Dysfunctional Variants Research Articles

Stress and the CRH System, Norepinephrine, Depression, and Type 2 Diabetes.

Major depressive disorder (MDD) increases the risk of type 2 diabetes (T2D) by 60% in untreated patients, and hypercortisolism is common in MDD as well as in some patients with T2D. Patients with MDD, despite hypercortisolism, show inappropriately normal levels of corticotropin-releasing hormone (CRH) and plasma adrenocorticotropin (ACTH) in the cerebrospinal fluid, which might implicate impaired negative feedback. Also, a positive feedback loop of the CRH-norepinephrine (NE)-CRH system may be involved in the hypercortisolism of MDD and T2D. Dysfunctional CRH receptor 1 (CRHR1) and CRH receptor 2 (CRHR2), both of which are involved in glucose regulation, may explain hypercortisolism in MDD and T2D, at least in a subgroup of patients. CRHR1 increases glucose-stimulated insulin secretion. Dysfunctional CRHR1 variants can cause hypercortisolism, leading to serotonin dysfunction and depression, which can contribute to hyperglycemia, insulin resistance, and increased visceral fat, all of which are characteristics of T2D. CRHR2 is implicated in glucose homeostasis through the regulation of insulin secretion and gastrointestinal functions, and it stimulates insulin sensitivity at the muscular level. A few studies show a correlation of the CRHR2 gene with depressive disorders. Based on our own research, we have found a linkage and association (i.e., linkage disequilibrium [LD]) of the genes CRHR1 and CRHR2 with MDD and T2D in families with T2D. The correlation of CRHR1 and CRHR2 with MDD appears stronger than that with T2D, and per our hypothesis, MDD may precede the onset of T2D. According to the findings of our analysis, CRHR1 and CRHR2 variants could modify the response to prolonged chronic stress and contribute to high levels of cortisol, increasing the risk of developing MDD, T2D, and the comorbidity MDD-T2D. We report here the potential links of the CRH system, NE, and their roles in MDD and T2D.

Functional validation of EIF2AK4 (GCN2) missense variants associated with pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a disorder with a large genetic component. Biallelic mutations of EIF2AK4, which encodes the kinase GCN2, are causal in two ultra-rare subtypes of PAH, pulmonary veno-occlusive disease and pulmonary capillary haemangiomatosis. EIF2AK4 variants of unknown significance have also been identified in patients with classical PAH, though their relationship to disease remains unclear. To provide patients with diagnostic information and enable family testing, the functional consequences of such rare variants must be determined, but existing computational methods are imperfect. We applied a suite of bioinformatic and experimental approaches to sixteen EIF2AK4 variants that had been identified in patients. By experimentally testing the functional integrity of the integrated stress response (ISR) downstream of GCN2, we determined that existing computational tools have insufficient sensitivity to reliably predict impaired kinase function. We determined experimentally that several EIF2AK4 variants identified in patients with classical PAH had preserved function and are therefore likely to be non-pathogenic. The dysfunctional variants of GCN2 that we identified could be subclassified into three groups: misfolded, kinase-dead, and hypomorphic. Intriguingly, members of the hypomorphic group were amenable to paradoxical activation by a type-1½ GCN2 kinase inhibitor. This experiment approach may aid in the clinical stratification of EIF2AK4 variants and potentially identify hypomorophic alleles receptive to pharmacological activation.

Probing of the reactive center loop region of alpha-1-antitrypsin by mutagenesis predicts new type-2 dysfunctional variants.

Lung disease in alpha-1-antitrypsin deficiency (AATD) mainly results from insufficient control of the serine proteases neutrophil elastase (NE) and proteinase-3 due to reduced plasma levels of alpha-1-antitrypsin (AAT) variants. Mutations in the specificity-determining reactive center loop (RCL) of AAT would be predicted to minimally affect protein folding and secretion by hepatocytes but can impair anti-protease activity or alter the target protease. These properly secreted but dysfunctional 'type-2' variants would not be identified by common diagnostic protocols that are predicated on a reduction in circulating AAT. This has potential clinical relevance: in addition to the dysfunctional Pittsburgh and Iners variants reported previously, several uncharacterized RCL variants are present in genome variation databases. To prospectively evaluate the impact of RCL variations on secretion and anti-protease activity, here we performed a systematic screening of amino acid substitutions occurring at the AAT-NE interface. Twenty-three AAT variants that can result from single nucleotide polymorphisms in this region, including 11 present in sequence variation databases, were expressed in a mammalian cell model. All demonstrated unaltered protein folding and secretion. However, when their ability to form stable complexes with NE was evaluated by western blot, enzymatic assays, and a novel ELISA developed to quantify AAT-NE complexes, substrate-like and NE-binding deficient dysfunctional variants were identified. This emphasizes the ability of the RCL to accommodate inactivating substitutions without impacting the integrity of the native molecule and demonstrates that this class of molecule violates a generally accepted paradigm that equates circulating levels with functional protection of lung tissue.

A Single nucleotide polymorphism in the ALDH2 gene modifies the risk of esophageal squamous cell carcinoma in BRCA2 p.K3326* carriers.

Esophageal squamous cell carcinoma (ESCC) has a very high incidence rate in northeastern Iran. Our team previously reported the BReast CAncer gene 2 (BRCA2) p.K3326* mutation as a moderately penetrant ESCC susceptibility variant in northern Iran (odds ratio (OR) = 3.64, 95% confidence interval (CI) = 1.74-7.59, P = 0.0003). Recently, it has been reported that aldehydes can induce BRCA2 haploinsufficiency in cells with a heterozygous pathogenic BRCA2 mutation and predispose them to carcinogenic effects. Based on this observation, we speculate that dysfunctional variants in Aldehyde Dehydrogenase 2 Family Member (ALDH2) may result in aldehyde-induced BRCA2 haploinsufficiency and increase cancer risk in BRCA2 mutation carriers. In support of this hypothesis, our team recently reported the breast cancer risk modifying effect of an ALDH2 common polymorphism, rs10744777, among Polish carriers of the BRCA2 p.K3326* mutation. In the current case-control study, we aimed to investigate the ESCC risk modifying effect of this ALDH2 polymorphism among BRCA2 p.K3326* mutation carriers. We assessed the interaction between the ALDH2 rs10744777 polymorphism and BRCA2 p.K3326* mutation in ESCC risk by genotyping this ALDH2 variant in the germline DNA of 746 ESCC cases and 1,373 controls from northern Iran who were previously genotyped for the BRCA2 p.K3326* mutation. Among a total of 464 individuals with TT genotype of the ALDH2 rs10744777 polymorphism, which is associated with lower ALDH2 expression, we found 9 of 164 cases versus 3 of 300 controls who carried the BRCA2 p.K3326* variant (OR = 5.66, 95% CI = 1.22-26.2, P = 0.018). This finding supports our hypothesis that the ALDH2-rs10744777 TT genotype may be a significant risk modifier of ESCC in individuals with a BRCA2 p.K3326* mutation.

Novel Loss of Function KCNA5 Variants in Pulmonary Arterial Hypertension.

Reduced expression and/or activity of Kv1.5 channels (encoded by KCNA5) is a common hallmark in human or experimental pulmonary arterial hypertension (PAH). Likewise, genetic variants in KCNA5 have been found in PAH patients, but their functional consequences and potential impact on the disease are largely unknown. Herein, we aimed to characterize the functional consequences of 7 KCNA5 variants found in a cohort of PAH patients. Potassium currents were recorded by patch-clamp technique in HEK293 cells transfected with WT or mutant Kv1.5 cDNA. Flow cytometry, western blot and confocal microscopy techniques were used for measuring protein expression and cell apoptosis in HEK293 and human pulmonary artery smooth muscle cells (hPASMC). KCNA5 variants found in PAH patients (namely, p.Arg184Pro and p.Gly384Arg) resulted in a clear loss of potassium channel function as assessed by electrophysiological and molecular modelling analyses. The p.Arg184Pro variant also resulted in a pronounced reduction of Kv1.5 expression. Transfection with p.Arg184Pro or p.Gly384Arg variants decreased apoptosis of hPASMCs compared with the WT, demonstrating that KCNA5 dysfunction in both variants affects cell viability. Thus, in addition to affecting channel activity, both variants were associated with impaired apoptosis, a crucial process linked to the disease. The estimated prevalence of dysfunctional KCNA5 variants in the PAH population analyzed was around 1 %. Our data indicate that some KCNA5 variants found PAH patients have critical consequences for channel function supporting the idea that KCNA5 pathogenic variants may be a causative or contributing factor for PAH.

Evaluation of ABCG2-mediated extra-renal urate excretion in hemodialysis patients

Two-thirds of urate is excreted via the renal pathway and the remaining one-third via the extra-renal pathway, the latter mainly via the intestine in healthy individuals. ABCG2, a urate exporter, is expressed in various tissues including the kidney and intestine, and its dysfunction leads to hyperuricemia and gout. ABCG2 is regarded as being responsible for most of the extra-renal urate excretion. However, the extra-renal urate excretion capacity via ABCG2 remains undefined in end-stage kidney diseases. Therefore, we evaluated the capacity of extra-renal ABCG2 using 123 anuric hemodialysis patients whose urate excretion depended on only the extra-renal pathway. ABCG2 function in each participant was estimated based on ABCG2 dysfunctional variants. We computed the uric acid pool (PoolUA) from bodyweight and serum urate level (SUA) using previously reported radio-isotopic data, and we analyzed the association between ABCG2 function and the PoolUA. SUA and PoolUA increased significantly with ABCG2 dysfunction, and extra-renal ABCG2 could excrete up to approximately 60% of the daily uric acid turnover in hemodialysis patients. Our findings indicate that the extra-renal urate excretion capacity can expand with renal function decline and highlight that the extra-renal pathway is particularly important in the uric acid homeostasis for patients with renal dysfunction.

Specific expression of alternatively spliced genes in the turkey (Meleagris gallopavo) reproductive tract revealed their function in spermatogenesis and post-testicular sperm maturation

The tissue-specific profile of alternatively spliced genes (ASGs) and their involvement in reproduction processes characteristic of turkey testis, epididymis, and ductus deferens were investigated for the first time in birds. Deep sequencing of male turkey reproductive tissue RNA samples (n=6) was performed using Illumina RNA-Seq with 2 independent methods, rMATs and SUPPA2, for differential alternative splicing (DAS) event prediction. The expression of selected ASGs was validated using quantitative real-time reverse transcriptase-polymerase chain reaction. The testis was found to be the site of the highest number of posttranscriptional splicing events within the reproductive tract, and skipping exons were the most frequently occurring class of alternative splicing (AS) among the reproductive tract. Statistical analysis revealed 86, 229, and 6 DAS events in the testis/epididymis, testis/ductus deferens, and epididymis/ductus deferens comparison, respectively. Alternative splicing was found to be a mechanism of gene expression regulation within the turkey reproduction tract. In testis, modification was observed for spermatogenesis specific genes; the changes in 5' UTR could act as regulator of MEIG1 expression (a player during spermatocytes meiosis), and modification of 3' UTR led to diversification of CREM mRNA (modulator of gene expression related to the structuring of mature spermatozoa). Sperm tail formation can be regulated by changes in the 5' UTR of testicular SLC9A3R1 and gene silencing by producing dysfunctional variants of ODF2 in the testis and ATP1B3 in the epididymis. Predicted differentially ASGs in the turkey reproductive tract seem to be involved in the regulation of spermatogenesis, including acrosome formation and sperm tail formation and binding of sperm to the zona pellucida. Several ASGs were classified as cilia by actin and microtubule cytoskeleton organization. Such genes may play a role in the organization of sperm flagellum and post-testicular motility development. To our knowledge, this is the first functional investigation of alternatively spliced genes associated with tissue-specific processes in the turkey reproductive tract.

Effects of the T337M and G391V disease-related variants on human phosphoglucomutase 1: structural disruptions large and small.

Phosphoglucomutase 1 (PGM1) plays a central role in glucose homeostasis in human cells. Missense variants of this enzyme cause an inborn error of metabolism, which is categorized as a congenital disorder of glycosylation. Here, two disease-related variants of PGM1, T337M and G391V, which are both located in domain 3 of the four-domain protein, were characterized via X-ray crystallography and biochemical assays. The studies show multiple impacts resulting from these dysfunctional variants, including both short- and long-range structural perturbations. In the T337M variant these are limited to a small shift in an active-site loop, consistent with reduced enzyme activity. In contrast, theG391V variant produces a cascade of structural perturbations, including displacement of both the catalytic phosphoserine and metal-binding loops. This work reinforces several themes that were found in prior studies of dysfunctional PGM1 variants, including increased structural flexibility and the outsized impacts of mutations affecting interdomain interfaces. The molecular mechanisms of PGM1 variants have implications for newly described inherited disorders of related enzymes.

Scinderin promotes fusion of electron transport chain dysfunctional muscle stem cells with myofibers

Muscle stem cells (MuSCs) experience age-associated declines in number and function, accompanied by mitochondrial electron transport chain (ETC) dysfunction and increased reactive oxygen species (ROS). The source of these changes, and how MuSCs respond to mitochondrial dysfunction, are unknown. We report here that in response to mitochondrial ROS, murine MuSCs directly fuse with neighboring myofibers; this phenomenon removes ETC-dysfunctional MuSCs from the stem cell compartment. MuSC–myofiber fusion is dependent on the induction of Scinderin, which promotes formation of actin-dependent protrusions required for membrane fusion. During aging, we find that the declining MuSC population accumulates mutations in the mitochondrial genome but selects against dysfunctional variants. In the absence of clearance by Scinderin, the decline in MuSC numbers during aging is repressed; however, ETC-dysfunctional MuSCs are retained and can regenerate dysfunctional myofibers. We propose a model in which ETC-dysfunctional MuSCs are removed from the stem cell compartment by fusing with differentiated tissue.

Renal Hypouricemia 1: Rare Disorder as Common Disease in Eastern Slovakia Roma Population.

Renal hypouricemia (RHUC) is caused by an inherited defect in the main reabsorption system of uric acid, SLC22A12 (URAT1) and SLC2A9 (GLUT9). RHUC is characterized by a decreased serum uric acid concentration and an increase in its excreted fraction. Patients suffer from hypouricemia, hyperuricosuria, urolithiasis, and even acute kidney injury. We report clinical, biochemical, and genetic findings in a cohort recruited from the Košice region of Slovakia consisting of 27 subjects with hypouricemia and relatives from 11 families, 10 of whom were of Roma ethnicity. We amplified, directly sequenced, and analyzed all coding regions and exon–intron boundaries of the SLC22A12 and SLC2A9 genes. Sequence analysis identified dysfunctional variants c.1245_1253del and c.1400C>T in the SLC22A12 gene, but no other causal allelic variants were found. One heterozygote and one homozygote for c.1245_1253del, nine heterozygotes and one homozygote for c.1400C>T, and two compound heterozygotes for c.1400C>T and c.1245_1253del were found in a total of 14 subjects. Our result confirms the prevalence of dysfunctional URAT1 variants in Roma subjects based on analyses in Slovak, Czech, and Spanish cohorts, and for the first time in a Macedonian Roma cohort. Although RHUC1 is a rare inherited disease, the frequency of URAT1-associated variants indicates that this disease is underdiagnosed. Our findings illustrate that there are common dysfunctional URAT1 allelic variants in the general Roma population that should be routinely considered in clinical practice as part of the diagnosis of Roma patients with hypouricemia and hyperuricosuria exhibiting clinical signs such as urolithiasis, nephrolithiasis, and acute kidney injury.

Substantial anti-gout effect conferred by common and rare dysfunctional variants of URAT1/SLC22A12.

ObjectivesGout, caused by chronic elevation of serum uric acid levels, is the commonest form of inflammatory arthritis. The causative effect of common and rare variants of ATP-binding cassette transporter G2 (ABCG2/BCRP) on gout risk has been studied, but little attention has been paid to the effect of common (rs121907892, p.W258X) and rare variants of urate transporter 1 (URAT1/SLC22A12) on gout, despite dysfunctional variants of URAT1 having been identified as pathophysiological causes of renal hypouricaemia.MethodsTo address this important but overlooked issue, we investigated the effects of these URAT1 variants on gout susceptibility, using targeted exon sequencing on 480 clinically defined gout cases and 480 controls of Japanese males in combination with a series of functional analyses of newly identified URAT1 variants.ResultsOur results show that both common and rare dysfunctional variants of URAT1 markedly decrease the risk of gout (OR 0.0338, reciprocal OR 29.6, P = 7.66 × 10−8). Interestingly, we also found that the URAT1-related protective effect on gout eclipsed the ABCG2-related causative effect (OR 2.30–3.32). Our findings reveal only one dysfunctional variant of URAT1 to have a substantial anti-gout effect, even in the presence of causative variants of ABCG2, a ‘gout gene’.ConclusionOur findings provide a better understanding of gout/hyperuricaemia and its aetiology that is highly relevant to personalized health care. The substantial anti-gout effect of common and rare variants of URAT1 identified in the present study support the genetic concept of a ‘Common Disease, Multiple Common and Rare Variant’ model.

Detection of Rare Germline Variants in the Genomes of Patients with B-Cell Neoplasms.

Simple SummaryThe global importance of rare variants in tumorigenesis has been addressed by some pan-cancer analysis, revealing significant enrichments in protein-truncating variants affecting genes such as ATM, BRCA1/2, BRIP1, and MSH6. Germline variants can influence treatment response and contribute to the development of treatment-related second neoplasms, especially in childhood leukemia. We aimed to analyze the genomes of patients with B-cell lymphoproliferative disorders for the discovery of genes enriched in rare pathogenic variants. We discovered a significant enrichment for two genes in germline rare and dysfunctional variants. Additionally, we detected rare and likely pathogenic variants associated with disease prognosis and potential druggability, indicating a relevant role of these events in the variability of cancer phenotypes.There is growing evidence indicating the implication of germline variation in cancer predisposition and prognostication. Here, we describe an analysis of likely disruptive rare variants across the genomes of 726 patients with B-cell lymphoid neoplasms. We discovered a significant enrichment for two genes in rare dysfunctional variants, both of which participate in the regulation of oxidative stress pathways (CHMP6 and GSTA4). Additionally, we detected 1675 likely disrupting variants in genes associated with cancer, of which 44.75% were novel events and 7.88% were protein-truncating variants. Among these, the most frequently affected genes were ATM, BIRC6, CLTCL1A, and TSC2. Homozygous or germline double-hit variants were detected in 28 cases, and coexisting somatic events were observed in 17 patients, some of which affected key lymphoma drivers such as ATM, KMT2D, and MYC. Finally, we observed that variants in six different genes were independently associated with shorter survival in CLL. Our study results support an important role for rare germline variation in the pathogenesis and prognosis of B-cell lymphoid neoplasms.

Identification of Two Dysfunctional Variants in the ABCG2 Urate Transporter Associated with Pediatric-Onset of Familial Hyperuricemia and Early-Onset Gout.

The ABCG2 gene is a well-established hyperuricemia/gout risk locus encoding a urate transporter that plays a crucial role in renal and intestinal urate excretion. Hitherto, p.Q141K—a common variant of ABCG2 exhibiting approximately one half the cellular function compared to the wild-type—has been reportedly associated with early-onset gout in some populations. However, compared with adult-onset gout, little clinical information is available regarding the association of other uricemia-associated genetic variations with early-onset gout; the latent involvement of ABCG2 in the development of this disease requires further evidence. We describe a representative case of familial pediatric-onset hyperuricemia and early-onset gout associated with a dysfunctional ABCG2, i.e., a clinical history of three generations of one Czech family with biochemical and molecular genetic findings. Hyperuricemia was defined as serum uric acid (SUA) concentrations 420 μmol/L for men or 360 μmol/L for women and children under 15 years on two measurements, performed at least four weeks apart. The proband was a 12-year-old girl of Roma ethnicity, whose SUA concentrations were 397–405 µmol/L. Sequencing analyses focusing on the coding region of ABCG2 identified two rare mutations—c.393G>T (p.M131I) and c.706C>T (p.R236X). Segregation analysis revealed a plausible link between these mutations and hyperuricemia and the gout phenotype in family relatives. Functional studies revealed that p.M131I and p.R236X were functionally deficient and null, respectively. Our findings illustrate why genetic factors affecting ABCG2 function should be routinely considered in clinical practice as part of a hyperuricemia/gout diagnosis, especially in pediatric-onset patients with a strong family history.

Utility of Zebrafish Models of Acquired and Inherited Long QT Syndrome.

Long-QT Syndrome (LQTS) is a cardiac electrical disorder, distinguished by irregular heart rates and sudden death. Accounting for ∼40% of cases, LQTS Type 2 (LQTS2), is caused by defects in the Kv11.1 (hERG) potassium channel that is critical for cardiac repolarization. Drug block of hERG channels or dysfunctional channel variants can result in acquired or inherited LQTS2, respectively, which are typified by delayed repolarization and predisposition to lethal arrhythmia. As such, there is significant interest in clear identification of drugs and channel variants that produce clinically meaningful perturbation of hERG channel function. While toxicological screening of hERG channels, and phenotypic assessment of inherited channel variants in heterologous systems is now commonplace, affordable, efficient, and insightful whole organ models for acquired and inherited LQTS2 are lacking. Recent work has shown that zebrafish provide a viable in vivo or whole organ model of cardiac electrophysiology. Characterization of cardiac ion currents and toxicological screening work in intact embryos, as well as adult whole hearts, has demonstrated the utility of the zebrafish model to contribute to the development of therapeutics that lack hERG-blocking off-target effects. Moreover, forward and reverse genetic approaches show zebrafish as a tractable model in which LQTS2 can be studied. With the development of new tools and technologies, zebrafish lines carrying precise channel variants associated with LQTS2 have recently begun to be generated and explored. In this review, we discuss the present knowledge and questions raised related to the use of zebrafish as models of acquired and inherited LQTS2. We focus discussion, in particular, on developments in precise gene-editing approaches in zebrafish to create whole heart inherited LQTS2 models and evidence that zebrafish hearts can be used to study arrhythmogenicity and to identify potential anti-arrhythmic compounds.

Urate Transporters in the Kidney: What Clinicians Need to Know.

Urate is produced in the liver by the degradation of purines from the diet and nucleotide turnover and excreted by the kidney and gut. The kidney is the major route of urate removal and has a pivotal role in the regulation of urate homeostasis. Approximately 10% of the glomerular filtered urate is excreted in the urine, and the remainder is reabsorbed by the proximal tubule. However, the transport of urate in the proximal tubule is bidirectional: reabsorption and secretion. Thus, an increase in reabsorption or a decrease in secretion may induce hyperuricemia. In contrast, a decrease in reabsorption or an increase in secretion may result in hyperuricosuria. In the proximal tubule, urate reabsorption is mainly mediated by apical URAT1 (SLC22A12) and basolateral GLUT9 (SLC2A9) transporter. OAT4 (SLC22A11) also acts in urate reabsorption in the apical membrane, and its polymorphism is associated with the risk of hyperuricemia. Renal hypouricemia is caused by SLC22A12 or SLC2A9 loss-of-function mutations, and it may be complicated by exercise-induced acute kidney injury. URAT1 and GLUT9 are also drug targets for uricosuric agents. Sodium-glucose cotransporter inhibitors may induce hyperuricosuria by inhibiting GLUT9b located in the apical plasma membrane. Urate secretion is mediated by basolateral OAT1 (SLC22A6) and OAT3 (SLC22A8) and apical ATP-binding cassette super-family G member 2 (ABCG2), NPT1 (SLC17A1), and NPT4 (SLC17A3) transporter in the proximal tubule. NPT1 and NPT4 may be key players in renal urate secretion in humans, and deletion of SLC22A6 and SLC22A8 in mice leads to decreased urate excretion. Dysfunctional variants of ABCG2 inhibit urate secretion from the gut and kidney and may cause gout. In summary, the net result of urate transport in the proximal tubule is determined by the dominance of transporters between reabsorption (URAT1, OAT4, and GLUT9) and secretion (ABCG2, NPT1, NPT4, OAT1, and OAT3).

P0169DYSFUNCTION OF ABCG2, URATE TRANSPORTER, IS RELATED WITH UROLITHIASIS

Abstract Background and Aims Urolithiasis is one of the rapidly increasing diseases in developed countries. It has been reported that urolithiasis is related with lifestyles, diet, obesity, climate and chronic diseases including hyperuricemia, diabetes mellitus, metabolic syndrome, chronic kidney disease et cetera. Among them, hyperuricemia is the leading cause of urolithiasis. ATP–binding cassette subfamily G member 2 (ABCG2), urate transporter, excretes uric acid in the kidney and the intestinal tract and ABCG2 dysfunction increases a risk of hyperuricemia. In this study, we investigated the estimated ABCG2 function by common two dysfunctional variants, Q126X (rs72552713) and Q141K (rs2231142), in patients with urolithiasis, and we evaluated the relation between urolithiasis and ABCG2 dysfunction. Method One hundred and ninety-seven urolithiasis patients without gout (150 males and 47 females) were enrolled. Q126X totally abolishes ABCG2 function, while Q141K reduces its function by about 50%. Since these two SNPs do not link each other and two SNPs do not exist on same chromosome, these two are regarded as independent risks. Namely, based on the genotype of Q126X and Q141K, it can be divided into three categories: a full functional group, a 75% functional group and a ≦50% functional group. Serum uric acid levels and amount of urinary uric acid were measured. Renal uric acid handling of the patients was classified into renal uric acid underexcretion type or renal uric acid overload type based on uric acid clearance and amount of urinary uric acid. Stone composition was analysed, if it had been possible. Results ABCG2 function of the patients was evaluated as follows: 82 patients, ABCG2 full function (41.6%); 78 patients, 75% function (39.6%); 37 patients, ≦50% function (18.8%). 155 patients were classified into renal uric acid underexcretion type, 13 patients were classified into renal uric acid overload type and 2 patients were classified into normal type (27 patients have not had 24-hour urine biochemical test). Stone composition analysis was performed for 136 patients; calcium oxalate stone was identified in 107 patients and uric acid stone in 29 patients. In this study, 58.4 % of the patients with urolithiasis had some ABCG2 dysfunction as against 49.8 % of the healthy individuals previously reported (p=0.0246). In addition, the ratio of subjects with urolithiasis having 50% or less of ABCG2 function was higher than healthy individuals significantly (OR=1.773, p=0.007). The high ratio of ABCG2 dysfunctional patients with urolithiasis suggested that ABCG2 dysfunction is a risk factor for urolithiasis. In the patients with urolithiasis, renal uric acid underexcretion type accounted for about 90% of the patients. This means that this type accelerated urolithiasis as well as hyperuricosuria. Conclusion ABCG2 dysfunction and renal uric acid underexcretion type were suggested to be a risk factor for urolithiasis.

Deciphering genetic signatures by whole exome sequencing in a case of co-prevalence of severe renal hypouricemia and diabetes with impaired insulin secretion

BackgroundRenal hypouricemia (RHUC) is a hereditary disorder where mutations in SLC22A12 gene and SLC2A9 gene cause RHUC type 1 (RHUC1) and RHUC type 2 (RHUC2), respectively. These genes regulate renal tubular reabsorption of urates while there exist other genes counterbalancing the net excretion of urates including ABCG2 and SLC17A1. Urate metabolism is tightly interconnected with glucose metabolism, and SLC2A9 gene may be involved in insulin secretion from pancreatic β-cells. On the other hand, a myriad of genes are responsible for the impaired insulin secretion independently of urate metabolism.Case presentationWe describe a 67 year-old Japanese man who manifested severe hypouricemia (0.7 mg/dl (3.8–7.0 mg/dl), 41.6 μmol/l (226–416 μmol/l)) and diabetes with impaired insulin secretion. His high urinary fractional excretion of urate (65.5%) and low urinary C-peptide excretion (25.7 μg/day) were compatible with the diagnosis of RHUC and impaired insulin secretion, respectively. Considering the fact that metabolic pathways regulating urates and glucose are closely interconnected, we attempted to delineate the genetic basis of the hypouricemia and the insulin secretion defect observed in this patient using whole exome sequencing. Intriguingly, we found homozygous Trp258* mutations in SLC22A12 gene causing RHUC1 while concurrent mutations reported to be associated with hyperuricemia were also discovered including ABCG2 (Gln141Lys) and SLC17A1 (Thr269Ile). SLC2A9, that also facilitates glucose transport, has been implicated to enhance insulin secretion, however, the non-synonymous mutations found in SLC2A9 gene of this patient were not dysfunctional variants. Therefore, we embarked on a search for causal mutations for his impaired insulin secretion, resulting in identification of multiple mutations in HNF1A gene (MODY3) as well as other genes that play roles in pancreatic β-cells. Among them, the Leu80fs in the homeobox gene NKX6.1 was an unreported mutation.ConclusionWe found a case of RHUC1 carrying mutations in SLC22A12 gene accompanied with compensatory mutations associated with hyperuricemia, representing the first report showing coexistence of the mutations with opposed potential to regulate urate concentrations. On the other hand, independent gene mutations may be responsible for his impaired insulin secretion, which contains novel mutations in key genes in the pancreatic β-cell functions that deserve further scrutiny.

Characterization of a Human ALDH2 Mutant that Causes Alcohol Flushing in Non‐east Asians

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is a ~55 kDa protein responsible for the metabolism of the alcohol derived intermediate, acetaldehyde, in addition to a variety of toxic aldehydes including 4‐hydroxynonenal (4HNE) and acrolein. A single nucleotide polymorphism (SNP) of the ALDH2 gene (rs671) is highly prevalent among East Asians with ~540 million people in the world with the ALDH2 genetic variant (ALDH2*2 or E504K) that causes facial flushing and tachycardia after alcohol consumption due inefficient acetaldehyde metabolism. However, anecdotal reports exist of non‐East Asians who flush after alcohol consumption. Therefore, we hypothesize that other dysfunctional human genetic variants exist, besides ALDH2*2, that cause inefficient acetaldehyde metabolism.To address our hypothesis, after receiving IRB approval, we recruited human volunteers with self‐reported alcohol‐induced facial flushing. Volunteers were genotyped in addition to being subjected to an alcohol challenge (0.25g/kg) where levels of acetaldehyde in the breath were quantified by gas mass spectrometry for the 2 hours after an alcohol challenge. In addition, to support these findings, we carried out site‐directed mutagenesis, expressed, and purified an ALDH2 mutant we identified in humans using a nickel affinity column for histidine tagged protein. We measured enzymatic activity of the ADLH2 mutant using wild type ALDH2 and ALDH2*2 as comparators. All data are presented as mean ± SEM. Statistical analysis was performed by ANOVA and Tukey’s post hoc with significance set at P<0.05.We discovered a novel non‐East Asian ALDH2 mutation (R114W) that also causes alcohol‐induced facial flushing and acetaldehyde accumulation. Acetaldehyde levels measured in human breath showed that a human heterozygous for ALDH2 R114W had ~30% more acetaldehyde accumulation compared to homozygote wild‐type ALDH2 human subjects. The heart rate of ALDH2 R114W was not affected by alcohol intake, whereas skin temperature increased after alcohol consumption. To confirm these findings in humans, enzymatic activity of the purified proteins showed that E504K and R114W had 49±3 and 603±11 nmol/min/mg protein, respectively compared to 1209±15 nmol/min/mg protein for wild‐type ALDH2 (n=3, p<0.05). When mixed in the 1:1 ratio with wild‐type enzyme to mimic the heterozygous condition, the activity of E504K and R114W increased to 683±6 and 838±16 nmol/min/mg of pure protein, respectively, but still significantly below the activity of the wild‐type ALDH2. The results presented here demonstrate that a newly found ALDH2 variant, R114W, is also responsible for alcohol‐induced acetaldehyde accumulation and facial flushing. In conclusion, individuals carrying this mutation are more potentially also more susceptible to esophageal cancer similar to those heterozygous for the ALDH2*2 genetic variant.Support or Funding InformationNIGMS HL119522 (ERG) and a Stanford ChEM‐H seed grant (ERG, DMR).

Dysfunctional ABCG2 gene polymorphisms are associated with serum uric acid levels and all-cause mortality in hemodialysis patients

Dysfunctional variants of ATP-binding cassette transporter subfamily G member 2 (ABCG2), a urate transporter in the kidney and intestine, are the major causes of hyperuricemia and gout. A recent study found that ABCG2 is a major transporter of uremic toxins; however, few studies have investigated the relationship between ABCG2 gene polymorphisms and mortality. This prospective cohort study of 1214 hemodialysis patients investigated the association between serum uric acid levels and ABCG2 genotype and mortality. Genotyping of dysfunctional ABCG2 variants, Q126X (rs72552713) and Q141K (rs2231142), was performed using the patients’ DNA. During the study period, 220 patients died. Lower serum uric acid levels were associated with higher mortality (hazard ratio [HR] 1.89, 95% confidence interval [CI] 1.14–3.10, P ≤ 0.001). ABCG2 dysfunction, estimated by genetic variants, had a significant positive association with serum uric acid levels (full function: 7.4 ± 1.2 mg/dl, 3/4 function: 7.9 ± 1.3 mg/dl, 1/2 function: 8.2 ± 1.4 mg/dl, ≤ 1/4 function: 8.7 ± 1.3 mg/dl, P ≤ 0.001). This association remained significant on multiple regression analysis. The Cox proportional hazard analysis indicated that the ABCG2 ≤ 1/4 function type was significantly associated with higher mortality (HR 6.66, 95% CI 2.49 to 17.8, P ≤ 0.001) than the other function types. These results showed that ABCG2 plays a physiologically important role in uric acid excretion, and that ABCG2 dysfunction is a risk factor for mortality in hemodialysis patients.

SERPING1 mutation update: Mutation spectrum and C1 Inhibitor phenotypes.

C1 inhibitor (C1Inh) deficiency is responsible for hereditary angioedema (C1-INH-HAE) and caused by variants of the SERPING1/C1INH/C1NH gene. C1Inh is the major control of kallikrein-kinin system. C1Inh deficiency leads to its uncontrolled activation, with subsequent generation of the vasoactive peptide bradykinin. This update documents 748 different SERPING1 variants, including published variants and additional 120 unpublished ones. They were identified as heterozygous variants (n = 729), as homozygous variants in 10 probands and as compound heterozygous variants (nine combinations). Six probands with heterozygous variants exhibited gonadal mosaicism. Probands with heterozygous (n = 72) and homozygous (n = 1) variants were identified as de novo cases. Overall, 58 variants were found at positions showing high residue conservation among serpins, and have been referred to as a mousetrap function of C1Inh: reactive center loop, gate, shutter, breach, and hinge. C1Inh phenotype analysis identified dysfunctional serpin variants with failed serpin-protease association and a residual 105-kDa species after incubation with target protease. Regarding this characteristic, in conditions with low antigenic C1Inh, 74 C1-INH-HAE probands presented with an additional so-called intermediate C1-INH-HAE phenotype. The present update addresses a comprehensive SERPING1 variant spectrum that facilitates genotype-phenotype correlations, highlighting residues of strategic importance for serpin function and for identification of C1Inh deficiency as serpinopathy.