The potassium-chloride co-transporter KCC2, encoded by SLC12A5, plays a fundamental role in fast synaptic inhibition by maintaining a hyperpolarizing gradient for chloride ions. KCC2 dysfunction has been implicated in human epilepsy, but to date, no monogenic KCC2-related epilepsy disorders have been described. Here we show recessive loss-of-function SLC12A5 mutations in patients with a severe infantile-onset pharmacoresistant epilepsy syndrome, epilepsy of infancy with migrating focal seizures (EIMFS). Decreased KCC2 surface expression, reduced protein glycosylation and impaired chloride extrusion contribute to loss of KCC2 activity, thereby impairing normal synaptic inhibition and promoting neuronal excitability in this early-onset epileptic encephalopathy.

Coenzyme Q is an essential mitochondrial electron carrier, redox cofactor and a potent antioxidant in the majority of cellular membranes. Coenzyme Q deficiency has been associated with a range of metabolic diseases, as well as with some drug treatments and ageing.

Complex chromosomal rearrangements (CCRs) are rearrangements involving more than two chromosomes or more than two breakpoints. Whole genome sequencing (WGS) allows for outstanding high resolution characterization on the nucleotide level in unique sequences of such rearrangements, but problems remain for mapping breakpoints in repetitive regions of the genome, which are known to be prone to rearrangements. Hence, multiple complementary WGS experiments are sometimes needed to solve the structures of CCRs. We have studied three individuals with CCRs: Case 1 and Case 2 presented with de novo karyotypically balanced, complex interchromosomal rearrangements (46,XX,t(2;8;15)(q35;q24.1;q22) and 46,XY,t(1;10;5)(q32;p12;q31)), and Case 3 presented with a de novo, extremely complex intrachromosomal rearrangement on chromosome 1. Molecular cytogenetic investigation revealed cryptic deletions in the breakpoints of chromosome 2 and 8 in Case 1, and on chromosome 10 in Case 2, explaining their clinical symptoms. In Case 3, 26 breakpoints were identified using WGS, disrupting five known disease genes. All rearrangements were subsequently analyzed using optical maps, linked-read WGS, and short-read WGS. In conclusion, we present a case series of three unique de novo CCRs where we by combining the results from the different technologies fully solved the structure of each rearrangement. The power in combining short-read WGS with long-molecule sequencing or optical mapping in these unique de novo CCRs in a clinical setting is demonstrated.

The current human reference sequence (GRCh38) is a foundation for large-scale sequencing projects. However, recent studies have suggested that GRCh38 may be incomplete and give a suboptimal representation of specific population groups. Here, we performed a de novo assembly of two Swedish genomes that revealed over 10 Mb of sequences absent from the human GRCh38 reference in each individual. Around 6 Mb of these novel sequences (NS) are shared with a Chinese personal genome. The NS are highly repetitive, have an elevated GC-content, and are primarily located in centromeric or telomeric regions. Up to 1 Mb of NS can be assigned to chromosome Y, and large segments are also missing from GRCh38 at chromosomes 14, 17, and 21. Inclusion of NS into the GRCh38 reference radically improves the alignment and variant calling from short-read whole-genome sequencing data at several genomic loci. A re-analysis of a Swedish population-scale sequencing project yields > 75,000 putative novel single nucleotide variants (SNVs) and removes > 10,000 false positive SNV calls per individual, some of which are located in protein coding regions. Our results highlight that the GRCh38 reference is not yet complete and demonstrate that personal genome assemblies from local populations can improve the analysis of short-read whole-genome sequencing data.

Clustered copy number variants (CNVs) as detected by chromosomal microarray analysis (CMA) are often reported as germline chromothripsis. However, such cases might need further investigations by massive parallel whole genome sequencing (WGS) in order to accurately define the underlying complex rearrangement, predict the occurrence mechanisms and identify additional complexities. Here, we utilized WGS to delineate the rearrangement structure of 21 clustered CNV carriers first investigated by CMA and identified a total of 83 breakpoint junctions (BPJs). The rearrangements were further sub-classified depending on the patterns observed: I) Cases with only deletions (n = 8) often had additional structural rearrangements, such as insertions and inversions typical to chromothripsis; II) cases with only duplications (n = 7) or III) combinations of deletions and duplications (n = 6) demonstrated mostly interspersed duplications and BPJs enriched with microhomology. In two cases the rearrangement mutational signatures indicated both a breakage-fusion-bridge cycle process and haltered formation of a ring chromosome. Finally, we observed two cases with Alu- and LINE-mediated rearrangements as well as two unrelated individuals with seemingly identical clustered CNVs on 2p25.3, possibly a rare European founder rearrangement. In conclusion, through detailed characterization of the derivative chromosomes we show that multiple mechanisms are likely involved in the formation of clustered CNVs and add further evidence for chromoanagenesis mechanisms in both "simple" and highly complex chromosomal rearrangements. Finally, WGS characterization adds positional information, important for a correct clinical interpretation and deciphering mechanisms involved in the formation of these rearrangements.

Highly penetrant cancer syndromes account for less than 5% of all cases with familial colorectal cancer (CRC), and other genetic contribution explains the majority of the genetic contribution to CRC. A CRC susceptibility locus on chromosome 9q has been suggested. In this study, families where risk of CRC was linked to the region, were used to search for predisposing mutations in all genes in the region. No disease-causing mutation was found. Next, haplotype association studies were performed in the region, comparing Swedish CRC cases (2664) and controls (4782). Two overlapping haplotypes were suggested. One 10-SNP haplotype was indicated in familial CRC (OR 1.4, p = 0.00005) and one 25-SNP haplotype was indicated in sporadic CRC (OR 2.2, p = 0.0000012). The allele frequencies of the 10-SNP and the 25-SNP haplotypes were 13.7% and 2.5% respectively and both included one RNA, RP11-332M4.1 and RP11-l80l4.2, in the non-overlapping regions. The sporadic 25-SNP haplotype could not be studied further, but the familial 10-SNP haplotype was analyzed in 61 additional CRC families, and 6 of them were informative for all markers and had the risk haplotype. Targeted sequencing of the 10-SNP region in the linked families identified one variant in RP11-332M4.1, suggestive to confer the increased CRC risk on this haplotype. Our results support the presence of two loci at 9q22.32, each with one RNA as the putative cause of increased CRC risk. These RNAs could exert their effect through the same, or different, genes/pathways, possibly through the regulation of neighboring genes, such as PTCH1, FANCC, DKFZP434H0512, ERCC6L2 or the processed transcript LINC00046.

A 160 kb minimal common region in Xp21 has been determined as the cause of XY gonadal dysgenesis, if duplicated. The region contains the MAGEB genes and the NR0B1 gene; this is the candidate for gonadal dysgenesis if overexpressed. Most patients present gonadal dysgenesis within a more complex phenotype. However, few independent cases have recently been described presenting with isolated XY gonadal dysgenesis caused by relatively small NR0B1 locus duplications. We have identified another NR0B1 duplication in two sisters with isolated XY gonadal dysgenesis with an X-linked inheritance pattern. We performed X-inactivation studies in three fertile female carriers of three different small NR0B1 locus duplications identified by our group. The carrier mothers did not show obvious skewing of X-chromosome inactivation, suggesting that NR0B1 overexpression does not impair ovarian function. We furthermore emphasize the importance to investigate the NR0B1 locus also in patients with isolated XY gonadal dysgenesis.

In Kinetoplastids, protein-coding genes are transcribed polycistronically by RNA polymerase II. Individual mature mRNAs are generated from polycistronic precursors by 5' trans splicing of a 39-nt capped leader RNA and 3' polyadenylation. It was previously known that trans splicing generally occurs at an AG dinucleotide downstream of a polypyrimidine tract, and that polyadenylation is coupled to downstream trans splicing. The few polyadenylation sites that had been examined were 100-400 nt upstream of the polypyrimidine tract which marked the adjacent trans splice site. We wished to define the sequence requirements for trypanosome mRNA processing more tightly and to generate a predictive algorithm. By scanning all available Trypanosoma brucei cDNAs for splicing and polyadenylation sites, we found that trans splicing generally occurs at the first AG following a polypyrimidine tract of 8-25 nt, giving rise to 5'-UTRs of a median length of 68 nt. We also found that in general, polyadenylation occurs at a position with one or more A residues located between 80 and 140 nt from the downstream polypyrimidine tract. These data were used to calibrate free parameters in a grammar model with distance constraints, enabling prediction of polyadenylation and trans splice sites for most protein-coding genes in the trypanosome genome. The data from the genome analysis and the program are available from: .

Temporal lobe resection for epilepsy involves a risk of damaging the anterior part of the optic radiation, Meyer's loop, causing a contralateral upper quadrant visual field defect. This study aims to assess the intersubject variability in the course of Meyer's loop in vivo by diffusion tensor imaging and tractography.

Trypanosomatids are widespread parasites that cause three major tropical diseases. In trypanosomatids, as in most other organisms, acetylation is a common protein modification that is important in multiple, diverse processes. This paper describes a new member of the Trypanosoma cruzi acetyltransferase family. The gene is single copy and orthologs are also present in the other two sequenced trypanosomatids, Trypanosoma brucei and Leishmania major. This protein (TcAT-1) has the essential motifs present in members of the GCN5-related acetyltransferase (GNAT) family, as well as an additional motif also found in some enzymes from plant and animal species. The protein is evolutionarily more closely related to this group of enzymes than to histone acetyltransferases. The native protein has a cytosolic cellular location and is present in all three life-cycle stages of the parasite. The recombinant protein was shown to have autoacetylation enzymatic activity.

Well-ordered HIV-1 envelope glycoprotein (Env) trimers are prioritized for clinical evaluation, and there is a need for an improved understanding about how elicited B cell responses evolve following immunization. To accomplish this, we prime-boosted rhesus macaques with clade C NFL trimers and identified 180 unique Ab lineages from ∼1,000 single-sorted Env-specific memory B cells. We traced all lineages in high-throughput heavy chain (HC) repertoire (Rep-seq) data generated from multiple immune compartments and time points and expressed several as monoclonal Abs (mAbs). Our results revealed broad dissemination and high levels of somatic hypermutation (SHM) of most lineages, including tier 2 virus neutralizing lineages, following boosting. SHM was highest in the Ab complementarity determining regions (CDRs) but also surprisingly high in the framework regions (FRs), especially FR3. Our results demonstrate the capacity of the immune system to affinity-mature large numbers of Env-specific B cell lineages simultaneously, supporting the use of regimens consisting of repeated boosts to improve each Ab, even those belonging to less expanded lineages.

Exome and genome sequencing is becoming the method of choice for rare disease diagnostics. One of the key challenges remaining is distinguishing the disease causing variants from the benign background variation. After analysis and annotation of the sequencing data there are typically thousands of candidate variants requiring further investigation. One of the most effective and least biased ways to reduce this number is to assess the rarity of a variant in any population. Currently, there are a number of reliable sources of information for major population frequencies when considering single nucleotide variants (SNVs) and small insertion and deletions (INDELs), with gnomAD as the most prominent public resource available. However, local variation or frequencies in sub-populations may be underrepresented in these public resources. In contrast, for structural variation (SV), the background frequency in the general population is more or less unknown mostly due to challenges in calling SVs in a consistent way. Keeping track of local variation is one way to overcome these problems and significantly reduce the number of potential disease causing variants retained for manual inspection, both for SNVs and SVs.

Analysis of cell-free tumour DNA, a liquid biopsy, is a promising biomarker for cancer. We have performed a proof-of principle study to test the applicability in the clinical setting, analysing copy number alterations (CNAs) in plasma and tumour tissue from 44 patients with gastro-oesophageal cancer.

Ataxia channelopathies share common features such as slow motor progression and variable degrees of cognitive dysfunction. Mutations in potassium voltage-gated channel subfamily D member 3 (KCND3), encoding the K+ channel, Kv4.3, are associated with spinocerebellar ataxia (SCA) 19, allelic with SCA22. Mutations in potassium voltage-gated channel subfamily C member 3 (KCNC3), encoding another K+ channel, Kv3.3, cause SCA13. First, a comprehensive phenotype assessment was carried out in a family with autosomal dominant ataxia harboring 2 genetic variants in KCNC3 and KCND3. To evaluate the physiological impact of these variants on channel currents, in vitro studies were performed.

Pathogenic mutations in mitochondrial (mt) tRNA genes that compromise oxidative phosphorylation (OXPHOS) exhibit heteroplasmy and cause a range of multisyndromic conditions. Although mitochondrial disease patients are known to suffer from abnormal immune responses, how heteroplasmic mtDNA mutations affect the immune system at the molecular level is largely unknown. Here, in mice carrying pathogenic C5024T in mt-tRNAAla and in patients with mitochondrial encephalomyopathy, lactic acidosis, stroke-like episodes (MELAS) syndrome carrying A3243G in mt-tRNALeu, we found memory T and B cells to have lower pathogenic mtDNA mutation burdens than their antigen-inexperienced naive counterparts, including after vaccination. Pathogenic burden reduction was less pronounced in myeloid compared with lymphoid lineages, despite C5024T compromising macrophage OXPHOS capacity. Rapid dilution of the C5024T mutation in T and B cell cultures could be induced by antigen receptor-triggered proliferation and was accelerated by metabolic stress conditions. Furthermore, we found C5024T to dysregulate CD8+ T cell metabolic remodeling and IFN-γ production after activation. Together, our data illustrate that the generation of memory lymphocytes shapes the mtDNA landscape, wherein pathogenic variants dysregulate the immune response.

Polyadenylation has well characterised roles in RNA turnover and translation in a variety of biological systems. While polyadenylation on mitochondrial transcripts has been suggested to be a two-step process required to complete translational stop codons, its involvement in mitochondrial RNA turnover is less well understood. We studied knockdown and knockout models of the mitochondrial poly(A) polymerase (MTPAP) in Drosophila melanogaster and demonstrate that polyadenylation of mitochondrial mRNAs is exclusively performed by MTPAP. Further, our results show that mitochondrial polyadenylation does not regulate mRNA stability but protects the 3' terminal integrity, and that despite a lack of functioning 3' ends, these trimmed transcripts are translated, suggesting that polyadenylation is not required for mitochondrial translation. Additionally, loss of MTPAP leads to reduced steady-state levels and disturbed maturation of tRNACys, indicating that polyadenylation in mitochondria might be important for the stability and maturation of specific tRNAs.

Recent advances in sequencing technology have created unprecedented opportunities for biological research. However, the increasing throughput of these technologies has created many challenges for data management and analysis. As the demand for sophisticated analyses increases, the development time of software and algorithms is outpacing the speed of traditional publication. As technologies continue to be developed, methods change rapidly, making publications less relevant for users. The SEQanswers wiki (SEQwiki) is a wiki database that is actively edited and updated by the members of the SEQanswers community (http://SEQanswers.com/). The wiki provides an extensive catalogue of tools, technologies and tutorials for high-throughput sequencing (HTS), including information about HTS service providers. It has been implemented in MediaWiki with the Semantic MediaWiki and Semantic Forms extensions to collect structured data, providing powerful navigation and reporting features. Within 2 years, the community has created pages for over 500 tools, with approximately 400 literature references and 600 web links. This collaborative effort has made SEQwiki the most comprehensive database of HTS tools anywhere on the web. The wiki includes task-focused mini-reviews of commonly used tools, and a growing collection of more than 100 HTS service providers. SEQwiki is available at: http://wiki.SEQanswers.com/.

Fertility in males with congenital adrenal hyperplasia (CAH) is reported from normal to severely impaired. Therefore, we investigated fertility/fecundity, social/sexual situation, and pituitary-gonadal function in CAH males.

The pathway for RNA interference is widespread in metazoans and participates in numerous cellular tasks, from gene silencing to chromatin remodeling and protection against retrotransposition. The unicellular eukaryote Trypanosoma cruzi is missing the canonical RNAi pathway and is unable to induce RNAi-related processes. To further understand alternative RNA pathways operating in this organism, we have performed deep sequencing and genome-wide analyses of a size-fractioned cDNA library (16-61 nt) from the epimastigote life stage. Deep sequencing generated 582,243 short sequences of which 91% could be aligned with the genome sequence. About 95-98% of the aligned data (depending on the haplotype) corresponded to small RNAs derived from tRNAs, rRNAs, snRNAs and snoRNAs. The largest class consisted of tRNA-derived small RNAs which primarily originated from the 3' end of tRNAs, followed by small RNAs derived from rRNA. The remaining sequences revealed the presence of 92 novel transcribed loci, of which 79 did not show homology to known RNA classes.

Four inborn errors of metabolism (IEMs) are known to cause hypermethioninemia by directly interfering with the methionine cycle. Hypermethioninemia is occasionally discovered incidentally, but it is often disregarded as an unspecific finding, particularly if liver disease is involved. In many individuals the hypermethioninemia resolves without further deterioration, but it can also represent an early sign of a severe, progressive neurodevelopmental disorder. Further investigation of unclear hypermethioninemia is therefore important. We studied two siblings affected by severe developmental delay and liver dysfunction. Biochemical analysis revealed increased plasma levels of methionine, S-adenosylmethionine (AdoMet), and S-adenosylhomocysteine (AdoHcy) but normal or mildly elevated homocysteine (Hcy) levels, indicating a block in the methionine cycle. We excluded S-adenosylhomocysteine hydrolase (SAHH) deficiency, which causes a similar biochemical phenotype, by using genetic and biochemical techniques and hypothesized that there was a functional block in the SAHH enzyme as a result of a recessive mutation in a different gene. Using exome sequencing, we identified a homozygous c.902C>A (p.Ala301Glu) missense mutation in the adenosine kinase gene (ADK), the function of which fits perfectly with this hypothesis. Increased urinary adenosine excretion confirmed ADK deficiency in the siblings. Four additional individuals from two unrelated families with a similar presentation were identified and shown to have a homozygous c.653A>C (p.Asp218Ala) and c.38G>A (p.Gly13Glu) mutation, respectively, in the same gene. All three missense mutations were deleterious, as shown by activity measurements on recombinant enzymes. ADK deficiency is a previously undescribed, severe IEM shedding light on a functional link between the methionine cycle and adenosine metabolism.