Integrating whole-genome sequencing in clinical genetics: a novel disruptive structural rearrangement identified in the dystrophin gene (DMD)

While in most patients the identification of genetic alterations causing dystrophinopathies is a relatively straightforward task, a significant number require genomic and transcriptomic approaches that go beyond a routine diagnostic set-up. In this work, we present a Becker Muscular Dystrophy patient with elevated creatinine kinase levels, progressive muscle weakness, mild intellectual disability and a muscle biopsy showing dystrophic features and irregular dystrophin labelling. Routine molecular techniques (Southern-blot analysis, multiplex PCR, MLPA and genomic DNA sequencing) failed to detect a defect in the DMD gene. Muscle DMD transcript analysis (RT-PCR and cDNA-MLPA) showed the absence of exons 75 to 79, seen to be present at the genomic level. These results prompted the application of low-coverage linked-read whole-genome sequencing (WGS), revealing a possible rearrangement involving DMD intron 74 and a region located upstream of the PRDX4 gene. Breakpoint PCR and Sanger sequencing confirmed the presence of a ~8 Mb genomic inversion. Aberrant DMD transcripts were subsequently identified, some of which contained segments from the region upstream of PRDX4. Besides expanding the mutational spectrum of the disorder, this study reinforces the importance of transcript analysis in the diagnosis of dystrophinopathies and shows how WGS has a legitimate role in clinical laboratory genetics.Molecular Technology and Informatics for Personalised Medicine and Healt

Anaerobic removal of 1-methoxy-2-propanol under ambient temperature in an EGSB reactor

Two laboratory-scale expanded granular sludge bed (EGSB) reactors were operated at 18 and 25 C, respectively, for the treatment of synthetic wastewater composed of ethanol and 1-methoxy-2-propanol (M2P) in a mass ratio of 4:1. Reactors were operated first with continuous wastewater supply and after with discontinuous substrate supply (5 days a week, 16 h a day) to simulate shift working conditions. Under continuous wastewater supply chemical oxygen demand (COD), removal efficiency higher than 95 % was achieved at the end of the trial applying organic loading rates (OLR) of 29 and 43 kg COD m-3 day-1 at 18 and 25 C; thus, corresponding to M2P OLR of 6.4 and 9.3 kg COD m-3 day-1, respectively. During intermittent supply of substrate, good performance was recorded at both temperatures with an OLR of 30 kg COD m-3 day-1 (M2P OLR of 6.6 kg COD m-3 day-1). After 56 h without substrate supply, a decline in methane yield of 15¿30 % was observed due to the deactivation of the biomass. Specific methanogenic activity (SMA) assays were carried out at the end of the experiments. SMA values using 1-methoxy-2-propanol as substrate were 24.3 and 7.8 ml CH4 gVSS-1 day-1 at 25 C and at 18 C, respectively. This is the first attempt to investigate the removal of 1-methoxy-2-propanol by EGSB reactors

A new methodology combining PCR, cloning, and sequencing of clones discriminated by RFLP for the study of microbial populations: application to an UASB reactor sample

This work describes a methodology combining DNA extraction, polymerase chain reaction amplification with primers targeting 16S ribosomal RNA genes, cloning, and sequencing of clones previously analyzed by restriction fragment length polymorphism (RFLP), which can be applied to study the microbial diversity in a given habitat. The methodology allows the minimization of the sequencing effort, which is particularly relevant when analyzing large numbers of clones. The methodology does not require particularly skilled personnel and can easily be adaptable to the molecular characterization of virtually any particular microbial population, provided that both adequate primers and suitable restriction enzymes for RFLP analysis of the clone library have been chosen. An example of application is presented, in which a sample taken from a continuously operating upflow anaerobic sludge blanket reactor was analyzed. RFLP analysis of the initial 162 clones with HaeIII allowed the identification of only 28 distinct profiles. As expected, identical RFLP profiles corresponded to identical nucleotide sequences.publishe

Impact of UASB reactors operation mode on the removal of estrone and 17α-ethinylestradiol from wastewaters

This work aims to compare the performance of the continuous operation (CO) and intermittent operation (IO) of upflow anaerobic sludge blanket (UASB) reactors for the removal of estrone (E1) and 17α-ethinylestradiol (EE2) from wastewaters. Results suggest that the IO contribute to the improvement of the overall removal of estrogens (above 95% for E1 and EE2) when compared to CO (49% for E1 and 39% for EE2). For both CO and IO, biodegradation was the main removal mechanism for E1, while for EE2, adsorption to sludge was the major removal pathway. Moreover, a higher biodegradation of estrogens was obtained with the IO compared to CO (69.4% vs. 43.3% for E1 and 21.8% vs. 8.0% for EE2). The favourable effect of IO can be justified by effluent recirculation during the feedless period which promotes the adaptation of microbial biomass to estrogens' biodegradation.publishe

Potential applications of porphyrins in photodynamic inactivation beyond the medical scope

Although the discovery of light-activated antimicrobial agents had been reported in the 1900s, only more recently research work has been developed toward the use of photodynamic process as an alternative to more conventional methods of inactivation of micro(organisms). The photoprocess causes cell death through irreversible oxidative damage by reactive oxygen species produced by the interaction between a photosensitizing compound and a light source. With great emphasis on the environmental area, photodynamic inactivation (PDI) has been tested in insect eradication and in water disinfection. Lately, other studies have been carried out concerning its possible use in aquaculture waters or to the control of food-borne pathogens. Other potential applications of PDI in household, industrial and hospital settings have been considered. In the last decade, scientific research in this area has gained importance not only due to great developments in the field of materials chemistry but also because of the serious problem of the increasing number of bacterial species resistant to common antibiotics. In fact, the design of antimicrobial surfaces or selfcleaning materials is a very appealing idea from the economic, social and public health standpoints. Thus, PDI of micro(organisms) represents a promising alternative. In this review, the efforts made in the last decade in the investigation of PDI of (micro)organisms with potential applications beyond the medical field will be discussed, focusing on porphyrins, free or immobilized on solid supports, as photosensitizing agents

Enhancing wastewater degradation and biogas production by intermittent operation of UASB reactors

The present work establishes intermittent operation of UASB reactors as a novel form of enhancing the anaerobic degradation of complex wastewaters and its conversion to usable biogas. Results show that the average methane production rate is 25% higher with the intermittent operation than with the continuous mode, meaning that it could produce 25% more electricity or heat. The methanization efficiency obtained in intermittent UASB reactors is around 20% higher than in the continuous systems, confirming a higher biological degradation of the substrates. It has been suggested that intermittent operation causes a forced adaptation of the biomass towards the degradation of complex substrates and results from morphological analyses of the biomass developed in intermittent and continuous UASB reactors showed marked differences between them. In order to gain a deeper knowledge on how microbial populations are affected by these operational parameters, a strategy involving the amplification, cloning, and analysis of the nucleotide sequences of genes encoding the 16S ribosomal RNA was undertaken and is described in this work. This strategy allowed the identification of a total of 49 different sequences. Results from the molecular characterization of the microbial populations are consistent with the higher methanization efficiency of the intermittent mode of operation.publishe

Integrating Whole-Genome Sequencing in Clinical Genetics: A Novel Disruptive Structural Rearrangement Identified in the Dystrophin Gene (DMD)

While in most patients the identification of genetic alterations causing dystrophinopathies is a relatively straightforward task, a significant number require genomic and transcriptomic approaches that go beyond a routine diagnostic set-up. In this work, we present a Becker Muscular Dystrophy patient with elevated creatinine kinase levels, progressive muscle weakness, mild intellectual disability and a muscle biopsy showing dystrophic features and irregular dystrophin labelling. Routine molecular techniques (Southern-blot analysis, multiplex PCR, MLPA and genomic DNA sequencing) failed to detect a defect in the DMD gene. Muscle DMD transcript analysis (RT-PCR and cDNA-MLPA) showed the absence of exons 75 to 79, seen to be present at the genomic level. These results prompted the application of low-coverage linked-read whole-genome sequencing (WGS), revealing a possible rearrangement involving DMD intron 74 and a region located upstream of the PRDX4 gene. Breakpoint PCR and Sanger sequencing confirmed the presence of a similar to 8 Mb genomic inversion. Aberrant DMD transcripts were subsequently identified, some of which contained segments from the region upstream of PRDX4. Besides expanding the mutational spectrum of the disorder, this study reinforces the importance of transcript analysis in the diagnosis of dystrophinopathies and shows how WGS has a legitimate role in clinical laboratory genetics.Molecular Technology and Informatics for Personalised Medicine and Healt

Integrating Whole-Genome Sequencing in Clinical Genetics: A Novel Disruptive Structural Rearrangement Identified in the Dystrophin Gene (DMD)

While in most patients the identification of genetic alterations causing dystrophinopathies is a relatively straightforward task, a significant number require genomic and transcriptomic approaches that go beyond a routine diagnostic set-up. In this work, we present a Becker Muscular Dystrophy patient with elevated creatinine kinase levels, progressive muscle weakness, mild intellectual disability and a muscle biopsy showing dystrophic features and irregular dystrophin labelling. Routine molecular techniques (Southern-blot analysis, multiplex PCR, MLPA and genomic DNA sequencing) failed to detect a defect in the DMD gene. Muscle DMD transcript analysis (RT-PCR and cDNA-MLPA) showed the absence of exons 75 to 79, seen to be present at the genomic level. These results prompted the application of low-coverage linked-read whole-genome sequencing (WGS), revealing a possible rearrangement involving DMD intron 74 and a region located upstream of the PRDX4 gene. Breakpoint PCR and Sanger sequencing confirmed the presence of a similar to 8 Mb genomic inversion. Aberrant DMD transcripts were subsequently identified, some of which contained segments from the region upstream of PRDX4. Besides expanding the mutational spectrum of the disorder, this study reinforces the importance of transcript analysis in the diagnosis of dystrophinopathies and shows how WGS has a legitimate role in clinical laboratory genetics