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Abstract Vitamin B6‐responsive epilepsies are a group of genetic disorders including ALDH7A1 deficiency, PNPO deficiency, and others, usually causing neonatal onset seizures resistant to treatment with common antiepileptic drugs. Recently, biallelic mutations in PLPBP were shown to be a novel cause of vitamin B6‐dependent epilepsy with a variable phenotype. The different vitamin B6‐responsive epilepsies can be detected and distinguished by their respective biomarkers and genetic analysis. Unfortunately, metabolic biomarkers for early detection and prognosis of PLPBP deficiency are currently still lacking. Here, we present data from two further patients with vitamin B6‐dependent seizures caused by variants in PLPBP, including a novel missense variant, and compare their genotype and phenotypic presentation to previously described cases. Hyperglycinemia and hyperlactatemia are the most consistently observed biochemical abnormalities in pyridoxal phosphate homeostasis protein (PLPHP) deficient patients and were present in both patients in this report within the first days of life. Lactic acidemia, the neuroradiological, and clinical presentation led to misdiagnosis of a mitochondrial encephalopathy in two previously published cases with an early fatal course. Similarly, on the background of glycine elevation in plasma, glycine encephalopathy was wrongly adopted as diagnosis for a patient in our report. In this regard, lactic acidemia as well as hyperglycinemia appear to be diagnostic pitfalls in patients with vitamin B6‐responsive epilepsies, including PLPHP deficiency. Synopsis In vitamin B6‐responsive epilepsies, including PLPHP deficiency, there are several diagnostic pitfalls, including lactic acidemia as well as hyperglycinemia, highlighting the importance of a pyridoxine trial, and genetic testing.

Abstract Background Since different types of genetic variants, from single nucleotide variants (SNVs) to large chromosomal rearrangements, underlie intellectual disability, we evaluated the use of whole-genome sequencing (WGS) rather than chromosomal microarray analysis (CMA) as a first-line genetic diagnostic test. Methods We analyzed three cohorts with short-read WGS: (i) a retrospective cohort with validated copy number variants (CNVs) (cohort 1, n = 68), (ii) individuals referred for monogenic multi-gene panels (cohort 2, n = 156), and (iii) 100 prospective, consecutive cases referred to our center for CMA (cohort 3). Bioinformatic tools developed include FindSV, SVDB, Rhocall, Rhoviz, and vcf2cytosure. Results First, we validated our structural variant (SV)-calling pipeline on cohort 1, consisting of three trisomies and 79 deletions and duplications with a median size of 850 kb (min 500 bp, max 155 Mb). All variants were detected. Second, we utilized the same pipeline in cohort 2 and analyzed with monogenic WGS panels, increasing the diagnostic yield to 8%. Next, cohort 3 was analyzed by both CMA and WGS. The WGS data was processed for large (> 10 kb) SVs genome-wide and for exonic SVs and SNVs in a panel of 887 genes linked to intellectual disability as well as genes matched to patient-specific Human Phenotype Ontology (HPO) phenotypes. This yielded a total of 25 pathogenic variants (SNVs or SVs), of which 12 were detected by CMA as well. We also applied short tandem repeat (STR) expansion detection and discovered one pathologic expansion in ATXN7. Finally, a case of Prader-Willi syndrome with uniparental disomy (UPD) was validated in the WGS data. Important positional information was obtained in all cohorts. Remarkably, 7% of the analyzed cases harbored complex structural variants, as exemplified by a ring chromosome and two duplications found to be an insertional translocation and part of a cryptic unbalanced translocation, respectively. Conclusion The overall diagnostic rate of 27% was more than doubled compared to clinical microarray (12%). Using WGS, we detected a wide range of SVs with high accuracy. Since the WGS data also allowed for analysis of SNVs, UPD, and STRs, it represents a powerful comprehensive genetic test in a clinical diagnostic laboratory setting.

Summary: Neurodegenerative disorders are an increasingly common and irreversible burden on society, often affecting the aging population, but their etiology and disease mechanisms are poorly understood. Studying monogenic neurodegenerative diseases with known genetic cause provides an opportunity to understand cellular mechanisms also affected in more complex disorders. We recently reported that loss-of-function mutations in the autophagy adaptor protein SQSTM1/p62 lead to a slowly progressive neurodegenerative disease presenting in childhood. To further elucidate the neuronal involvement, we studied the cellular consequences of loss of p62 in a neuroepithelial stem cell (NESC) model and differentiated neurons derived from reprogrammed p62 patient cells or by CRISPR/Cas9-directed gene editing in NESCs. Transcriptomic and proteomic analyses suggest that p62 is essential for neuronal differentiation by controlling the metabolic shift from aerobic glycolysis to oxidative phosphorylation required for neuronal maturation. This shift is blocked by the failure to sufficiently downregulate lactate dehydrogenase expression due to the loss of p62, possibly through impaired Hif-1α downregulation and increased sensitivity to oxidative stress. The findings imply an important role for p62 in neuronal energy metabolism and particularly in the regulation of the shift between glycolysis and oxidative phosphorylation required for normal neurodifferentiation. : SQSTM1/p62 is a known autophagy adaptor that, if lost, causes childhood-onset neurodegeneration. Data from Wredenberg et al. show that loss of p62 in a neuronal stem cell model does not affect mitophagy but instead leads to impaired differentiation. The authors suggest p62 finely tunes LDHA expression and thus controls the metabolic shift to OXPHOS required for proper differentiation. Keywords: SQSTM1, p62, hypoxia, mitochondria, neurodifferentiation, neuroepithelial-like stem cells, neuronal development, oxidative stress, mitophagy, neurodegeneration

Myoglobin is a hemeprotein that reversibly binds oxygen and gives muscle its red color. Here, the authors report a genetic variant in the MB gene that associates with myoglobinopathy, an autosomal dominant progressive myopathy, and altered oxygen binding properties of the mutant protein.

Summary: Here, we present a revised protocol to derive neuroepithelial stem (NES) cells from human induced pluripotent stem cells. NES cells can be further differentiated into a culture of neurons (90%) and glia (10%). We describe how to derive and maintain NES cells in culture and how to differentiate them. In addition, we show the potential use of NES cells to study the role of reactive oxygen species in neuronal differentiation and a guideline for NES cell transfection.For complete details on the use and execution of this protocol, please refer to Calvo-Garrido et al. (2019); Falk et al. (2012).

Leber hereditary optic neuropathy (LHON) is a mitochondrial disease causing severe bilateral visual loss, typically in young adults. The disorder is commonly caused by one of three primary point mutations in mitochondrial DNA, but a number of other rare mutations causing or associated with the clinical syndrome of LHON have been reported. The mutations in LHON are almost exclusively located in genes encoding subunits of complex I in the mitochondrial respiratory chain. Here we report two patients, a mother and her son, with the typical LHON phenotype. Genetic investigations for the three common mutations were negative, instead we found a new and previously unreported mutation in mitochondrial DNA. This homoplasmic mutation, m.13345G>A, is located in the MT-ND5 gene, encoding a core subunit in complex I in the mitochondrial respiratory chain. Investigation of the patients mitochondrial respiratory chain in muscle found a mild defect in the combined activity of complex I+III. In the literature six other mutations in the MT-ND5 gene have been associated with LHON and by this report a new putative mutation in the MT-ND5 can be added.

Coverage analysis is essential when analysing massive parallel sequencing (MPS) data. The analysis indicates existence of false negatives or positives in a region of interest or poorly covered genomic regions. There are several tools that have excellent performance when doing coverage analysis on a few samples with predefined regions. However, there is no current tool for collecting samples over a longer period of time for aggregated coverage analysis of multiple samples or sequencing methods. Furthermore, current coverage analysis tools do not generate customized coverage reports or enable exploratory coverage analysis without extensive bioinformatic skill and access to the original alignment files. We present Chanjo, a user friendly coverage analysis tool for persistent storage of coverage data, that, accompanied with Chanjo Report, produces coverage reports that summarize coverage data for predefined regions in an elegant manner. Chanjo Report can produce both structured coverage reports and dynamic reports tailored to a subset of genomic regions, coverage cut-offs or samples. Chanjo stores data in an SQL database where thousands of samples can be added over time, which allows for aggregate queries to discover problematic regions. Chanjo is well tested, supports whole exome and genome sequencing, and follows common UNIX standards, allowing for easy integration into existing pipelines. Chanjo is easy to install and operate, and provides a solution for persistent coverage analysis and clinical-grade reporting. It makes it easy to set up a local database and automate the addition of multiple samples and report generation. To our knowledge there is no other tool with matching capabilities. Chanjo handles the common file formats in genetics, such as BED and BAM, and makes it easy to produce PDF coverage reports that are highly valuable for individuals with limited bioinformatic expertise. We believe Chanjo to be a vital tool for clinicians and researchers performing MPS analysis.

Mutations in mitochondrially-encoded tRNA genes can lead to mitochondrial disorders. Here the authors use next generation RNA sequencing to reveal the role of a N1 -methyladenosine modification in tRNALys MERR patients for translation elongation and the stability of selected nascent chains.

BackgroundThe interaction between the Blue Moon butterfly, Hypolimnas bolina, and Wolbachia has attracted interest because of the high prevalence of male-killing achieved within the species, the ecological consequences of this high prevalence, the intensity of selection on the host to suppress the infection, and the presence of multiple Wolbachia infections inducing different phenotypes. We examined diversity in the co-inherited marker, mtDNA, and the partitioning of this between individuals of different infection status, as a means to investigate the population biology and evolutionary history of the Wolbachia infections.ResultsPart of the mitochondrial COI gene was sequenced from 298 individuals of known infection status revealing ten different haplotypes. Despite very strong biological evidence that the sample represents a single species, the ten haplotypes did not fall within a monophyletic clade within the Hypolimnas genus, with one haplotype differing by 5% from the other nine. There were strong associations between infection status and mtDNA haplotype. The presence of wBol1 infection in association with strongly divergent haplotypes prompted closer examination of wBol1 genetic variation. This revealed the existence of two cryptic subtypes, wBol1a and wBol1b. The wBol1a infection, by far the most common, was in strict association with the single divergent mtDNA haplotype. The wBol1b infection was found with two haplotypes that were also observed in uninfected specimens. Finally, the wBol2 infection was associated with a large diversity of mtDNA haplotypes, most often shared with uninfected sympatric butterflies.ConclusionThis data overall supports the hypothesis that high prevalence of male-killing Wolbachia (wBol1) in H. bolina is associated with very high transmission efficiency rather than regular horizontal transmission. It also suggests this infection has undergone a recent selective sweep and was introduced in this species through introgression. In contrast, the sharing of haplotypes between wBol2-infected and uninfected individuals indicates that this strain is not perfectly transmitted and/or shows a significant level of horizontal transmission.

Pyruvate dehydrogenase complex deficiency (PDCD) is a defect of aerobic carbohydrate metabolism that causes neurological disorders with varying degrees of severity. We report the clinical, biochemical, and molecular findings in patients with primary and secondary PDCD caused by novel atypical genetic variants. Whole-genome sequencing (WGS) identified the synonymous variants c.447A>G, p.(Lys149=) and c.570C>T, p.(Cys190=) in pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1), the deep intronic variants c.1023+2267G>A and c.1023+2302A>G in pyruvate dehydrogenase complex component X (PDHX), and c.185+15054G>A in thiamine pyrophosphokinase (TPK1). Analysis by Sanger and RNA sequencing of cDNA from patient blood and/or cultured fibroblasts showed that the synonymous variants in PDHA1 lead to aberrant splicing and skipping of exons 5 and 5-6 in one of the patients and transcripts lacking exon 6 in the other. The deep intronic variants in PDHX and TPK1 lead to insertion of intronic sequence in the corresponding transcripts. The splice defects in PDHA1 were more pronounced in cultured fibroblasts than in blood. Our findings expand the spectrum of pathogenic variants causing PDCD and highlight the importance of atypical variants leading to aberrant splicing. The severity of the splice defects and resulting biochemical dysfunction varied between tissues, stressing the importance of performing biochemical and transcript analysis in affected tissues. The two males with hemizygous synonymous PDHA1 variants have a mild phenotype and higher PDH enzyme activity than expected, which is consistent with aberrant but leaky splicing with a proportion of the transcripts remaining correctly spliced. [ABSTRACT FROM AUTHOR]

Male-killing bacteria are widespread in arthropods, and can profoundly alter the reproductive biology of their host species. Here we detail the first case of complete suppression of a male killer. The nymphalid butterfly Hypolimnas bolina is infected with a strain of the bacterium Wolbachia, wBol1, which kills male host embryos in Polynesian populations, but does not do so in many areas of Southeast Asia, where both males and female adults are naturally infected, and wBol1-infected females produce a 1:1 sex ratio. We demonstrate that absence of male killing by wBol1 is associated with dominant zygotic suppression of the action of the male killer. Simulations demonstrate host suppressors of male-killer action can spread very rapidly, and historical data indicating the presence of male killing in Southeast Asia in the very recent past suggests suppressor spread has been a very recent occurrence. Thus, male killer/host interactions are much more dynamic than previously recognised, with rapid and dramatic loss of the phenotype. Our results also indicate that suppression can render male killers completely quiescent, leading to the conclusion that some species that do not currently express a male killer may have done so in the past, and thus that more species have had their biology affected by these parasites than previously believed.