Rare variants in IFFO1, DTNB, NLRC3 and SLC22A10 associate with Alzheimer's disease CSF profile of neuronal injury and inflammation.

Alzheimer's disease (AD) biomarkers represent several neurodegenerative processes, such as synaptic dysfunction, neuronal inflammation and injury, as well as amyloid pathology. We performed an exome-wide rare variant analysis of six AD biomarkers (ß-amyloid, total/phosphorylated tau, NfL, YKL-40, and Neurogranin) to discover genes associated with these markers. Genetic and biomarker information was available for 480 participants from two studies: EMIF-AD and ADNI. We applied a principal component (PC) analysis to derive biomarkers combinations, which represent statistically independent biological processes. We then tested whether rare variants in 9576 protein-coding genes associate with these PCs using a Meta-SKAT test. We also tested whether the PCs are intermediary to gene effects on AD symptoms with a SMUT test. One PC loaded on NfL and YKL-40, indicators of neuronal injury and inflammation. Four genes were associated with this PC: IFFO1, DTNB, NLRC3, and SLC22A10. Mediation tests suggest, that these genes also affect dementia symptoms via inflammation/injury. We also observed an association between a PC loading on Neurogranin, a marker for synaptic functioning, with GABBR2 and CASZ1, but no mediation effects. The results suggest that rare variants in IFFO1, DTNB, NLRC3, and SLC22A10 heighten susceptibility to neuronal injury and inflammation, potentially by altering cytoskeleton structure and immune activity disinhibition, resulting in an elevated dementia risk. GABBR2 and CASZ1 were associated with synaptic functioning, but mediation analyses suggest that the effect of these two genes on synaptic functioning is not consequential for AD development.

Whole-exome rare-variant analysis of Alzheimer's disease and related biomarker traits.

INTRODUCTION: Despite increasing evidence of a role of rare genetic variation in the risk of Alzheimer's disease (AD), limited attention has been paid to its contribution to AD-related biomarker traits indicative of AD-relevant pathophysiological processes. METHODS: We performed whole-exome gene-based rare-variant association studies (RVASs) of 17 AD-related traits on whole-exome sequencing (WES) data generated in the European Medical Information Framework for Alzheimer's Disease Multimodal Biomarker Discovery (EMIF-AD MBD) study (n = 450) and whole-genome sequencing (WGS) data from ADNI (n = 808). RESULTS: Mutation screening revealed a novel probably pathogenic mutation (PSEN1 p.Leu232Phe). Gene-based RVAS revealed the exome-wide significant contribution of rare coding variation in RBKS and OR7A10 to cognitive performance and protection against left hippocampal atrophy, respectively. DISCUSSION: The identification of these novel gene-trait associations offers new perspectives into the role of rare coding variation in the distinct pathophysiological processes culminating in AD, which may lead to identification of novel therapeutic and diagnostic targets.

Mitochondrial GpC and CpG DNA Hypermethylation Cause Metabolic Stress-Induced Mitophagy and Cholestophagy.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by a constant accumulation of lipids in the liver. This hepatic lipotoxicity is associated with a dysregulation of the first step in lipid catabolism, known as beta oxidation, which occurs in the mitochondrial matrix. Eventually, this dysregulation will lead to mitochondrial dysfunction. To evaluate the possible involvement of mitochondrial DNA methylation in this lipid metabolic dysfunction, we investigated the functional metabolic effects of mitochondrial overexpression of CpG (MSssI) and GpC (MCviPI) DNA methyltransferases in relation to gene expression and (mito)epigenetic signatures. Overall, the results show that mitochondrial GpC and, to a lesser extent, CpG methylation increase bile acid metabolic gene expression, inducing the onset of cholestasis through mito-nuclear epigenetic reprogramming. Moreover, both increase the expression of metabolic nuclear receptors and thereby induce basal overactivation of mitochondrial respiration. The latter promotes mitochondrial swelling, favoring lipid accumulation and metabolic-stress-induced mitophagy and autophagy stress responses. In conclusion, both mitochondrial GpC and CpG methylation create a metabolically challenging environment that induces mitochondrial dysfunction, which may contribute to the progression of MASLD.

Structural variants identified by Oxford Nanopore PromethION sequencing of the human genome.

We sequenced the genome of the Yoruban reference individual NA19240 on the long-read sequencing platform Oxford Nanopore PromethION for evaluation and benchmarking of recently published aligners and germline structural variant calling tools, as well as a comparison with the performance of structural variant calling from short-read sequencing data. The structural variant caller Sniffles after NGMLR or minimap2 alignment provides the most accurate results, but additional confidence or sensitivity can be obtained by a combination of multiple variant callers. Sensitive and fast results can be obtained by minimap2 for alignment and a combination of Sniffles and SVIM for variant identification. We describe a scalable workflow for identification, annotation, and characterization of tens of thousands of structural variants from long-read genome sequencing of an individual or population. By discussing the results of this well-characterized reference individual, we provide an approximation of what can be expected in future long-read sequencing studies aiming for structural variant identification.

Loss of DPP6 in neurodegenerative dementia: a genetic player in the dysfunction of neuronal excitability.

Emerging evidence suggested a converging mechanism in neurodegenerative brain diseases (NBD) involving early neuronal network dysfunctions and alterations in the homeostasis of neuronal firing as culprits of neurodegeneration. In this study, we used paired-end short-read and direct long-read whole genome sequencing to investigate an unresolved autosomal dominant dementia family significantly linked to 7q36. We identified and validated a chromosomal inversion of ca. 4 Mb, segregating on the disease haplotype and disrupting the coding sequence of dipeptidyl-peptidase 6 gene (DPP6). DPP6 resequencing identified significantly more rare variants-nonsense, frameshift, and missense-in early-onset Alzheimer's disease (EOAD, p value = 0.03, OR = 2.21 95% CI 1.05-4.82) and frontotemporal dementia (FTD, p = 0.006, OR = 2.59, 95% CI 1.28-5.49) patient cohorts. DPP6 is a type II transmembrane protein with a highly structured extracellular domain and is mainly expressed in brain, where it binds to the potassium channel Kv4.2 enhancing its expression, regulating its gating properties and controlling the dendritic excitability of hippocampal neurons. Using in vitro modeling, we showed that the missense variants found in patients destabilize DPP6 and reduce its membrane expression (p < 0.001 and p < 0.0001) leading to a loss of protein. Reduced DPP6 and/or Kv4.2 expression was also detected in brain tissue of missense variant carriers. Loss of DPP6 is known to cause neuronal hyperexcitability and behavioral alterations in Dpp6-KO mice. Taken together, the results of our genomic, genetic, expression and modeling analyses, provided direct evidence supporting the involvement of DPP6 loss in dementia. We propose that loss of function variants have a higher penetrance and disease impact, whereas the missense variants have a variable risk contribution to disease that can vary from high to low penetrance. Our findings of DPP6, as novel gene in dementia, strengthen the involvement of neuronal hyperexcitability and alteration in the homeostasis of neuronal firing as a disease mechanism to further investigate.

miRVaS: a tool to predict the impact of genetic variants on miRNAs.

Genetic variants in or near miRNA genes can have profound effects on miRNA expression and targeting. As user-friendly software for the impact prediction of miRNA variants on a large scale is still lacking, we created a tool called miRVaS. miRVaS automates this prediction by annotating the location of the variant relative to functional regions within the miRNA hairpin (seed, mature, loop, hairpin arm, flanks) and by annotating all predicted structural changes within the miRNA due to the variant. In addition, the tool defines the most important region that is predicted to have structural changes and calculates a conservation score that is indicative of the reliability of the structure prediction. The output is presented in a tab-separated file, which enables fast screening, and in an html file, which allows visual comparison between wild-type and variant structures. All separate images are provided for downstream use. Finally, we tested two different approaches on a small test set of published functionally validated genetic variants for their capacity to predict the impact of variants on miRNA expression.

Novel mutations in genes causing hereditary spastic paraplegia and Charcot-Marie-Tooth neuropathy identified by an optimized protocol for homozygosity mapping based on whole-exome sequencing.

PURPOSE: Homozygosity mapping is an effective approach for detecting molecular defects in consanguineous families by delineating stretches of genomic DNA that are identical by descent. Constant developments in next-generation sequencing created possibilities to combine whole-exome sequencing (WES) and homozygosity mapping in a single step. METHODS: Basic optimization of homozygosity mapping parameters was performed in a group of families with autosomal-recessive (AR) mutations for which both single-nucleotide polymorphism (SNP) array and WES data were available. We varied the criteria for SNP extraction and PLINK thresholds to estimate their effect on the accuracy of homozygosity mapping based on WES. RESULTS: Our protocol showed high specificity and sensitivity for homozygosity detection and facilitated the identification of novel mutations in GAN, GBA2, and ZFYVE26 in four families affected by hereditary spastic paraplegia or Charcot-Marie-Tooth disease. Filtering and mapping with optimized parameters was integrated into the HOMWES (homozygosity mapping based on WES analysis) tool in the GenomeComb package for genomic data analysis. CONCLUSION: We present recommendations for detection of homozygous regions based on WES data and a bioinformatics tool for their identification, which can be widely applied for studying AR disorders.Genet Med 18 6, 600-607.

CMT-associated mutations in glycyl- and tyrosyl-tRNA synthetases exhibit similar pattern of toxicity and share common genetic modifiers in Drosophila.

Aminoacyl-tRNA synthetases are ubiquitously expressed proteins that charge tRNAs with their cognate amino acids. By ensuring the fidelity of protein synthesis, these enzymes are essential for the viability of every cell. Yet, mutations in six tRNA synthetases specifically affect the peripheral nerves and cause Charcot-Marie-Tooth (CMT) disease. The CMT-causing mutations in tyrosyl- and glycyl-tRNA synthetases (YARS and GARS, respectively) alter the activity of the proteins in a range of ways (some mutations do not impact charging function, while others abrogate it), making a loss of function in tRNA charging unlikely to be the cause of disease pathology. It is currently unknown which cellular mechanisms are triggered by the mutant enzymes and how this leads to neurodegeneration. Here, by expressing two pathogenic mutations (G240R, P234KY) in Drosophila, we generated a model for GARS-associated neuropathy. We observed compromised viability, and behavioral, electrophysiological and morphological impairment in flies expressing the cytoplasmic isoform of mutant GARS. Their features recapitulated several hallmarks of CMT pathophysiology and were similar to the phenotypes identified in our previously described Drosophila model of YARS-associated neuropathy. Furthermore, CG8316 and CG15599 - genes identified in a retinal degeneration screen to modify mutant YARS, also modified the mutant GARS phenotypes. Our study presents genetic evidence for common mutant-specific interactions between two CMT-associated aminoacyl-tRNA synthetases, lending support for a shared mechanism responsible for the synthetase-induced peripheral neuropathies.

Combinatorial optimization of gene expression through recombinase-mediated promoter and terminator shuffling in yeast.

Microbes are increasingly employed as cell factories to produce biomolecules. This often involves the expression of complex heterologous biosynthesis pathways in host strains. Achieving maximal product yields and avoiding build-up of (toxic) intermediates requires balanced expression of every pathway gene. However, despite progress in metabolic modeling, the optimization of gene expression still heavily relies on trial-and-error. Here, we report an approach for in vivo, multiplexed Gene Expression Modification by LoxPsym-Cre Recombination (GEMbLeR). GEMbLeR exploits orthogonal LoxPsym sites to independently shuffle promoter and terminator modules at distinct genomic loci. This approach facilitates creation of large strain libraries, in which expression of every pathway gene ranges over 120-fold and each strain harbors a unique expression profile. When applied to the biosynthetic pathway of astaxanthin, an industrially relevant antioxidant, a single round of GEMbLeR improved pathway flux and doubled production titers. Together, this shows that GEMbLeR allows rapid and efficient gene expression optimization in heterologous biosynthetic pathways, offering possibilities for enhancing the performance of microbial cell factories.

Identification of rare copy number variants in high burden schizophrenia families.

Over the last years, genome-wide studies consistently showed an increased burden of rare copy number variants (CNVs) in schizophrenia patients, supporting the "common disease, rare variant" hypothesis in at least a subset of patients. We hypothesize that in families with a high burden of disease, and thus probably a high genetic load influencing disease susceptibility, rare CNVs might be involved in the etiology of schizophrenia. We performed a genome-wide CNV analysis in the index patients of eight families with multiple schizophrenia affected members, and consecutively performed a detailed family analysis for the most relevant CNVs. One index patient showed a DRD5 containing duplication. A second index patient presented with an NRXN1 containing deletion and two adjacent duplications containing MYT1L and SNTG2. Detailed analysis in the subsequent families showed segregation of the identified CNVs. With this study we show the importance of screening high burden families for rare CNVs, which will not only broaden our knowledge concerning the molecular genetic mechanisms involved in schizophrenia but also allow the use of the obtained genetic data to provide better clinical care to these families in general and to non-symptomatic causal CNV carriers in particular.

Mitochondrial DNA methylation in metabolic associated fatty liver disease.

Introduction: Hepatic lipid accumulation and mitochondrial dysfunction are hallmarks of metabolic associated fatty liver disease (MAFLD), yet molecular parameters underlying MAFLD progression are not well understood. Differential methylation within the mitochondrial DNA (mtDNA) has been suggested to be associated with dysfunctional mitochondria, also during progression to Metabolic Steatohepatitis (MeSH). This study further investigates whether mtDNA methylation is associated with hepatic lipid accumulation and MAFLD. Methods: HepG2 cells were constructed to stably express mitochondria-targeted viral and prokaryotic cytosine DNA methyltransferases (mtM.CviPI or mtM.SssI for GpC or CpG methylation, respectively). A catalytically inactive variant (mtM.CviPI-Mut) was constructed as a control. Mouse and human patients' samples were also investigated. mtDNA methylation was assessed by pyro- or nanopore sequencing. Results and discussion: Differentially induced mtDNA hypermethylation impaired mitochondrial gene expression and metabolic activity in HepG2-mtM.CviPI and HepG2-mtM.SssI cells and was associated with increased lipid accumulation, when compared to the controls. To test whether lipid accumulation causes mtDNA methylation, HepG2 cells were subjected to 1 or 2 weeks of fatty acid treatment, but no clear differences in mtDNA methylation were detected. In contrast, hepatic Nd6 mitochondrial gene body cytosine methylation and Nd6 gene expression were increased in mice fed a high-fat high cholesterol diet (HFC for 6 or 20 weeks), when compared to controls, while mtDNA content was unchanged. For patients with simple steatosis, a higher ND6 methylation was confirmed using Methylation Specific PCR, but no additional distinctive cytosines could be identified using pyrosequencing. This study warrants further investigation into a role for mtDNA methylation in promoting mitochondrial dysfunction and impaired lipid metabolism in MAFLD.

SCAMP5, NBEA and AMISYN: three candidate genes for autism involved in secretion of large dense-core vesicles.

Autism is a neurodevelopmental disorder characterized by impaired social reciprocity, impaired communication and stereotypical behaviors. Despite strong evidence for a genetic basis, few susceptibility genes have been identified. Here, we describe the positional cloning of SCAMP5, CLIC4 and PPCDC as candidate genes for autism, starting from a person with idiopathic, sporadic autism carrying a de novo chromosomal translocation. One of these genes, SCAMP5 is silenced on the derivative chromosome, and encodes a brain-enriched protein involved in membrane trafficking, similar to the previously identified candidate genes NBEA and AMISYN. Gene silencing of Nbea, Amisyn and Scamp5 in mouse beta-TC3 cells resulted in a 2-fold increase in stimulated secretion of large dense-core vesicles (LDCVs), while overexpression suppressed secretion. Moreover, ultrastructural analysis of blood platelets from the patients with haploinsufficieny of one of the three candidate genes, showed morphological abnormalities of dense-core granules, which closely resemble LDCVs. Taken together, this study shows that in three independent patients with autism three different negative regulators of LDCV secretion are affected, respectively, suggesting that in at least a subgroup of patients the regulation of neuronal vesicle trafficking may be involved in the pathogenesis of autism.

Less cognitive and neurological deficits in schizophrenia patients carrying risk variant in ZNF804A.

BACKGROUND: The rs1344706 single nucleotide polymorphism in the ZNF804A gene is a common variant with strong evidence for association with schizophrenia. Recent studies show an association of rs1344706 with cognitive functioning, and there is some evidence suggesting that the risk allele may increase susceptibility for a subtype of schizophrenia with relatively spared cognition. METHODS: We tested the effect of rs1344706 genotype in 89 schizophrenia patients on 3 basic cognitive domains (working memory, processing speed and attention) shown to be severely impaired in schizophrenia. Also we investigated the effect of rs1344706 on the severity of neurological soft signs, subtle impairments in motor and sensory functions highly frequent in schizophrenia patients. Neurological soft signs and cognitive deficits are central features of schizophrenia and are tightly linked with clinical, social and functional outcome. RESULTS: Our results show an association of higher rs1344706 risk allele load with improved performance on processing speed and with fewer neurological soft signs. CONCLUSIONS: Together with other studies, our findings suggest that ZNF804A is associated with a subtype of schizophrenia with better cognitive and neurological functioning. Discovery of the specific pathways through which ZNF804A is exerting this effect may lead to better prevention, diagnosis and treatment for a specific group of schizophrenia patients.

Rare copy number variants in neuropsychiatric disorders: Specific phenotype or not?

From a number of genome-wide association studies it was shown that de novo and/or rare copy number variants (CNVs) are found at an increased frequency in neuropsychiatric diseases. In this study we examined the prevalence of CNVs in six genomic regions (1q21.1, 2p16.3, 3q29, 15q11.2, 15q13.3, and 16p11.2) previously implicated in neuropsychiatric diseases. Hereto, a cohort of four neuropsychiatric disorders (schizophrenia, bipolar disorder, major depressive disorder, and intellectual disability) and control individuals from three different populations was used in combination with Multilpex Amplicon Quantifiaction (MAQ) assays, capable of high resolution (kb range) and custom-tailored CNV detection. Our results confirm the etiological candidacy of the six selected CNV regions for neuropsychiatric diseases. It is possible that CNVs in these regions can result in disturbed brain development and in this way lead to an increased susceptibility for different neuropsychiatric disorders, dependent on additional genetic and environmental factors. Our results also suggest that the neurodevelopmental component is larger in the etiology of schizophrenia and intellectual disability than in mood disorders. Finally, our data suggest that deletions are in general more pathogenic than duplications. Given the high frequency of the examined CNVs (1-2%) in patients of different neuropsychiatric disorders, screening of large cohorts with an affordable and feasible method like the MAQ assays used in this study is likely to result in important progress in unraveling the genetic factors leading to an increased susceptibility for several psychiatric disorders.

Identification of a CACNA2D4 deletion in late onset bipolar disorder patients and implications for the involvement of voltage-dependent calcium channels in psychiatric disorders.

The GWAS-based association of CACNA1C with bipolar disorder (BPD) is one of the strongest genetic findings to date. CACNA1C belongs to the family of CACN genes encoding voltage-dependent calcium channels (VDCCs). VDCCs are involved in brain circuits and cognitive processes implicated in BPD and schizophrenia (SZ). Recently, it was shown that rare copy number variations (CNVs) are found at an increased frequency in SZ and to a lesser extent also in BPD, suggesting the involvement of CNVs in the causation of these diseases. We hypothesize that CNVs in CACN genes can influence the susceptibility to BPD, SZ, and/or schizoaffective disorder (SZA). A search for CNVs in eight CACN genes in a patient-control sample of European decent was performed. A total of 709 BP patients, 645 SZ patients, 189 SZA patients, and 1,470 control individuals were screened using the Multiplex Amplicon Quantification (MAQ) method. We found a rare, partial deletion of 35.7 kb in CACNA2D4 in two unrelated late onset bipolar I patients and in one control individual. All three deletions shared the same breakpoints removing exons 17-26 of CACNA2D4, comprising part of the CACHE domain. Based on the data we cannot claim causality to BPD of the identified CACNA2D4 deletion but nevertheless this deletion can be important in unraveling the underlying processes leading to psychiatric diseases in general and BPD in particular.

Dissection of TAF1 neuronal splicing and implications for neurodegeneration in X-linked dystonia-parkinsonism.

X-linked dystonia-parkinsonism (XDP) is a monogenic neurodegenerative disorder of the basal ganglia, which presents as a combination of hyperkinetic movements and parkinsonian features. The underlying genetic mechanism involves the insertion of a SINE-VNTR-Alu retrotransposon within the TAF1 gene. Interestingly, alterations of TAF1 have been involved in multiple neurological diseases. In XDP, the SINE-VNTR-Alu insertion in TAF1 has been proposed to result in alternative splicing defects, including the decreased incorporation of a neuron-specific microexon annotated as 34'. This mechanism has become controversial as recent studies failed to provide support. In order to resolve this conundrum, we examined the alternative splicing patterns of TAF1 mRNAs in XDP and control brains. The impact of the disease-associated SINE-VNTR-Alu on alternative splicing of microexon 34' was further investigated in cellular assays. Subsequently, microexon 34' incorporation was explored by RT-PCR and Nanopore long-read sequencing of TAF1 mRNAs from XDP and control brains tissues. Using cell-based splicing assays, we demonstrate that presence of the disease-associated SINE-VNTR-Alu does not affect the inclusion of microexon 34'. In addition, we show that (1) microexon 34'-containing TAF1 mRNAs are detected at similar levels in XDP as in controls and that (2) the architecture of TAF1 transcripts is remarkably similar between XDP and controls brains. These results indicate that microexon 34' incorporation into TAF1 mRNA is not affected in XDP brains. Our findings shift the current paradigm of XDP by discounting alternative splicing of TAF1 microexon 34' as the molecular basis for this disease.

Long-Read Sequencing to Unravel Complex Structural Variants of CEP78 Leading to Cone-Rod Dystrophy and Hearing Loss.

Inactivating variants as well as a missense variant in the centrosomal CEP78 gene have been identified in autosomal recessive cone-rod dystrophy with hearing loss (CRDHL), a rare syndromic inherited retinal disease distinct from Usher syndrome. Apart from this, a complex structural variant (SV) implicating CEP78 has been reported in CRDHL. Here we aimed to expand the genetic architecture of typical CRDHL by the identification of complex SVs of the CEP78 region and characterization of their underlying mechanisms. Approaches used for the identification of the SVs are shallow whole-genome sequencing (sWGS) combined with quantitative polymerase chain reaction (PCR) and long-range PCR, or ExomeDepth analysis on whole-exome sequencing (WES) data. Targeted or whole-genome nanopore long-read sequencing (LRS) was used to delineate breakpoint junctions at the nucleotide level. For all SVs cases, the effect of the SVs on CEP78 expression was assessed using quantitative PCR on patient-derived RNA. Apart from two novel canonical CEP78 splice variants and a frameshifting single-nucleotide variant (SNV), two SVs affecting CEP78 were identified in three unrelated individuals with CRDHL: a heterozygous total gene deletion of 235 kb and a partial gene deletion of 15 kb in a heterozygous and homozygous state, respectively. Assessment of the molecular consequences of the SVs on patient's materials displayed a loss-of-function effect. Delineation and characterization of the 15-kb deletion using targeted LRS revealed the previously described complex CEP78 SV, suggestive of a recurrent genomic rearrangement. A founder haplotype was demonstrated for the latter SV in cases of Belgian and British origin, respectively. The novel 235-kb deletion was delineated using whole-genome LRS. Breakpoint analysis showed microhomology and pointed to a replication-based underlying mechanism. Moreover, data mining of bulk and single-cell human and mouse transcriptional datasets, together with CEP78 immunostaining on human retina, linked the CEP78 expression domain with its phenotypic manifestations. Overall, this study supports that the CEP78 locus is prone to distinct SVs and that SV analysis should be considered in a genetic workup of CRDHL. Finally, it demonstrated the power of sWGS and both targeted and whole-genome LRS in identifying and characterizing complex SVs in patients with ocular diseases.

Critical length in long-read resequencing.

Long-read sequencing has substantial advantages for structural variant discovery and phasing of variants compared to short-read technologies, but the required and optimal read length has not been assessed. In this work, we used long reads simulated from human genomes and evaluated structural variant discovery and variant phasing using current best practice bioinformatics methods. We determined that optimal discovery of structural variants from human genomes can be obtained with reads of minimally 20 kb. Haplotyping variants across genes only reaches its optimum from reads of 100 kb. These findings are important for the design of future long-read sequencing projects.

Simultaneous mutation and copy number variation (CNV) detection by multiplex PCR-based GS-FLX sequencing.

We evaluated multiplex PCR amplification as a front-end for high-throughput sequencing, to widen the applicability of massive parallel sequencers for the detailed analysis of complex genomes. Using multiplex PCR reactions, we sequenced the complete coding regions of seven genes implicated in peripheral neuropathies in 40 individuals on a GS-FLX genome sequencer (Roche). The resulting dataset showed highly specific and uniform amplification. Comparison of the GS-FLX sequencing data with the dataset generated by Sanger sequencing confirmed the detection of all variants present and proved the sensitivity of the method for mutation detection. In addition, we showed that we could exploit the multiplexed PCR amplicons to determine individual copy number variation (CNV), increasing the spectrum of detected variations to both genetic and genomic variants. We conclude that our straightforward procedure substantially expands the applicability of the massive parallel sequencers for sequencing projects of a moderate number of amplicons (50-500) with typical applications in resequencing exons in positional or functional candidate regions and molecular genetic diagnostics.

Unusual resistance patterns in macrolide-resistant Streptococcus pyogenes harbouring erm(A).

BACKGROUND: We identified erm(A)-harbouring Streptococcus pyogenes that expressed three variant phenotypes: (1) low-level resistance to erythromycin (MICs 1-4 mg/L) but high azithromycin MICs in absolute terms (16-64 mg/L; n=6); (2) same as (1) but with a high clindamycin MIC (256 mg/L; n=1); and (3) high-level constitutive MLS (cMLS) resistance (n=1). Here we analysed the genetic basis of these novel phenotypes. METHODS: The presence of erm(A) and the absence of macrolide/lincosamide resistance genes erm(B), mef and cfr were confirmed by PCR. erm(A), 23S rRNA, L4 and L22 genes were sequenced. Mutant erm(A) genes were cloned and electrotransformed into the macrolide-susceptible Escherichia coli AG100A. Clonality was determined by emm typing and PFGE. Effects of the identified mutations on free energy changes (DeltaG) and putative configurations of the leader sequence were studied in silico. RESULTS: Point mutations (G98A, A137C, C140T and G205A) were observed in the erm(A) regulatory region of all eight erm(A)-harbouring S. pyogenes. Five and two isolates belonged to emm77 and emm89 clones, respectively, and one isolate was an emm1. E. coli transformed with mutant erm(A) harbouring G98A, A137C or C140T mutations (phenotypes 1 and 2) did not express high-level azithromycin or clindamycin resistance. However, cMLS resistance was clearly observed in transformants with erm(A) harbouring both A137C and G205A mutations (phenotype 3). In silico analysis showed that DeltaG was minor except for the G205A mutation. Secondary structure predictions further showed that the A137C and G205A mutations together abolished the hairpin sequestering the ribosome-binding and initiation sites of the erm(A) gene, explaining the cMLS phenotype 3. CONCLUSIONS: We report point mutations in the erm(A) regulatory region leading to constitutive methylase expression and the presence of additional, as yet unidentified mechanisms mediating high-level azithromycin and clindamycin resistance in erm(A)-harbouring S. pyogenes.