Syndrome-associated Mutations Research Articles

Genome mining yields putative disease-associated ROMK variants with distinct defects.

Bartter syndrome is a group of rare genetic disorders that compromise kidney function by impairing electrolyte reabsorption. Left untreated, the resulting hyponatremia, hypokalemia, and dehydration can be fatal, and there is currently no cure. Bartter syndrome type II specifically arises from mutations in KCNJ1, which encodes the renal outer medullary potassium channel, ROMK. Over 40 Bartter syndrome-associated mutations in KCNJ1 have been identified, yet their molecular defects are mostly uncharacterized. Nevertheless, a subset of disease-linked mutations compromise ROMK folding in the endoplasmic reticulum (ER), which in turn results in premature degradation via the ER associated degradation (ERAD) pathway. To identify uncharacterized human variants that might similarly lead to premature degradation and thus disease, we mined three genomic databases. First, phenotypic data in the UK Biobank were analyzed using a recently developed computational platform to identify individuals carrying KCNJ1 variants with clinical features consistent with Bartter syndrome type II. In parallel, we examined genomic data in both the NIH TOPMed and ClinVar databases with the aid of Rhapsody, a verified computational algorithm that predicts mutation pathogenicity and disease severity. Subsequent phenotypic studies using a yeast screen to assess ROMK function-and analyses of ROMK biogenesis in yeast and human cells-identified four previously uncharacterized mutations. Among these, one mutation uncovered from the two parallel approaches (G228E) destabilized ROMK and targeted it for ERAD, resulting in reduced cell surface expression. Another mutation (T300R) was ERAD-resistant, but defects in channel activity were apparent based on two-electrode voltage clamp measurements in X. laevis oocytes. Together, our results outline a new computational and experimental pipeline that can be applied to identify disease-associated alleles linked to a range of other potassium channels, and further our understanding of the ROMK structure-function relationship that may aid future therapeutic strategies to advance precision medicine.

MLH1 Loss in Primary Prostate Cancer.

Among mismatch repair-deficient (MMRd) prostate cancers, loss of MLH1 is relatively uncommon and few cases have been reported in detail. Here, we describe the molecular features of two cases of primary prostate cancer with MLH1 loss detected by immunohistochemistry, and in one case, confirmed via transcriptomic profiling. Both cases were microsatellite stable on standard polymerase chain reaction (PCR)-based microsatellite instability (MSI) testing, but showed evidence of MSI on a newer PCR-based long mononucleotide repeat (LMR) assay and by next-generation sequencing. Germline testing was negative for Lynch syndrome-associated mutations in both cases. Targeted or whole-exome tumor sequencing using multiple commercial/academic platforms (Foundation, Tempus, JHU, and UW-OncoPlex) showed modestly elevated, though variable, tumor mutation burden estimates (2.3-10 mutations/Mb) consistent with MMRd, but without identifiable pathogenic single-nucleotide or indel mutations in MLH1. Copy-number analysis confirmed biallelic MLH1 loss in one case and monoallelic MLH1 loss in the second case, without evidence of MLH1 promoter hypermethylation in either. The second patient was treated with single-agent pembrolizumab and demonstrated a short-lived prostate-specific antigen response. These cases highlight the challenges in identifying MLH1-deficient prostate cancers using standard MSI testing and commercial sequencing panels, and support the utility of immunohistochemical assays and LMR- or sequencing-based MSI testing for detection of MMRd prostate cancers.

Unexpected thermodynamic signature for the interaction of hydroxymethylated DNA with MeCP2

Hydroxymethylated cytosine (5hmC) is a stable DNA epigenetic mark recognized by methyl-CpG binding protein 2 (MeCP2), which acts as a transcriptional regulator and a global chromatin-remodeling element. Because 5hmC triggers a gene regulation response markedly different from that produced by methylated cytosine (5mC), both modifications must affect DNA structure and/or DNA interaction with MeCP2 differently. MeCP2 is a six-domain intrinsically disordered protein (IDP) with two domains responsible for dsDNA binding: methyl-CpG binding domain (MBD) and intervening domain (ID). Here we report the detailed thermodynamic characterization of the interaction of hmCpG-DNA with MeCP2. We find that hmCpG-DNA interacts with MeCP2 in a distinctly different mode with a particular thermodynamic signature, compared to methylated or unmethylated DNA. In addition, we find evidence for Rett syndrome-associated mutations altering the interaction of MeCP2 with dsDNA in a cytosine modification-specific manner which may correlate with disease onset time and clinical severity score.

Risk of Gastric Cancer and Utility of Endoscopic Screening for Lynch Syndrome Patients

Background: Lynch syndrome (LS) is an autosomal dominant hereditary condition associated with a genetic predisposition to several cancers. Prior studies have shown an increased risk of gastric cancer (GC) in LS patients. The aim of this study was to synthesize the evidence for gastric cancer risk across Lynch syndrome-associated mutations and to assess the diagnostic utility of endoscopic screening for gastric cancer in these patients. Methods: A systematic literature review and meta-analysis of LS-associated gastric cancer risk and the diagnostic utility of endoscopic gastric cancer screening was conducted in accordance with PRISMA guidelines and GRADE methodology. Results: The results of this study found an elevated risk of GC in LS patients. Cumulative lifetime risk of GC in LS patients ranged from 2.6% to 38.7% when assessing across all LS mutations. Cumulative incidence ranged from 2.0% to 41.0%. When assessing cumulative incidence across specific LS mutations, MSH2 mutation carriers showed the highest cumulative incidence. RR across specific mutation carriers was generally highest in MSH2 carriers. A meta-analysis of 5 studies reporting diagnostic utility of endoscopic screening for gastric cancer in LS patients revealed a 2% incidence of GC (95% CI, 1.4%−2.9%). Conclusions: Gastric cancer risk in LS patients is greater than in the general population. Endoscopic screening yields low rates of GC in LS patients. Higher quality studies including RCTs are required to more accurately define GC risk and the yield of endoscopic surveillance in LS patients.

Crystal structure of Ankyrin-G in complex with a fragment of Neurofascin reveals binding mechanisms required for integrity of the axon initial segment

The axon initial segment (AIS) has characteristically dense clustering of voltage-gated sodium channels (Nav), cell adhesion molecule Neurofascin 186 (Nfasc), and neuronal scaffold protein Ankyrin-G (AnkG) in neurons, which facilitates generation of an action potential and maintenance of axonal polarity. However, the mechanisms underlying AIS assembly, maintenance, and plasticity remain poorly understood. Here, we report the high-resolution crystal structure of the AnkG ankyrin repeat (ANK repeat) domain in complex with its binding site in the Nfasc cytoplasmic tail that shows, in conjunction with binding affinity assays with serial truncation variants, the molecular basis of AnkG–Nfasc binding. We confirm AnkG interacts with the FIGQY motif in Nfasc, and we identify another region required for their high affinity binding. Our structural analysis revealed that ANK repeats form 4 hydrophobic or hydrophilic layers in the AnkG inner groove that coordinate interactions with essential Nfasc residues, including F1202, E1204, and Y1212. Moreover, we show disruption of the AnkG–Nfasc complex abolishes Nfasc enrichment at the AIS in cultured mouse hippocampal neurons. Finally, our structural and biochemical analysis indicated that L1 syndrome-associated mutations in L1CAM, a member of the L1 immunoglobulin family proteins including Nfasc, L1CAM, NrCAM, and CHL1, compromise binding with ankyrins. Taken together, these results define the mechanisms underlying AnkG–Nfasc complex formation and show that AnkG-dependent clustering of Nfasc is required for AIS integrity.

Acceleration of the DNA methylation clock among lynch syndrome-associated mutation carriers

BackgroundDNA methylation (DNAm) age metrics have been widely accepted as an epigenetic biomarker for biological aging and disease. The purpose of this study is to assess whether or not individuals carrying Lynch Syndrome-associated mutations are affected in their rate of biological aging, as measured by the epigenetic clock.MethodsGenome-wide bisulfite DNA sequencing data were generated using DNA from CD4 + T-cells obtained from peripheral blood using 27 patient samples from Lynch syndrome families. Horvath’s DNAm age model based on penalized linear regression was applied to estimate DNAm age from patient samples with distinct clinical and genetic characteristics to investigate cancer mutation-related aging effects.ResultsBoth Lynch mutation carriers and controls exhibited high variability in their estimated DNAm age, but regression analysis showed steeper slope for the Lynch mutation carriers. Remarkably, six Lynch Syndrome-associated mutation carriers showed a strong correlation to the control group, and two sisters carrying Lynch Syndrome-associated mutations, with no significant difference in lifestyle and similar chronological age, were assigned very different DNAm age.ConclusionsFuture studies will be required to explore, in larger patient populations, whether specific epigenetic age acceleration is predictive of time-to-cancer development, treatment response, and survival. Epigenetic clock DNAm metrics may be affected by the presence of cancer mutations in the germline, and thus show promise of potential clinical utility for stratified surveillance strategies based on the relative risk for imminent emergence of tumor lesions in otherwise healthy Lynch Syndrome-associated mutation carriers.

SPECC1L Mutations Are Not Common in Sporadic Cases of Opitz G/BBB Syndrome.

Opitz G/BBB syndrome (OS) is a rare genetic developmental condition characterized by congenital defects along the midline of the body. The main clinical signs are represented by hypertelorism, laryngo–tracheo–esophageal defects and hypospadias. The X-linked form of the disease is associated with mutations in the MID1 gene located in Xp22 whereas mutations in the SPECC1L gene in 22q11 have been linked to few cases of the autosomal dominant form of this disorder, as well as to other genetic syndromes. In this study, we have undertaken a mutation screening of the SPECC1L gene in samples of sporadic OS cases in which mutations in the MID1 gene were excluded. The heterozygous missense variants identified are already reported in variant databases raising the issue of their pathogenetic meaning. Recently, it was reported that some clinical manifestations peculiar to OS signs are not observed in patients carrying mutations in the SPECC1L gene, leading to the proposal of the designation of ‘SPECC1L syndrome’ to refer to this disorder. Our study confirms that patients with diagnosis of OS, mainly characterized by the presence of hypospadias and laryngo–tracheo–esophageal defects, do not carry pathogenic SPECC1L mutations. In addition, SPECC1L syndrome-associated mutations are clustered in two specific domains of the protein, whereas the missense variants detected in our work lies elsewhere and the impact of these variants in the function of this protein is difficult to ascertain with the current knowledge and will require further investigations. Nonetheless, our study provides further insight into the SPECC1L syndrome classification.

Disease-associated mutations in topoisomerase IIβ result in defective NK cells

Hoffman syndrome is a syndromic, inborn error of immunity due to autosomal-dominant mutations in TOP2B, an essential gene required to alleviate topological stress during DNA replication and gene transcription. Although mutations identified in patients lead to a block in B-cell development and the absence of circulating B cells, an effect on natural killer (NK) cells was not previously examined. We sought to determine whether disease-associated mutations in TOP2B impact NK-cell development and function. Using a knockin murine model and patient-derived induced pluripotent stem cells (iPSCs), we investigated NK-cell development in mouse bone marrow and spleen, and performed immunophenotyping by flow cytometry, gene expression, and functional assessment of cytotoxic activity in murine NK cells, and human IPSC-derived NK cells. Mature NK cells were reduced in the periphery of TOP2B knockin mice consistent with patient reports, with reduced cytotoxicity toward target cell lines. IPSCs were successfully derived from patients with Hoffman syndrome, but under optimal conditions showed reduced cytotoxicity compared with iPSC-derived NK cells from healthy controls. Hoffman syndrome-associated mutations in TOP2B impact NK-cell development and function in murine and human models.

Xbx-4, a homolog of the Joubert syndrome gene FAM149B1, acts via the CCRK and RCK kinase cascade to regulate cilia morphology

Primary cilia are microtubule (MT)-based organelles that mediate sensory functions in multiple cell types. Disruption of cilia structure or function leads to a diverse collection of diseases termed ciliopathies.1-3 The highly conserved CCRK and RCK kinases (ICK/MOK/MAK) negatively regulate cilia length and structure in Chlamydomonas, C.elegans, and mammalian cells.4-10 How the activity of this kinase cascade is tuned to precisely regulate cilia architecture is unclear. Mutations in the Domain of Unknown Function 3719 (DUF3719)-containing protein FAM149B1 have recently been shown to elongate cilia via unknown mechanisms and result in the ciliopathy Joubert syndrome.11 Here we identify XBX-4, a DUF3719-containing protein related to human FAM149B1, as a regulator of the DYF-18 CCRK and DYF-5 MAK kinase pathway in C.elegans. As in dyf-18 and dyf-5 mutants,10 sensory neuron cilia are elongated in xbx-4 mutants and exhibit stabilized axonemal MTs. XBX-4 promotes DYF-18 CCRK function to regulate localization and function of DYF-5 MAK. We find that Joubert syndrome-associated mutations in the XBX-4 DUF3719 domain also elongate cilia in C.elegans. Our results identify a new metazoan-specific regulator of this highly conserved kinase pathway and suggest that FAM149B1 may similarly act via the CCRK/RCK kinase pathway to regulate ciliary homeostasis in humans.

Cornelia\xa0de Lange syndrome-associated mutations cause a DNA damage signalling and repair defect

Cornelia de Lange syndrome is a multisystem developmental disorder typically caused by mutations in the gene encoding the cohesin loader NIPBL. The associated phenotype is generally assumed to be the consequence of aberrant transcriptional regulation. Recently, we identified a missense mutation in BRD4 associated with a Cornelia de Lange-like syndrome that reduces BRD4 binding to acetylated histones. Here we show that, although this mutation reduces BRD4-occupancy at enhancers it does not affect transcription of the pluripotency network in mouse embryonic stem cells. Rather, it delays the cell cycle, increases DNA damage signalling, and perturbs regulation of DNA repair in mutant cells. This uncovers a role for BRD4 in DNA repair pathway choice. Furthermore, we find evidence of a similar increase in DNA damage signalling in cells derived from NIPBL-deficient individuals, suggesting that defective DNA damage signalling and repair is also a feature of typical Cornelia de Lange syndrome.

Dravet syndrome-associated mutations in GABRA1, GABRB2 and GABRG2 define the genetic landscape of defects of GABAA receptors.

Dravet syndrome is a rare, catastrophic epileptic encephalopathy that begins in the first year of life, usually with febrile or afebrile hemiclonic or generalized tonic–clonic seizures followed by status epilepticus. De novo variants in genes that mediate synaptic transmission such as SCN1A and PCDH19 are often associated with Dravet syndrome. Recently, GABAA receptor subunit genes (GABRs) encoding α1 (GABRA1), β3 (GABRB3) and γ2 (GABRG2), but not β2 (GABRB2) or β1 (GABRB1), subunits are frequently associated with Dravet syndrome or Dravet syndrome-like phenotype. We performed next generation sequencing on 870 patients with Dravet syndrome and identified nine variants in three different GABRs. Interestingly, the variants were all in genes encoding the most common GABAA receptor, the α1β2γ2 receptor. Mutations in GABRA1 (c.644T>C, p. L215P; c.640C>T, p. R214C; c.859G>A; V287I; c.641G>A, p. R214H) and GABRG2 (c.269C>G, p. T90R; c.1025C>T, p. P342L) presented as de novo cases, while in GABRB2 two variants were de novo (c.992T>C, p. F331S; c.542A>T, p. Y181F) and one was autosomal dominant and inherited from the maternal side (c.990_992del, p.330_331del). We characterized the effects of these GABR variants on GABAA receptor biogenesis and channel function. We found that defects in receptor gating were the common deficiency of GABRA1 and GABRB2 Dravet syndrome variants, while mainly trafficking defects were found with the GABRG2 (c.269C>G, p. T90R) variant. It seems that variants in α1 and β2 subunits are less tolerated than in γ2 subunits, since variant α1 and β2 subunits express well but were functionally deficient. This suggests that all of these GABR variants are all targeting GABR genes that encode the assembled α1β2γ2 receptor, and regardless of which of the three subunits are mutated, variants in genes coding for α1, β2 and γ2 receptor subunits make them candidate causative genes in the pathogenesis of Dravet syndrome.

Molecular Context-Dependent Effects Induced by Rett Syndrome-Associated Mutations in MeCP2.

Methyl-CpG binding protein 2 (MeCP2) is a transcriptional regulator and a chromatin-binding protein involved in neuronal development and maturation. Loss-of-function mutations in MeCP2 result in Rett syndrome (RTT), a neurodevelopmental disorder that is the main cause of mental retardation in females. MeCP2 is an intrinsically disordered protein (IDP) constituted by six domains. Two domains are the main responsible elements for DNA binding (methyl-CpG binding domain, MBD) and recruitment of gene transcription/silencing machinery (transcription repressor domain, TRD). These two domains concentrate most of the RTT-associated mutations. R106W and R133C are associated with severe and mild RTT phenotype, respectively. We have performed a comprehensive characterization of the structural and functional impact of these substitutions at molecular level. Because we have previously shown that the MBD-flanking disordered domains (N-terminal domain, NTD, and intervening domain, ID) exert a considerable influence on the structural and functional features of the MBD (Claveria-Gimeno, R. et al. Sci Rep. 2017, 7, 41635), here we report the biophysical study of the influence of the protein scaffold on the structural and functional effect induced by these two RTT-associated mutations. These results represent an example of how a given mutation may show different effects (sometimes opposing effects) depending on the molecular context.

A Lynch syndrome-associated mutation at a Bergerat ATP-binding fold destabilizes the structure of the DNA mismatch repair endonuclease MutL

In humans, mutations in genes encoding homologs of the DNA mismatch repair endonuclease MutL cause a hereditary cancer that is known as Lynch syndrome. Here, we determined the crystal structures of the N-terminal domain (NTD) of MutL from the thermophilic eubacterium Aquifex aeolicus (aqMutL) complexed with ATP analogs at 1.69-1.73 Å. The structures revealed significant structural similarities to those of a human MutL homolog, postmeiotic segregation increased 2 (PMS2). We introduced five Lynch syndrome-associated mutations clinically found in human PMS2 into the aqMutL NTD and investigated the protein stability, ATPase activity, and DNA-binding ability of these protein variants. Among the mutations studied, the most unexpected results were obtained for the residue Ser34. Ser34 (Ser46 in PMS2) is located at a previously identified Bergerat ATP-binding fold. We found that the S34I aqMutL NTD retains ATPase and DNA-binding activities. Interestingly, CD spectrometry and trypsin-limited proteolysis indicated the disruption of a secondary structure element of the S34I NTD, destabilizing the overall structure of the aqMutL NTD. In agreement with this, the recombinant human PMS2 S46I NTD was easily digested in the host Escherichia coli cells. Moreover, other mutations resulted in reduced DNA-binding or ATPase activity. In summary, using the thermostable aqMutL protein as a model molecule, we have experimentally determined the effects of the mutations on MutL endonuclease; we discuss the pathological effects of the corresponding mutations in human PMS2.

SLX4 interacts with RTEL1 to prevent transcription-mediated DNA replication perturbations.

The SLX4 tumor suppressor is a scaffold that plays a pivotal role in several aspects of genome protection, including homologous recombination, interstrand DNA crosslink repair and the maintenance of common fragile sites and telomeres. Here, we unravel an unexpected direct interaction between SLX4 and the DNA helicase RTEL1, which, until now, were viewed as having independent and antagonistic functions. We identify cancer and Hoyeraal-Hreidarsson syndrome-associated mutations in SLX4 and RTEL1, respectively, that abolish SLX4-RTEL1 complex formation. We show that both proteins are recruited to nascent DNA, tightly co-localize with active RNA pol II, and that SLX4, in complex with RTEL1, promotes FANCD2/RNA pol II co-localization. Importantly, disrupting the SLX4-RTEL1 interaction leads to DNA replication defects in unstressed cells, which are rescued by inhibiting transcription. Our data demonstrate that SLX4 and RTEL1 interact to prevent replication-transcription conflicts and provide evidence that this is independent of the nuclease scaffold function of SLX4.

Complementary computational and experimental evaluation of missense variants in the ROMK potassium channel.

The renal outer medullary potassium (ROMK) channel is essential for potassium transport in the kidney, and its dysfunction is associated with a salt-wasting disorder known as Bartter syndrome. Despite its physiological significance, we lack a mechanistic understanding of the molecular defects in ROMK underlying most Bartter syndrome-associated mutations. To this end, we employed a ROMK-dependent yeast growth assay and tested single amino acid variants selected by a series of computational tools representative of different approaches to predict each variants’ pathogenicity. In one approach, we used in silico saturation mutagenesis, i.e. the scanning of all possible single amino acid substitutions at all sequence positions to estimate their impact on function, and then employed a new machine learning classifier known as Rhapsody. We also used two additional tools, EVmutation and Polyphen-2, which permitted us to make consensus predictions on the pathogenicity of single amino acid variants in ROMK. Experimental tests performed for selected mutants in different classes validated the vast majority of our predictions and provided insights into variants implicated in ROMK dysfunction. On a broader scope, our analysis suggests that consolidation of data from complementary computational approaches provides an improved and facile method to predict the severity of an amino acid substitution and may help accelerate the identification of disease-causing mutations in any protein.

Associations Between Mutations in MSH6 and PMS2 and Risk of Surveillance-detected Colorectal Cancer

Lynch syndrome is the most common inherited cause of colorectal cancer (CRC). Contemporary and mutation-specific estimates of CRC-risk in patients undergoing colonoscopy would optimize surveillance strategies. We performed a prospective national cohort study, using data from New Zealand, to assess overall and mutation-specific risk of CRC in patients with Lynch syndrome undergoing surveillance. We performed a prospective study of 381 persons with Lynch syndrome in New Zealand (98 with Lynch-syndrome associated variants in MLH1, 159 in MSH2, 103 in MSH6, and 21 in PMS2). Participants were offered annual colonoscopy starting at age 25 y, and those who underwent 2 or more colonoscopies before December 31, 2017 were included in the final analysis. Patients with previous colonic resection, history of CRC or diagnosis of CRC at index colonoscopy were excluded. Study participants underwent 2061 colonoscopies during 2296 person-y; the median observation-period was 4.43 y and mean-age at enrollment was 43 y. Eighteen patients developed CRC (8 with variants in MLH1, 8 in MSH2, and 2 in MSH6) after a median follow-up period of 6.5 y (range 1-16 y). Eighty-three percent of patients had a surveillance colonoscopy in preceding 24 months before diagnosis of CRC; 94% were diagnosed with stage 0-II CRC and there was no CRC-related mortality. The overall-risk of developing CRC in the 5 y after first surveillance colonoscopy was 2.49% (95% CI, 1.18-5.23); cumulative risks for CRC in patients with Lynch syndrome-associated variants in MLH1, MSH2, or MSH6 by age 70 y were 17.7%, 17.8%, and 8.5%, respectively. Age-adjusted CRC-risk in patients with variants in MSH6 was lower than in MLH1 (hazard ratio, 0.2; 95% CI, 0.04-0.94; P = .02). Of patients with CRC, 33% had an adenomatous polyp resected from same segment in which a colorectal tumor later developed. The risk of CRC in patients with Lynch syndrome-associated mutations in MSH6 or PMS2 was significantly lower than in patients with mutations in MLH1. Incomplete adenomatous polyp resection might be responsible for one third of surveillance-detected CRCs.

Impacts of deletion and ichthyosis prematurity syndrome-associated mutations in fatty acid transport protein 4 on the function of RPE65.

The retinal pigment epithelium-specific 65kDa (RPE65) isomerase plays a pivotal role in photoreceptor survival and function. RPE65-catalyzed synthesis of 11-cis-retinol from all-trans-retinyl esters in the visual cycle is negatively regulated, through a heretofore unknown mechanism, by the fatty acid transport protein FATP4, mutations in which are associated with ichthyosis prematurity syndrome (IPS). Here, we analyzed the interaction between deletion mutants of FATP4 and RPE65 and the impacts of IPS-associated FATP4 mutations on RPE65 expression, 11-cis-retinol synthesis, and all-trans-retinyl ester synthesis. Our results suggest that the interaction between FATP4 and RPE65 contributes to the inhibition of RPE65 function and that IPS-associated nonsense and missense mutations in FATP4 have different effects on the visual cycle.

Clinical Factors Associated With Gastric Cancer in Individuals With Lynch Syndrome

Lynch syndrome is the most common inherited cause of gastrointestinal cancer and increases risk for a variety of malignancies, including gastric cancer. We aimed to identify clinical factors associated with gastric cancer in carriers of germline variants causing Lynch syndrome. We collected data from 52,758 consecutive individuals tested for genetic variants associated with Lynch syndrome from June 2006 through July 2013 at a commercial laboratory. We obtained clinical and demographic data, as well as information on personal and family histories of cancer (first- and second-degree relatives) from forms completed by ordering providers. We performed multivariate logistic regression to identify clinical factors associated with gastric cancer in carriers of mutations that cause Lynch syndrome (pathogenic mutations). After we excluded individuals with missing clinical data (n= 1664) or with multiple pathogenic mutations (n= 8), we analyzed data from 51,086 individuals. Of these, 3828 persons carried pathogenic mutations (1346 with mutations in MLH1, 1639 with mutations in MSH2, 670 with mutations in MSH6, 145 with mutations in PMS2, and 28 with mutations in EPCAM). Of the 3828 carriers of pathogenic mutations, 41 (1.1%) had a previous gastric cancer and 350 (9.1%) had 1 or more first- or second-degree relatives with gastric cancer. In multivariate analysis, male sex (odds ratio [OR], 2.82; 95% CI, 1.48-5.38), older age (OR, 2.07 per 10 years; 95% CI, 1.64-2.61), mutations in MLH1 (OR, 6.53; 95% CI, 1.50-28.42) or MSH2 (OR, 5.23 compared to mutations in MSH6, PMS2, or EPCAM; 95% CI, 1.21-22.71), and number of first-degree relatives with gastric cancer (OR, 2.52; 95% CI, 1.42-4.45), but not second-degree relatives (OR, 1.12; 95% CI, 0.40-3.18) were independently associated with gastric cancer among carriers of pathogenic mutations. In an analysis of data from almost 4000 carriers of Lynch syndrome-associated mutations, we found history of gastric cancer to be independently associated with male sex, older age, mutations in MLH1 or MSH2, and number of first-degree relatives with gastric cancer. These findings suggest that personalized, risk-stratified approaches to gastric cancer surveillance may be appropriate for individuals with Lynch syndrome-associated mutations.

Atomic Mechanisms of Timothy Syndrome-Associated Mutations in Calcium Channel Cav1.2.

Timothy syndrome (TS) is a very rare multisystem disorder almost exclusively associated with mutations G402S and G406R in helix IS6 of Cav1.2. Recently, mutations R518C/H in helix IIS0 of the voltage sensing domain II (VSD-II) were described as a cause of cardiac-only TS. The three mutations are known to decelerate voltage-dependent inactivation (VDI). Here, we report a case of cardiac-only TS caused by mutation R518C. To explore possible impact of the three mutations on interdomain contacts, we modeled channel Cav1.2 using as templates Class Ia and Class II cryo-EM structures of presumably inactivated channel Cav1.1. In both models, R518 and several other residues in VSD-II donated H-bonds to the IS6-linked α1-interaction domain (AID). We further employed steered Monte Carlo energy minimizations to move helices S4–S5, S5, and S6 from the inactivated-state positions to those seen in the X-ray structures of the open and closed NavAb channel. In the open-state models, positions of AID and VSD-II were similar to those in Cav1.1. In the closed-state models, AID moved along the β subunit (Cavβ) toward the pore axis and shifted AID-bound VSD-II. In all the models R518 retained strong contacts with AID. Our calculations suggest that conformational changes in VSD-II upon its deactivation would shift AID along Cavβ toward the pore axis. The AID-linked IS6 would bend at flexible G402 and G406, facilitating the activation gate closure. Mutations R518C/H weakened the IIS0-AID contacts and would retard the AID shift. Mutations G406R and G402S stabilized the open state and would resist the pore closure. Several Cav1.2 mutations associated with long QT syndromes are consistent with this proposition. Our results provide a mechanistic rationale for the VDI deceleration caused by TS-associated mutations and suggest targets for further studies of calcium channelopathies.

Molecular Mechanism of Polyunsaturated Fatty Acid Analogues as KV7.1-Channel Modulators

Negatively charged analogues of polyunsaturated fatty acids (PUFAs) have been shown to activate the human KV7.1+KCNE1 channel, which generates the repolarizing cardiac IKs current. Specific PUFA analogues activate both wild type KV7.1+KCNE1 and KV7.1+KCNE1 containing Long QT syndrome-associated mutations. Therefore, PUFA analogues represent a new class of KV7.1+KCNE1 channel activators that may aid in development of novel anti-arrhythmic drugs. To determine the molecular mechanism of how PUFA analogues activate KV7.1+KCNE1, we here combine electrophysiology recordings and site-directed mutagenesis with a panel of PUFA analogues with distinct properties. We identify two main effects of PUFA analogues on KV7.1 that are dependent on discrete channel motifs. The first effect is a shift in the voltage dependence of channel opening that is critically dependent on specific S4 arginines in the voltage sensor domain of KV7.1. Intriguingly, different S4 arginines are important in KV7.1 and KV7.1+KCNE1. The second effect is alteration in the maximum conductance that is critically dependent on the S6 residue K326 in the pore domain of KV7.1. Both effects appear to be primarily electrostatic. Our findings provide novel insights into how KV7.1+KCNE1 channel activators, such as PUFA analogues, simultaneously may interact with the voltage sensor domain and the pore domain to alter channel activity. Our findings also contribute pharmacological support to the hypothesis that KV7.1 and KV7.1+KCNE1 open from different conformational states.