Biology subjects

Van Poucke, M.

Publications and source records attributed to Van Poucke, M..

3 recordsLinked to original sources

bioRxiv2022

Association of spinocerebellar ataxia related variants with myokymia and neuromyotonia in dogs

BackgroundKCNJ10 and CAPN1 variants have been shown to cause spinocerebellar ataxia (SCA) in Jack Russell, Parson Russell and Fox terriers (JRT, PRT and FT). However, their association with the clinical manifestation of myokymia and neuromyotonia (M/NM), often reported alongside SCA, remains unclear. AimsTo investigate the association between M/NM and SCA-related variants in 34 with SCA and/or M/NM affected dogs (30 JRTs; 1 PRT; 1 Yorkshire terrier, YT; 1 Dachshund; 1 crossbreed). MethodsKCNJ10 XM_038448705.1:c.627C>G (p.(Ile209Met)) and CAPN1 XM_038425033.1:c.344G>A (p.(Cys115Tyr)) variants, and the complete coding sequence (CDS) of KCNA1, KCNA2, KCNA6, KCNJ10 and HINT1, were analysed via Sanger sequencing. ResultsThe KCNJ10 c.627C>G variant was homozygously present in 16 JRTs, 1 Dachshund and 1 crossbreed with SCA and M/NM, and in 9 JRTs with SCA but without M/NM. The CAPN1 c.344G>A variant was homozygously present in 1 PRT with SCA but without M/NM. Both variants were not found in 2 JRTs with SCA but without M/NM, neither in 3 JRTs and 1 YT without SCA but with M/NM. No other causal variants were found in the coding sequence of the investigated candidate genes in these latter 6 dogs. ConclusionsThe KCNJ10 c.627C>G or CAPN1 c.344G>A variant was confirmed to be the causal variant in 28 of the 34 affected dogs (including 1 Dachshund with the KCNJ10 variant). The fact that 10 of these 28 dogs did not suffer from M/NM (all of them suffered from SCA) and that these variants were not found in 4 dogs suffering from M/NM without SCA, suggests that M/NM is caused by another genetic variant or that these patients suffer from an acquired (immune-mediated) syndrome, similar to humans. Another SCA-causing variant is probably also segregating in JRTs (but not in the CDS of the investigated candidate genes), because no causal variant was found in 2 JRTs suffering from SCA without M/NM.

genetics↗

bioRxiv2017

An optimized strategy for cloning-based locus-specific bisulfite sequencing PCR

In this methods paper, we describe a successful strategy to investigate locus-specific methylation by cloning-based bisulfite sequencing. We cover sample handling, DNA isolation, DNA quality control before bisulfite conversion, bisulfite conversion, DNA quality control after bisulfite conversion, in silico identification of CpG islands, methylation-independent bisulfite sequencing PCR (BSP) assay design, methylation-independent BSP, cloning strategy, sequencing and data analysis. Methods that are described nicely elsewhere will not be covered in detail. Instead, the focus will be on tips/tricks and new methods/strategies used in this protocol, including quality control assessment of the DNA before and after bisulfite conversion and a pooled cloning strategy to reduce time, costs and effort during this step. In addition we comment on dealing with bias and improving overall protocol efficiency.

genomics↗

bioRxiv2017

Flexible, multi-use, PCR-based nucleic acid integrity assays based on the ubiquitin C gene

Nucleic acid integrity assessment is an important aspect of quality control for many applications in molecular biology. A number of methods exist (electrophoresis- or PCR-based), but they are not universally applicable. Some of them need huge amounts of input, process certain amount of samples, require expensive equipment, only work specifically on (c)DNA, (m)RNA, certain species or certain tissues, or produce fragments covering a small length range. We investigated if the ubiquitin C gene (UBC) could be used to develop flexible, multi-use PCR-based (deoxy)ribonucleic acid integrity assays. UBC gene analysis (in human, mouse, pig, cow, horse, sheep, dog and cat) shows that UBC is a highly conserved and ubiquitously expressed gene (reference gene in RT-qPCR), that encodes a polyubiquitin precursor (containing tandem repeats of at least 5 ubiquitin monomers of 228 bp) in a single exon. On average, ubiquitin monomers show a nucleic acid sequence identity of 96% at intraspecies level and 93% at interspecies level. Based on a multiple alignment of all monomer ubiquitin sequences of all investigated species, we could design a single degenerated primer pair generating PCR amplicons of 137, 365, 593 and 821 bp on low amounts of high quality DNA of all investigated species (down to 10 pg) and on cDNA reverse transcribed from high quality RNA from different tissues (e.g. heart, liver, brain, kidney). Increasing levels of nucleic acid degradation resulted in a decrease of amplification products starting with the longer amplicons. We conclude that UBC is suited to develop a single, cheap, universal assay to estimate the presence, integrity and amplificability of native mammalian nucleic acids. In addition, we used the same strategy to design a similar assay to check the quality of bisulfite treated mammalian DNA.

genetics↗