Unexpected complexity in the molecular diagnosis of spastic paraplegia 11.

BACKGROUND: Spastic paraplegia 11 (SPG11) is the most prevalent form of autosomal recessive hereditary spastic paraplegia, resulting from biallelic pathogenic variants in the SPG11 gene (MIM *610844). METHODS: The proband is a 36-year-old female referred for genetic evaluation due to cognitive dysfunction, gait impairment, and corpus callosum atrophy (brain MRI was normal at 25-years-old). Diagnostic approaches included CGH array, next-generation sequencing, and whole transcriptome sequencing. RESULTS: CGH array revealed a 180 kb deletion located upstream of SPG11. Sequencing of SPG11 uncovered two rare single nucleotide variants: the novel variant c.3143C>T in exon 17 (in cis with the deletion), and the previously reported pathogenic variant c.6409C>T in exon 34 (in trans). Whole transcriptome sequencing revealed that the variant c.3143C>T caused exon 17 skipping. CONCLUSION: We report a novel sequence variant in the SPG11 gene resulting in exon 17 skipping, which, along with a nonsense variant, causes Spastic Paraplegia 11 in our proband. In addition, a deletion upstream of SPG11 was identified in the patient, whose implication in the phenotype remains uncertain. Nonetheless, the deletion apparently affects cis-regulatory elements of the gene, suggesting a potential new pathogenic mechanism underlying the disease in a subset of undiagnosed patients. Our findings further support the hypothesis that the origin of thin corpus callosum in patients with SPG11 is of progressive nature.

Targeted phasing of 2-200 kilobase DNA fragments with a short-read sequencer and a single-tube linked-read library method.

In the human genome, heterozygous sites refer to genomic positions with a different allele or nucleotide variant on the maternal and paternal chromosomes. Resolving these allelic differences by chromosomal copy, also known as phasing, is achievable on a short-read sequencer when using a library preparation method that captures long-range genomic information. TELL-Seq is a library preparation that captures long-range genomic information with the aid of molecular identifiers (barcodes). The same barcode is used to tag the reads derived from the same long DNA fragment within a range of up to 200 kilobases (kb), generating linked-reads. This strategy can be used to phase an entire genome. Here, we introduce a TELL-Seq protocol developed for targeted applications, enabling the phasing of enriched loci of varying sizes, purity levels, and heterozygosity. To validate this protocol, we phased 2-200 kb loci enriched with different methods: CRISPR/Cas9-mediated excision coupled with pulse-field electrophoresis for the longest fragments, CRISPR/Cas9-mediated protection from exonuclease digestion for mid-size fragments, and long PCR for the shortest fragments. All selected loci have known clinical relevance: BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, PMS2, SCN5A-SCN10A, and PKI3CA. Collectively, the analyses show that TELL-Seq can accurately phase 2-200 kb targets using a short-read sequencer.

Targeted Phasing of 2-200 Kilobase DNA Fragments with a Short-Read Sequencer and a Single-Tube Linked-Read Library Method.

In the human genome, heterozygous sites are genomic positions with different alleles inherited from each parent. On average, there is a heterozygous site every 1-2 kilobases (kb). Resolving whether two alleles in neighboring heterozygous positions are physically linked-that is, phased-is possible with a short-read sequencer if the sequencing library captures long-range information. TELL-Seq is a library preparation method based on millions of barcoded micro-sized beads that enables instrument-free phasing of a whole human genome in a single PCR tube. TELL-Seq incorporates a unique molecular identifier (barcode) to the short reads generated from the same high-molecular-weight (HMW) DNA fragment (known as 'linked-reads'). However, genome-scale TELL-Seq is not cost-effective for applications focusing on a single locus or a few loci. Here, we present an optimized TELL-Seq protocol that enables the cost-effective phasing of enriched loci (targets) of varying sizes, purity levels, and heterozygosity. Targeted TELL-Seq maximizes linked-read efficiency and library yield while minimizing input requirements, fragment collisions on microbeads, and sequencing burden. To validate the targeted protocol, we phased seven 180-200 kb loci enriched by CRISPR/Cas9-mediated excision coupled with pulse-field electrophoresis, four 20 kb loci enriched by CRISPR/Cas9-mediated protection from exonuclease digestion, and six 2-13 kb loci amplified by PCR. The selected targets have clinical and research relevance (BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, PMS2, SCN5A-SCN10A, and PKI3CA). These analyses reveal that targeted TELL-Seq provides a reliable way of phasing allelic variants within targets (2-200 kb in length) with the low cost and high accuracy of short-read sequencing.

Analysis of Brugada syndrome loci reveals that fine-mapping clustered GWAS hits enhances the annotation of disease-relevant variants.

Genome-wide association studies (GWASs) are instrumental in identifying loci harboring common single-nucleotide variants (SNVs) that affect human traits and diseases. GWAS hits emerge in clusters, but the focus is often on the most significant hit in each trait- or disease-associated locus. The remaining hits represent SNVs in linkage disequilibrium (LD) and are considered redundant and thus frequently marginally reported or exploited. Here, we interrogate the value of integrating the full set of GWAS hits in a locus repeatedly associated with cardiac conduction traits and arrhythmia, SCN5A-SCN10A. Our analysis reveals 5 common 7-SNV haplotypes (Hap1-5) with 2 combinations associated with life-threatening arrhythmia-Brugada syndrome (the risk Hap1/1 and protective Hap2/3 genotypes). Hap1 and Hap2 share 3 SNVs; thus, this analysis suggests that assuming redundancy among clustered GWAS hits can lead to confounding disease-risk associations and supports the need to deconstruct GWAS data in the context of haplotype composition.

Epigenetic Changes Governing Scn5a Expression in Denervated Skeletal Muscle.

The SCN5A gene encodes the α-subunit of the voltage-gated cardiac sodium channel (NaV1.5), a key player in cardiac action potential depolarization. Genetic variants in protein-coding regions of the human SCN5A have been largely associated with inherited cardiac arrhythmias. Increasing evidence also suggests that aberrant expression of the SCN5A gene could increase susceptibility to arrhythmogenic diseases, but the mechanisms governing SCN5A expression are not yet well understood. To gain insights into the molecular basis of SCN5A gene regulation, we used rat gastrocnemius muscle four days following denervation, a process well known to stimulate Scn5a expression. Our results show that denervation of rat skeletal muscle induces the expression of the adult cardiac Scn5a isoform. RNA-seq experiments reveal that denervation leads to significant changes in the transcriptome, with Scn5a amongst the fifty top upregulated genes. Consistent with this increase in expression, ChIP-qPCR assays show enrichment of H3K27ac and H3K4me3 and binding of the transcription factor Gata4 near the Scn5a promoter region. Also, Gata4 mRNA levels are significantly induced upon denervation. Genome-wide analysis of H3K27ac by ChIP-seq suggest that a super enhancer recently described to regulate Scn5a in cardiac tissue is activated in response to denervation. Altogether, our experiments reveal that similar mechanisms regulate the expression of Scn5a in denervated muscle and cardiac tissue, suggesting a conserved pathway for SCN5A expression among striated muscles.

Role of Non-Coding Variants in Brugada Syndrome.

Brugada syndrome (BrS) is an inherited electrical heart disease associated with a high risk of sudden cardiac death (SCD). The genetic characterization of BrS has always been challenging. Although several cardiac ion channel genes have been associated with BrS, SCN5A is the only gene that presents definitive evidence for causality to be used for clinical diagnosis of BrS. However, more than 65% of diagnosed cases cannot be explained by variants in SCN5A or other genes. Therefore, in an important number of BrS cases, the underlying mechanisms are still elusive. Common variants, mostly located in non-coding regions, have emerged as potential modulators of the disease by affecting different regulatory mechanisms, including transcription factors (TFs), three-dimensional organization of the genome, or non-coding RNAs (ncRNAs). These common variants have been hypothesized to modulate the interindividual susceptibility of the disease, which could explain incomplete penetrance of BrS observed within families. Altogether, the study of both common and rare variants in parallel is becoming increasingly important to better understand the genetic basis underlying BrS. In this review, we aim to describe the challenges of studying non-coding variants associated with disease, re-examine the studies that have linked non-coding variants with BrS, and provide further evidence for the relevance of regulatory elements in understanding this cardiac disorder.

Do sodium channel proteolytic fragments regulate sodium channel expression?

The cardiac voltage-gated sodium channel (gene: SCN5A, protein: NaV1.5) is responsible for the sodium current that initiates the cardiomyocyte action potential. Research into the mechanisms of SCN5A gene expression has gained momentum over the last few years. We have recently described the transcriptional regulation of SCN5A by GATA4 transcription factor. In this addendum to our study, we report our observations that 1) the linker between domains I and II (LDI-DII) of NaV1.5 contains a nuclear localization signal (residues 474-481) that is necessary to localize LDI-DII into the nucleus, and 2) nuclear LDI-DII activates the SCN5A promoter in gene reporter assays using cardiac-like H9c2 cells. Given that voltage-gated sodium channels are known targets of proteases such as calpain, we speculate that NaV1.5 degradation is signaled to the cell transcriptional machinery via nuclear localization of LDI-DII and subsequent stimulation of the SCN5A promoter.

SMYD2-Mediated Histone Methylation Contributes to HIV-1 Latency.

Transcriptional latency of HIV is a last barrier to viral eradication. Chromatin-remodeling complexes and post-translational histone modifications likely play key roles in HIV-1 reactivation, but the underlying mechanisms are incompletely understood. We performed an RNAi-based screen of human lysine methyltransferases and identified the SET and MYND domain-containing protein 2 (SMYD2) as an enzyme that regulates HIV-1 latency. Knockdown of SMYD2 or its pharmacological inhibition reactivated latent HIV-1 in T cell lines and in primary CD4+ T cells. SMYD2 associated with latent HIV-1 promoter chromatin, which was enriched in monomethylated lysine 20 at histone H4 (H4K20me1), a mark lost in cells lacking SMYD2. Further, we find that lethal 3 malignant brain tumor 1 (L3MBTL1), a reader protein with chromatin-compacting properties that recognizes H4K20me1, was recruited to the latent HIV-1 promoter in a SMYD2-dependent manner. We propose that a SMYD2-H4K20me1-L3MBTL1 axis contributes to HIV-1 latency and can be targeted with small-molecule SMYD2 inhibitors.

Transcriptional regulation of the sodium channel gene (SCN5A) by GATA4 in human heart.

Aberrant expression of the sodium channel gene (SCN5A) has been proposed to disrupt cardiac action potential and cause human cardiac arrhythmias, but the mechanisms of SCN5A gene regulation and dysregulation still remain largely unexplored. To gain insight into the transcriptional regulatory networks of SCN5A, we surveyed the promoter and first intronic regions of the SCN5A gene, predicting the presence of several binding sites for GATA transcription factors (TFs). Consistent with this prediction, chromatin immunoprecipitation (ChIP) and sequential ChIP (Re-ChIP) assays show co-occupancy of cardiac GATA TFs GATA4 and GATA5 on promoter and intron 1 SCN5A regions in fresh-frozen human left ventricle samples. Gene reporter experiments show GATA4 and GATA5 synergism in the activation of the SCN5A promoter, and its dependence on predicted GATA binding sites. GATA4 and GATA6 mRNAs are robustly expressed in fresh-frozen human left ventricle samples as measured by highly sensitive droplet digital PCR (ddPCR). GATA5 mRNA is marginally but still clearly detected in the same samples. Importantly, GATA4 mRNA levels are strongly and positively correlated with SCN5A transcript levels in the human heart. Together, our findings uncover a novel mechanism of GATA TFs in the regulation of the SCN5A gene in human heart tissue. Our studies suggest that GATA5 but especially GATA4 are main contributors to SCN5A gene expression, thus providing a new paradigm of SCN5A expression regulation that may shed new light into the understanding of cardiac disease.

Large Genomic Imbalances in Brugada Syndrome.

PURPOSE: Brugada syndrome (BrS) is a form of cardiac arrhythmia which may lead to sudden cardiac death. The recommended genetic testing (direct sequencing of SCN5A) uncovers disease-causing SNVs and/or indels in ~20% of cases. Limited information exists about the frequency of copy number variants (CNVs) in SCN5A in BrS patients, and the role of CNVs in BrS-minor genes is a completely unexplored field. METHODS: 220 BrS patients with negative genetic results were studied to detect CNVs in SCN5A. 63 cases were also screened for CNVs in BrS-minor genes. Studies were performed by Multiplex ligation-dependent probe amplification or Next-Generation Sequencing (NGS). RESULTS: The detection rate for CNVs in SCN5A was 0.45% (1/220). The detected imbalance consisted of a duplication from exon 15 to exon 28, and could potentially explain the BrS phenotype. No CNVs were found in BrS-minor genes. CONCLUSION: CNVs in current BrS-related genes are uncommon among BrS patients. However, as these rearrangements may underlie a portion of cases and they undergo unnoticed by traditional sequencing, an appealing alternative to conventional studies in these patients could be targeted NGS, including in a single experiment the study of SNVs, indels and CNVs in all the known BrS-related genes.

The HIV-1 Tat Protein Is Monomethylated at Lysine 71 by the Lysine Methyltransferase KMT7.

The HIV-1 transactivator protein Tat is a critical regulator of HIV transcription primarily enabling efficient elongation of viral transcripts. Its interactions with RNA and various host factors are regulated by ordered, transient post-translational modifications. Here, we report a novel Tat modification, monomethylation at lysine 71 (K71). We found that Lys-71 monomethylation (K71me) is catalyzed by KMT7, a methyltransferase that also targets lysine 51 (K51) in Tat. Using mass spectrometry, in vitro enzymology, and modification-specific antibodies, we found that KMT7 monomethylates both Lys-71 and Lys-51 in Tat. K71me is important for full Tat transactivation, as KMT7 knockdown impaired the transcriptional activity of wild type (WT) Tat but not a Tat K71R mutant. These findings underscore the role of KMT7 as an important monomethyltransferase regulating HIV transcription through Tat.

Contribution of Cardiac Sodium Channel ß-Subunit Variants to Brugada Syndrome.

BACKGROUND: Brugada syndrome (BrS) is an inheritable cardiac disease associated with syncope, malignant ventricular arrhythmias and sudden cardiac death. The largest proportion of mutations in BrS is found in the SCN5A gene encoding the α-subunit of cardiac sodium channels (Nav1.5). Causal SCN5A mutations are present in 18-30% of BrS patients. The additional genetic diagnostic yield of variants in cardiac sodium channel ß-subunits in BrS patients was explored and functional studies on 3 novel candidate variants were performed. METHODS AND RESULTS: TheSCN1B-SCN4B genes were screened, which encode the 5 sodium channel ß-subunits, in a SCN5A negative BrS population (n=74). Five novel variants were detected; in silico pathogenicity prediction classified 4 variants as possibly disease causing. Three variants were selected for functional study. These variants caused only limited alterations of Nav1.5 function. Next generation sequencing of a panel of 88 arrhythmia genes could not identify other major causal mutations. CONCLUSIONS: It was hypothesized that the studied variants are not the primary cause of BrS in these patients. However, because small functional effects of these ß-subunit variants can be discriminated, they might contribute to the BrS phenotype and be considered a risk factor. The existence of these risk factors can give an explanation to the reduced penetrance and variable expressivity seen in this syndrome. We therefore recommend including the SCN1-4B genes in a next generation sequencing-based gene panel.

Comprehensive Genetic Characterization of a Spanish Brugada Syndrome Cohort.

BACKGROUND: Brugada syndrome (BrS) is a rare genetic cardiac arrhythmia that can lead to sudden cardiac death in patients with a structurally normal heart. Genetic variations in SCN5A can be identified in approximately 20-25% of BrS cases. The aim of our work was to determine the spectrum and prevalence of genetic variations in a Spanish cohort diagnosed with BrS. METHODOLOGY/PRINCIPAL FINDINGS: We directly sequenced fourteen genes reported to be associated with BrS in 55 unrelated patients clinically diagnosed. Our genetic screening allowed the identification of 61 genetic variants. Of them, 20 potentially pathogenic variations were found in 18 of the 55 patients (32.7% of the patients, 83.3% males). Nineteen of them were located in SCN5A, and had either been previously reported as pathogenic variations or had a potentially pathogenic effect. Regarding the sequencing of the minority genes, we discovered a potentially pathogenic variation in SCN2B that was described to alter sodium current, and one nonsense variant of unknown significance in RANGRF. In addition, we also identified 40 single nucleotide variations which were either synonymous variants (four of them had not been reported yet) or common genetic variants. We next performed MLPA analysis of SCN5A for the 37 patients without an identified genetic variation, and no major rearrangements were detected. Additionally, we show that being at the 30-50 years range or exhibiting symptoms are factors for an increased potentially pathogenic variation discovery yield. CONCLUSIONS: In summary, the present study is the first comprehensive genetic evaluation of 14 BrS-susceptibility genes and MLPA of SCN5A in a Spanish BrS cohort. The mean pathogenic variation discovery yield is higher than that described for other European BrS cohorts (32.7% vs 20-25%, respectively), and is even higher for patients in the 30-50 years age range.

Interplay between R513 methylation and S516 phosphorylation of the cardiac voltage-gated sodium channel.

Arginine methylation is a novel post-translational modification within the voltage-gated ion channel superfamily, including the cardiac sodium channel, NaV1.5. We show that NaV1.5 R513 methylation decreases S516 phosphorylation rate by 4 orders of magnitude, the first evidence of protein kinase A inhibition by arginine methylation. Reciprocally, S516 phosphorylation blocks R513 methylation. NaV1.5 p.G514C, associated to cardiac conduction disease, abrogates R513 methylation, while leaving S516 phosphorylation rate unchanged. This is the first report of methylation-phosphorylation cross-talk of a cardiac ion channel.

Identification of N-terminal protein acetylation and arginine methylation of the voltage-gated sodium channel in end-stage heart failure human heart.

The α subunit of the cardiac voltage-gated sodium channel, NaV1.5, provides the rapid sodium inward current that initiates cardiomyocyte action potentials. Here, we analyzed for the first time the post-translational modifications of NaV1.5 purified from end-stage heart failure human cardiac tissue. We identified R526 methylation as the major post-translational modification of any NaV1.5 arginine or lysine residue. Unexpectedly, we found that the N terminus of NaV1.5 was: 1) devoid of the initiation methionine, and 2) acetylated at the resulting initial alanine residue. This is the first evidence for N-terminal acetylation in any member of the voltage-gated ion channel superfamily. Our results open the door to explore NaV1.5 N-terminal acetylation and arginine methylation levels as drivers or markers of end-stage heart failure.

Protein arginine methyl transferases-3 and -5 increase cell surface expression of cardiac sodium channel.

The α-subunit of the cardiac voltage-gated sodium channel (NaV1.5) plays a central role in cardiomyocyte excitability. We have recently reported that NaV1.5 is post-translationally modified by arginine methylation. Here, we aimed to identify the enzymes that methylate NaV1.5, and to describe the role of arginine methylation on NaV1.5 function. Our results show that protein arginine methyl transferase (PRMT)-3 and -5 methylate NaV1.5 in vitro, interact with NaV1.5 in human embryonic kidney (HEK) cells, and increase NaV1.5 current density by enhancing NaV1.5 cell surface expression. Our observations are the first evidence of regulation of a voltage-gated ion channel, including calcium, potassium, sodium and TRP channels, by arginine methylation.

A missense mutation in the sodium channel ß2 subunit reveals SCN2B as a new candidate gene for Brugada syndrome.

Brugada Syndrome (BrS) is a familial disease associated with sudden cardiac death. A 20%-25% of BrS patients carry genetic defects that cause loss-of-function of the voltage-gated cardiac sodium channel. Thus, 70%-75% of patients remain without a genetic diagnosis. In this work, we identified a novel missense mutation (p.Asp211Gly) in the sodium ß2 subunit encoded by SCN2B, in a woman diagnosed with BrS. We studied the sodium current (INa ) from cells coexpressing Nav 1.5 and wild-type (ß2WT) or mutant (ß2D211G) ß2 subunits. Our electrophysiological analysis showed a 39.4% reduction in INa density when Nav 1.5 was coexpressed with the ß2D211G. Single channel analysis showed that the mutation did not affect the Nav 1.5 unitary channel conductance. Instead, protein membrane detection experiments suggested that ß2D211G decreases Nav 1.5 cell surface expression. The effect of the mutant ß2 subunit on the INa strongly suggests that SCN2B is a new candidate gene associated with BrS.

Activation of HIV transcription by the viral Tat protein requires a demethylation step mediated by lysine-specific demethylase 1 (LSD1/KDM1).

The essential transactivator function of the HIV Tat protein is regulated by multiple posttranslational modifications. Although individual modifications are well characterized, their crosstalk and dynamics of occurrence during the HIV transcription cycle remain unclear.We examine interactions between two critical modifications within the RNA-binding domain of Tat: monomethylation of lysine 51 (K51) mediated by Set7/9/KMT7, an early event in the Tat transactivation cycle that strengthens the interaction of Tat with TAR RNA, and acetylation of lysine 50 (K50) mediated by p300/KAT3B, a later process that dissociates the complex formed by Tat, TAR RNA and the cyclin T1 subunit of the positive transcription elongation factor b (P-TEFb). We find K51 monomethylation inhibited in synthetic Tat peptides carrying an acetyl group at K50 while acetylation can occur in methylated peptides, albeit at a reduced rate. To examine whether Tat is subject to sequential monomethylation and acetylation in cells, we performed mass spectrometry on immunoprecipitated Tat proteins and generated new modification-specific Tat antibodies against monomethylated/acetylated Tat. No bimodified Tat protein was detected in cells pointing to a demethylation step during the Tat transactivation cycle. We identify lysine-specific demethylase 1 (LSD1/KDM1) as a Tat K51-specific demethylase, which is required for the activation of HIV transcription in latently infected T cells. LSD1/KDM1 and its cofactor CoREST associates with the HIV promoter in vivo and activate Tat transcriptional activity in a K51-dependent manner. In addition, small hairpin RNAs directed against LSD1/KDM1 or inhibition of its activity with the monoamine oxidase inhibitor phenelzine suppresses the activation of HIV transcription in latently infected T cells.Our data support the model that a LSD1/KDM1/CoREST complex, normally known as a transcriptional suppressor, acts as a novel activator of HIV transcription through demethylation of K51 in Tat. Small molecule inhibitors of LSD1/KDM1 show therapeutic promise by enforcing HIV latency in infected T cells.

The cardiac sodium channel is post-translationally modified by arginine methylation.

The α subunit of the cardiac sodium channel (Na(v)1.5) is an essential protein in the initial depolarization phase of the cardiomyocyte action potential. Post-translational modifications such as phosphorylation are known to regulate Na(v)1.5 function. Here, we used a proteomic approach for the study of the post-translational modifications of Na(v)1.5 using tsA201 cells as a model system. We generated a stable cell line expressing Na(v)1.5, purified the sodium channel, and analyzed Na(v)1.5 by MALDI-TOF and LC-MS/MS. We report the identification of arginine methylation as a novel post-translational modification of Na(v)1.5. R513, R526, and R680, located in the linker between domains I and II in Na(v)1.5, were found in mono- or dimethylated states. The functional relevance of arginine methylation in Na(v)1.5 is underscored by the fact that R526H and R680H are known Na(v)1.5 mutations causing Brugada and long QT type 3 syndromes, respectively. Our work describes for the first time arginine methylation in the voltage-gated ion channel superfamily.

Characterization of HIV Tat modifications using novel methyl-lysine-specific antibodies.

Modification-specific antibodies are important tools to examine the dynamics and functions of posttranslational protein modifications in cells. Here, we describe in detail the generation of polyclonal antibodies specific for mono-, di-, and trimethylated lysine 51 within the HIV transactivator Tat. Lysine 51 is a highly conserved residue located in the RNA-binding region of Tat and the target of lysine methyltransferases KMT1E (SETDB1) and KMT7 (Set7/9). Using affinity-purified methyl-specific antibodies of Tat, we find that cellular Tat is predominantly monomethylated at lysine 51, a modification enhanced by coexpression of KMT7.