gout patients; and 274 normouricemic controls.Results:We identified 39 non-synonymous allelic variants in the 10 genes of urate transportosome in hyperuricemia/gout cohort. For 22 variants, a European MAF <0.0001 is documented. From the total of 39 identified variants we selected 23 variants for functional characterization based on a) finding of a newly identified variant, b) MAF variant was significantly different in the group of patients with hyperuricemia/gout, c) with high probability, in silico predictions showed devastating influence of variant on protein function.Conclusion:Although further analyzes are needed to elucidate the contribution of urate transportosome to urate homeostasis, our results clearly show that ABCG2 transporter analysis has significant clinical potential. Of the identified non-synonymous allelic variants of the urate transportosome, rs2231142 (p.Q141K) in the ABCG2 gene, proved to be the most clinically significant on the age onset (P < 0.0002, Kruskal-Wallis test).References:[1]Toyoda Y, et al. Cells. 2019 Apr 18;8(4). 2. Stiburkova B, et al. Rheumatology (Oxford). 2016 Jan;55(1):191-4. 3. Toyoda Y, et al. Arthritis Res Ther. 2019 Oct 28;21(1):219.Acknowledgments:This study was supported by the grant from the Czech Republic Ministry of Health AZV 15-26693A and RVO 00023728.Disclosure of Interests: :Blanka Stiburkova: None declared, Katerina Pavelcova: None declared, Jana Bohata: None declared, Marketa Pavlikova: None declared, Tappei Takada: None declared, Yu Toyoda: None declared, Lenka Hasikova: None declared, Jakub Zavada Speakers bureau: Abbvie, UCB, Sanofi, Elli-Lilly, Novartis, Zentiva, Accord, Karel Pavelka Consultant of: Abbvie, MSD, BMS, Egis, Roche, UCB, Medac, Pfizer, Biogen, Speakers bureau: Abbvie, MSD, BMS, Egis, Roche, UCB, Medac, Pfizer, Biogen

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Germline Variants of CYBA and TRPM4 Predispose to Familial Colorectal Cancer

10.20944/preprints202112.0354.v1 ◽

2021 ◽

Author(s):

Lizhen Zhu ◽

Beiping Miao ◽

Dagmara Dymerska ◽

Magdalena Kuświk ◽

Elena Bueno-Martínez ◽

...

Keyword(s):

Colorectal Cancer ◽

Stop Codon ◽

Bacterial Colonization ◽

Functional Characterization ◽

Premature Stop Codon ◽

Familial Colorectal Cancer ◽

Intestinal Mucus ◽

Healthy Elderly ◽

Predisposing Genes ◽

Mucus Barrier

Familial colorectal cancer (CRC) is only partially explained by known germline predisposing genes. We performed whole genome sequencing in 15 Polish families of many affected individuals, without mutations in known CRC predisposing genes. We focused on loss-of-function variants and functionally characterized them. We identified a frameshift variant in the CYBA gene (c.246delC) in one family and a splice site variant in the TRPM4 gene (c.25-1 G>T) in another family. While both variants were absent or extremely rare in gene variant databases, we identified four additional Polish familial CRC cases and two healthy elderly individuals with the CYBA variant (odds ratio 2.46, 95% confidence interval 0.48-12.69). Both variants led to a premature stop codon and to a truncated protein. Functional characterization of the variants showed that knockdown of CYBA or TRPM4 depressed generation of reactive oxygen species (ROS) in LS174T and HT-29 cell lines. Knockdown of TRPM4 resulted in decreased MUC2 protein production. CYBA encodes a component in the NADPH oxidase system which generates ROS and controls, e.g., bacterial colonization in the gut. Germline CYBA variants are associated with early onset inflammatory bowel disease, supported with experimental evidence on loss of intestinal mucus barrier function due to ROS deficiency. TRPM4 encodes a calcium-activated ion channel, which in a human colonic cancer cell line controls calcium-mediated secretion of MUC2, a major component of intestinal mucus barrier. We suggest that the gene defects in CYBA and TRPM4 mechanistically involve intestinal barrier integrity through ROS and mucus biology, which converges in chronic bowel inflammation.

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A Novel Splice Site Variant of hOCT-1 and Response to Imatinib.

Blood ◽

10.1182/blood.v120.21.2555.2555 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2555-2555

Author(s):

Jacob Grinfeld ◽

Juan M Alonso-Dominguez ◽

Mary Alikian ◽

Mikel Valgañón ◽

Georgios Nteliopoulos ◽

...

Keyword(s):

Splice Site ◽

Base Pair ◽

Research Funding ◽

Stop Codon ◽

Premature Stop Codon ◽

Rank Test ◽

Mrna Levels ◽

Nucleotide Polymorphisms ◽

The Impact ◽

Base Pair Insertion

Abstract Abstract 2555 Background: The prognosis for patients with chronic myeloid leukaemia (CML) has vastly improved through the use of tyrosine kinase inhibitors (TKIs), such as imatinib. However, a proportion of patients will not respond, or will lose their initial response, and the biological mechanisms underlying this heterogeneity are poorly understood. Human organic cation transporter-1 (hOCT-1 or SLC22A1), the main transporter for imatinib, has been proposed as one determinant. This study set out to assess the prognostic value of hOCT-1 expression and the presence of polymorphisms in the hOCT-1 gene. Methods: hOCT-1 mRNA levels in 153 diagnostic whole blood samples from two different patient cohorts (one from a trial population, and the other from patients treated only at our institution) were measured by RT-qPCR (normalised against two control genes). hOCT-1 exon 7 DNA (and cDNA transcripts) were sequenced in 156 patients, and four cells lines, to identify insertions/deletions or single nucleotide polymorphisms (SNPs). Fragment length analysis using gene-scanner technology was also used to identify patients with insertions/deletions and to correlate genotypes with cDNA transcript lengths. Time to each endpoint (remission/imatinib failure) was compared according to the level of expression for each gene, or according to genotype, using Kaplan-Meier analyses and the log-rank test. Results and Conclusions: No significant differences in outcomes were found when comparing patients with high or low hOCT-1 expression (whether defined using the median or other cut-offs). The 408V>M (g.1222G>A) SNP in hOCT-1 exon 7 was found in 102/156 patients (22 homozygotes and 80 heterozygotes) and was associated in all cases with an eight base-pair insertion at the exon-intron boundary (rs113569197). This insertion was found to create an alternative splice site, leading to the transcription of an additional RNA/cDNA transcript in these patients, the sequence of which contains a premature stop codon soon after the splice site. Additionally, M420del was found in 52 patients (three homozygotes and 49 heterozygotes) and was not found in alleles containing the eight base pair insertion. The six possible combinations of these three alleles (N=no insertion/deletion, 8+=8bp insertion and 3−=3bp deletion) were found to give rise to five possible combinations of RNA transcript lengths, since patients with 8+8+ produce identical transcript lengths to those with 8+N (i.e. the normal transcript and one with an additional 8bp and premature stop). In the trial cohort (n=109), significant differences in time to 10% (p=0.01), 1% (p=0.0002) and 0.1% (p=0.0003) molecular responses (by the international scale) and time to imatinib failure (p=0.02) were seen when patients with 8+8+/8+N were compared to those with the remaining four genotypes (NN/N3−/3−8+/3−3−). However, this association was not replicated in the internal cohort, which was smaller (n=47) and more heterogeneous in terms of baseline characteristics and management. These findings may explain discrepancies in the results of previous studies that have examined the association between hOCT-1 expression and outcome, since a number used primer/probe sets that would be affected by the presence/absence of these polymorphisms. Our results suggest that while hOCT-1 expression is not a determinant of response, alterations in splice sites or amino acid sequence due to insertions/deletions may be. Further work is required to clarify the impact of these polymorphisms on hOCT-1 protein levels, function and responses to imatinib. Disclosures: White: Novartis Oncology: Honoraria, Research Funding; BMS: Research Funding; CSL: Research Funding. Marin:Novartis: Research Funding; BMS: Research Funding. Apperley:Novartis, Bristol Myers-Squibb, and ARIAD: Honoraria, Research Funding. Goldman:Novartis, Bristol Myers-Squibb, and Amgen: Honoraria.

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Functional Characterization of Clinically-Relevant Rare Variants in ABCG2 Identified in a Gout and Hyperuricemia Cohort

Cells ◽

10.3390/cells8040363 ◽

2019 ◽

Vol 8 (4) ◽

pp. 363 ◽

Cited By ~ 14

Author(s):

Toyoda ◽

Mančíková ◽

Krylov ◽

Morimoto ◽

Pavelcová ◽

...

Keyword(s):

Genetic Risk ◽

Rare Variants ◽

Direct Sequencing ◽

Functional Characterization ◽

Cellular Proteins ◽

European Descent ◽

Primary Gout ◽

Abcg2 Protein ◽

Functional Analyses

ATP-binding cassette subfamily G member 2 (ABCG2) is a physiologically important urate transporter. Accumulating evidence demonstrates that congenital dysfunction of ABCG2 is an important genetic risk factor in gout and hyperuricemia; recent studies suggest the clinical significance of both common and rare variants of ABCG2. However, the effects of rare variants of ABCG2 on the risk of such diseases are not fully understood. Here, using a cohort of 250 Czech individuals of European descent (68 primary hyperuricemia patients and 182 primary gout patients), we examined exonic non-synonymous variants of ABCG2. Based on the results of direct sequencing and database information, we experimentally characterized nine rare variants of ABCG2: R147W (rs372192400), T153M (rs753759474), F373C (rs752626614), T421A (rs199854112), T434M (rs769734146), S476P (not annotated), S572R (rs200894058), D620N (rs34783571), and a three-base deletion K360del (rs750972998). Functional analyses of these rare variants revealed a deficiency in the plasma membrane localization of R147W and S572R, lower levels of cellular proteins of T153M and F373C, and null urate uptake function of T434M and S476P. Accordingly, we newly identified six rare variants of ABCG2 that showed lower or null function. Our findings contribute to deepening the understanding of ABCG2-related gout/hyperuricemia risk and the biochemical characteristics of the ABCG2 protein.

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Association Analysis and Meta-Analysis of Multi-allelic Variants for Large Scale Sequence Data

10.1101/197913 ◽

2017 ◽

Author(s):

Xiaowei Zhan ◽

Sai Chen ◽

Yu Jiang ◽

Mengzhen Liu ◽

William G. Iacono ◽

...

Keyword(s):

Large Scale ◽

Rare Variants ◽

Sequence Data ◽

Meta Analysis ◽

Joint Modeling ◽

Allelic Variants ◽

Association Analyses ◽

Link Type ◽

Gene Level ◽

The Impact

AbstractMotivation:There is great interest to understand the impact of rare variants in human diseases using large sequence datasets. In deep sequences datasets of >10,000 samples, ∼10% of the variant sites are observed to be multi-allelic. Many of the multi-allelic variants have been shown to be functional and disease relevant. Proper analysis of multi-allelic variants is critical to the success of a sequencing study, but existing methods do not properly handle multi-allelic variants and can produce highly misleading association results.Results:We propose novel methods to encode multi-allelic sites, conduct single variant and gene-level association analyses, and perform meta-analysis for multi-allelic variants. We evaluated these methods through extensive simulations and the study of a large meta-analysis of ∼18,000 samples on the cigarettes-per-day phenotype. We showed that our joint modeling approach provided an unbiased estimate of genetic effects, greatly improved the power of single variant association tests, and enhanced gene-level tests over existing approaches.Availability:Software packages implementing these methods are available at (https://github.com/zhanxw/rvtestshttp://genome.sph.umich.edu/wiki/RareMETAL).Contact:[email protected]; [email protected]

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Integrated Genomics Analysis Identifies Recessive Ciliopathy Mutations in Primary Endocardial Fibroelastosis: a Rare Neonatal Cardiomyopathy

10.21203/rs.3.rs-327918/v1 ◽

2021 ◽

Author(s):

Yan Zhao ◽

Wang Lee-Kai ◽

Ascia Eskin ◽

Xuedong Kang ◽

Viviana M. Fajardo ◽

...

Keyword(s):

Stop Codon ◽

Novel Mutation ◽

Premature Stop Codon ◽

Genomic Analysis ◽

Dermal Fibroblasts ◽

Cellular Migration ◽

Endocardial Fibroelastosis ◽

Rna Seq ◽

Mesenchymal Transition ◽

The Impact

Abstract Among neonatal cardiomyopathies, primary endocardial fibroelastosis (pEFE) remains a mysterious disease of the endomyocardium, affecting 1/5000 live births and accounting for 25% of the entire pediatric dilated cardiomyopathy with a devastating course and grave prognosis. To investigate the potential genetic cause that underlies pEFE, we performed integrative genomic analysis, using whole exome sequencing (WES) and RNA-seq in a female infant with confirmed pathological diagnosis of pEFE. Within regions of homozygosity in the proband genome, WES analysis revealed novel parent-transmitted homozygous mutations affecting three genes with known roles in cilia assembly or function. Among them, a novel homozygous variant [c.1938delA] of uncertain significance in ALMS1 was prioritized for functional genomic and mechanistic analysis. Loss of function mutations of ALMS1 have been implicated in Alstrom syndrome (AS) [OMIM 203800], a rare recessive ciliopathy that has been associated with cardiomyopathy. The variant of interest results in a frameshift introducing a premature stop codon. RNA-seq of the proband’s dermal fibroblasts confirmed the impact of novel ALMS1 variant on RNA-seq reads and revealed dysregulated cellular signaling and function, including the induction of epithelial mesenchymal transition (EMT) and activation of TGFβ signaling. ALMS1 loss enhanced cellular migration in patient fibroblasts as well as neonatal cardiac fibroblasts, while ALMS1-depleted cardiomyocytes exhibited enhanced proliferation activity. Herein we present the unique pathological features of pEFE compared to DCM and utilize integrated genomic analysis to elucidate the molecular impact of a novel mutation in ALMS1 gene in an AS case. Our report provides insights into pEFE etiology and suggests, for the first time to our knowledge, ciliopathy as a potential underlying mechanism for this poorly understood and incurable form of neonatal cardiomyopathy.

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Review Article: URIC ACID HOMEOSTASIS AND DISTURBANCES

Folia Medica Indonesiana ◽

10.20473/fmi.v53i4.7164 ◽

2017 ◽

Vol 53 (4) ◽

pp. 292 ◽

Cited By ~ 1

Author(s):

Ferry Fredy Karwur ◽

Dwi Rahayu Pujiastuti

Keyword(s):

Uric Acid ◽

Renal Excretion ◽

The Body ◽

Uric Acid Concentration ◽

Dietary Consumption ◽

History Of Disease ◽

Blood Uric Acid ◽

History Of ◽

The Impact ◽

Abcg2 Gene

This review examined the homeostasis of uric acid in human body and analyzed recent studies of the affecting major variables. Normal uric acid concentration in male is 3.5-7.2 mg/dL and in female is 2.6-6 mg/dL. Daily turnover of normal uric acid ranges from 498-1392 mg/day, miscible pool is 767-1650 mg, reabsorption is 8064 mg/day, renal excretion is 262-620 mg/day and intestine 186-313 mg/day. The dynamics of uric acid is influenced by factors of food, drink, age, history of disease, and genetic. High purine dietary consumption increases blood uric acid by 1-2 mg/dL, 213-290 g/day fructose drinks increases 0.52-1.7 mg/dL, 1.5 g/kgBW sucrose increases 0.61 mg/dL, and 10-20 ml/kgBW beer increases 0.50-0.92 mg/dL. The ABCG2 gene plays a role in bringing uric acid out of the body by 114.31-162.73 mg/dL, SLC2A9 of 5.43-20.17 mg/dL, and SLC22A12 of 5.77-6.71 mg/dL. The data described the homeostasis of uric acid and the magnitude of the impact of environmental (consumption of food, beverages, and lifestyle) and genetic factors. Understanding uric acid homeostasis and its disturbances is important in managing diseases as a consequence of hyperuricemia and hypouryscemia

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Expression of Sequence Variants Identified in Type 1 VWD Subjects in the Zimmerman Program Study Reveals Defects in VWF Secretion and Multimerization

Blood ◽

10.1182/blood.v128.22.1398.1398 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1398-1398

Author(s):

Azza Abdelaal ◽

David Jakab ◽

Pamela A Christopherson ◽

Robert R Montgomery ◽

Sandra L Haberichter

Keyword(s):

Conflicts Of Interest ◽

Stop Codon ◽

Premature Stop Codon ◽

Von Willebrand Disease ◽

Sequence Variants ◽

Cell Lysate ◽

Expression Studies ◽

Von Willebrand ◽

The Impact

Abstract von Willebrand Disease (VWD) is the most prevalent inherited bleeding disorder. Type 1 is the most common form of VWD and results in a partial quantitative deficiency of von Willebrand Factor (VWF). The mechanisms underlying type 1 VWD are still not very well understood although reduced VWF secretion and increased VWF clearance have been implicated in causing VWD. We aimed to characterize novel sequence variants (SV) identified in the VWF gene in type 1 VWD patients recruited through the Zimmerman Program for the Molecular and Clinical Biology of VWD in order to define the underlying mechanism and explore if SV in a particular domain are mechanistically similar. We utilized homozygous expression in human embryonic kidney cells (HEK-293T) to study the effect of VWF SV on VWF secretion, intracellular retention, multimerization, and function. Novel SV have been identified throughout the entire VWF protein. We introduced the following variants into a VWF-mycHis plasmid vector: V86M, W199X, C524Y, M947V, R960P, G994D, C996W, R1204W, Q1353X, E1660X, R1763Q, C2199Y, Q2256H, T2282I, P2524L, A2569E, C2693F, C2701Y, and C2754Y. Sequence variants were confirmed by Sanger sequencing. Variant VWF cDNA is transfected homozygously into HEK-293T cells. The supernatants and cell lysates from 3 independent transfections are collected and analyzed by ELISA for VWF:Ag and VWF binding to collagen type III (VWF:CB). VWF multimer structure is analyzed by SDS-agarose gel electrophoresis and western blotting. The genotype-phenotype patient data is correlated with the data from expression studies to explore a model to predict the impact of the SV on the VWD phenotype. Variants V86M, M947V, R1204W, R1763Q, Q2256H, T2282I, P2524L, A2569E, and C2693F demonstrated secretion comparable to that of wild type (WT)-VWF. In contrast, VWF variants R960P and C2701Y showed reduced VWF secretion (<50% of WT) with increased VWF in the cell lysate. VWF variants W199X, C524Y, G994D, C996W, Q1353X, E1660X, C2199Y, and C2754Y demonstrated a complete absence of secreted VWF. Not unexpectedly, homozygous expression of stop codon variants W199X, Q1353X, and E1660X demonstrated no VWF in the cell lysate. However, non-secreted VWF variants C524Y, G994D, C996W, C2199Y, and C2754Y showed intracellular retention with detectable VWF in the cell lysate. SV occurring at cysteine residues (C524Y, C996W, C2199Y, C2701Y, and C2754Y) all had reduced secretion and increased intracellular retention, consistent with altered conformation leading to increased intracellular chaperone interaction and proteasomal degradation. VWF binding to collagen is dependent on the presence of high molecular weight multimers (HMWM). VWF:CB/VWF:Ag is used to predict multimer structure with VWF:CB/VWF:Ag < 0.7 indicative of loss of the HMWM. VWF variants V86M, M947V, R1763Q, Q2256H, P2524L, C2701Y had VWF:CB/VWF:Ag ≥ 0.7 consistent with normal multimer structure, while variants R960P, R1204W, T2282I, A2569E, and C2693F had VWF:CB/VWF:Ag < 0.7 indicating abnormal multimer structure. 47.3% of the 19 VWF variants studied had normal VWF secretion, 10.5% had reduced secretion with increased intracellular retention, and 26.3% revealed absent secretion with intracellular retention. Variants with a premature stop codon did not synthesize VWF at all. Some SV had normal secretion and multimerization (V86M, M947V, R1204W, R1763Q, Q2256H, T2282I, P2524L, A2569E, and C2693F) implying that the VWD phenotype in these patients results from yet unidentified mechanisms and may not be associated with these SV. Reduced plasma survival is unlikely as these patients had normal VWFpp/VWF:Ag level consistent with normal VWF clearance. Among the VWF variants with normal or decreased secretion, 45.4% had reduced VWF:CB/VWF:Ag consistent with abnormal multimer structure. Heterozygous expression, as observed in the patient, is expected to normalize these multimerization defects. The decreased or absent secretion observed for 52.7% of the variants studied correlates with the patient phenotype, indicating reduced secretion is the mechanism underlying these patients' type 1 VWD phenotype. No domain-specific correlation of VWF secretion or multimer abnormality was observed. In summary, VWF expression studies confirmed the causative nature of many, but not all of the novel sequence variants identified in type 1 VWD subjects in the Zimmerman Program. Disclosures No relevant conflicts of interest to declare.

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Association Analysis and Meta-Analysis of Multi-Allelic Variants for Large-Scale Sequence Data

Genes ◽

10.3390/genes11050586 ◽

2020 ◽

Vol 11 (5) ◽

pp. 586

Author(s):

Yu Jiang ◽

Sai Chen ◽

Xingyan Wang ◽

Mengzhen Liu ◽

William G. Iacono ◽

...

Keyword(s):

Large Scale ◽

Rare Variants ◽

Sequence Data ◽

Meta Analysis ◽

Joint Modeling ◽

Allelic Variants ◽

Association Analyses ◽

Software Packages ◽

Gene Level ◽

The Impact

There is great interest in understanding the impact of rare variants in human diseases using large sequence datasets. In deep sequence datasets of >10,000 samples, ~10% of the variant sites are observed to be multi-allelic. Many of the multi-allelic variants have been shown to be functional and disease-relevant. Proper analysis of multi-allelic variants is critical to the success of a sequencing study, but existing methods do not properly handle multi-allelic variants and can produce highly misleading association results. We discuss practical issues and methods to encode multi-allelic sites, conduct single-variant and gene-level association analyses, and perform meta-analysis for multi-allelic variants. We evaluated these methods through extensive simulations and the study of a large meta-analysis of ~18,000 samples on the cigarettes-per-day phenotype. We showed that our joint modeling approach provided an unbiased estimate of genetic effects, greatly improved the power of single-variant association tests among methods that can properly estimate allele effects, and enhanced gene-level tests over existing approaches. Software packages implementing these methods are available online.

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