Loss-of-Function (LoF) variants in the filaggrin (FLG) gene are the strongest known genetic risk factor for atopic dermatitis (AD), but the impact of these variants on AD outcomes is poorly understood. We comprehensively identified genetic variants through targeted region sequencing of FLG in children (n = 438) participating in the Mechanisms of Progression of Atopic Dermatitis to Asthma in Children (MPAACH) cohort. Twenty FLG LoF variants were identified, including one novel variant and nine variants not previously associated with AD. FLG LoF variants were found in 13.6% of the cohort. Among these children, the presence of one or more FLG LoF variants was associated with moderate/severe AD (odds ratio (OR) = 2.00 (95% CI, 1.23-3.68) compared to those with mild AD. Children with FLG LoF variants had a higher SCORAD (SCORing for Atopic Dermatitis (SCORAD); P = 0.012) and higher likelihood of food allergy within the first 2.5 years of life (OR = 2.81, 1.50-5.26). LoF variants were associated with higher transepidermal Water Loss (TEWL) in both lesional (P = 0.018) and non-lesional skin (P = 0.015). Collectively, our study identifies established and novel AD-associated FLG LoF variants and associates FLG LoF with higher TEWL in lesional and non-lesional skin.

Filaggrin loss-of-function variants are associated with atopic dermatitis phenotypes in a diverse, early life prospective cohort.

X-linked genetic disorders typically affect females less severely than males due to the presence of a second X Chromosome not carrying the deleterious variant. However, the phenotypic expression in females is highly variable, which may be explained by an allelic skew in X-Chromosome inactivation. Accurate measurement of X inactivation skew is crucial to understand and predict disease phenotype in carrier females, with prediction especially relevant for degenerative conditions. We propose a novel approach using nanopore sequencing to quantify skewed X inactivation accurately. By phasing sequence variants and methylation patterns, this single assay reveals the disease variant, X inactivation skew, its directionality, and is applicable to all patients and X-linked variants. Enrichment of X Chromosome reads through adaptive sampling enhances cost-efficiency. Our study includes a cohort of 16 X-linked variant carrier females affected by two X-linked inherited retinal diseases: choroideremia and RPGR-associated retinitis pigmentosa. As retinal DNA cannot be readily obtained, we instead determine the skew from peripheral samples (blood, saliva and buccal mucosa), and correlate it to phenotypic outcomes. This revealed a strong correlation between X inactivation skew and disease presentation, confirming the value in performing this assay and its potential as a way to prioritise patients for early intervention, such as gene therapy currently in clinical trials for these conditions. Our method of assessing skewed X inactivation is applicable to all long-read genomic datasets, providing insights into disease risk and severity and aiding in the development of individualised strategies for X-linked variant carrier females. 

Measuring X inactivation skew for X-linked diseases with adaptive nanopore sequencing

Sequencing of the low-complexity ORF15 exon of RPGR, a gene correlated with retinitis pigmentosa and cone dystrophy, is difficult to achieve with NGS and Sanger sequencing. False results could lead to the inaccurate annotation of genetic variants in dbSNP and ClinVar databases, tools on which HGMD and Ensembl rely, finally resulting in incorrect genetic variants interpretation. This paper aims to propose PacBio sequencing as a feasible method to correctly detect genetic variants in low-complexity regions, such as the ORF15 exon of RPGR, and interpret their pathogenicity by structural studies. Biological samples from 75 patients affected by retinitis pigmentosa or cone dystrophy were analyzed with NGS and repeated with PacBio. The results showed that NGS has a low coverage of the ORF15 region, while PacBio was able to sequence the region of interest and detect eight genetic variants, of which four are likely pathogenic. Furthermore, molecular modeling and dynamics of the RPGR Glu-Gly repeats binding to TTLL5 allowed for the structural evaluation of the variants, providing a way to predict their pathogenicity. Therefore, we propose PacBio sequencing as a standard procedure in diagnostic research for sequencing low-complexity regions such as RPGRORF15, aiding in the correct annotation of genetic variants in online databases.

Towards a Long-Read Sequencing Approach for the Molecular Diagnosis of RPGRORF15 Genetic Variants

Considering the limited literature and the difficulty of quantifying 1-μm micro-nanoplastics (1-μm MNP) in complex aqueous matrices such as wastewater and sludge, the removal rate of these very small particles in wastewater treatment plants (WWTP) represents a major challenge. In this study, coagulation-flocculation-sedimentation (CFS) with aluminum salts was investigated to evaluate the removal of 1-μm MNPs spiked in tap water, raw wastewater, pre-settled wastewater, and activated sludge. Quantification of 1-μm MNP was performed using the high-throughput flow cytometry (FCM) analysis which takes only a few minutes and produces results with high accuracy and reproducibly. The results indicated that the 1-μm MNPs were highly stable in pure water and unable to settle rapidly. In raw wastewater, sedimentation without coagulants removed less than 4% of 1-μm MNP. Conversely, CFS treatment showed a significant improvement in the removal of 1-μm MNP from wastewater. At dosages of 0.3-3 mg Al3+/L, the removal of MNPs in wastewater reached 30% and no flocs were observed, while floc formation was visible with increased dosages of 3-12 mg Al3+/L, obtaining MNP removal greater than 90%. CFS in activated sludge with a solids content of 6800 mg MLSS/L registered the highest removal efficiency (95-99%) even for dosages of 0.3-60 mg Al3+/L and pH dropping to 5. However, activated sludge showed extremely high removal efficiency of MNPs (97.3±0.9%) even without coagulants. The large, dense flocs that constitute activated sludge appear particularly efficient in capturing 1-μm MNPs during the sedimentation process even in the absence of coagulants. 

Flow cytometry as a tool for the rapid enumeration of 1-μm microplastics spiked in wastewater and activated sludge after coagulation-flocculation-sedimentation

Genome-resolved metagenomics from short-read sequencing data in the era of artificial intelligence.

The filaggrin (FLG) protein, encoded by the FLG gene, is an intermediate filament-associated protein that plays a crucial role in the terminal stages of human epidermal differentiation. Loss-of-function mutations in the FLG exon 3 have been associated with skin diseases. The identification of causative mutations is challenging, due to the high sequence homology within its exon 3 (12,753 bp), which includes 10 to 12 filaggrin tandem repeats. With this study we aimed to obtain the whole FLG exon 3 sequence through PacBio technology, once 13-kb amplicons have been generated.For the preparation of SMRTbell libraries to be sequenced using PacBio technology, we focused on optimizing a 2-step long-range PCR protocol to generate 13-kb amplicons covering the whole FLG exon 3 sequence. The performance of three long-range DNA polymerases was assessed in an attempt to improve the PCR conditions required for the enzymes to function properly. We focused on optimization of the input template DNA concentration and thermocycling parameters to correctly amplify the entire FLG exon 3 sequence, minimizing non-specific amplification.Taken together, our findings suggested that the PrimeSTAR protocol is suitable for producing the amplicons of the 13-kb FLG whole exon 3 to prepare SMRTbell libraries. We suggest that sequencing the generated amplicons may be useful for identifying LoF variants that are causative of the patients' disorders. 

/pdf/optimization-of-long-range-pcr-protocol-to-prepare-filaggrin-12ld0lg0.pdf

Optimization of long-range PCR protocol to prepare filaggrin exon 3 libraries for PacBio long-read sequencing

The microbiome of water springs is gaining increasing interest, especially in water intended for human consumption. However, the knowledge about large-scale patterns in water springs microbiome is still incomplete. The presence of bacteria in water sources used for human consumption is a major concern for health authorities; nonetheless, the standard microbiological quality checks are focused only on pathogenic species and total microbial load. Using 16S rRNA high throughput sequencing, we characterized the microbiome from 38 water springs in Trentino (Northern Italy) for 2 consecutive years in order to gain precious insights on the microbiome composition of these unexplored yet hardly exploited environments. The microbiological studies were integrated with standard measurements of physico-chemical parameters performed by the Provincial Office for Environmental Monitoring in order to highlight some of the dynamics influencing the microbial communities of these waters. We found that alpha diversity showed consistent patterns of variation overtime, and showed a strong positive correlation with the water nitrate concentration and negatively with fixed residue, electrical conductivity, and calcium concentration. Surprisingly, alpha diversity did not show any significant correlation with neither pH nor temperature. We found that despite their remarkable stability, different water springs display different coefficients of variation in alpha diversity, and that springs used for similar purposes showed similar microbiomes. Furthermore, the springs could be grouped according to the number of shared species into three major groups: low, mid, and high number of shared taxa, and those three groups of springs were consistent with the spring usage. Species belonging to the phyla Planctomycetes and Verrucomicrobia were prevalent and at relatively high abundance in springs classified as low number of shared species, whereas the phylum Lentisphaerae and the Candidate Phyla radiation were prevalent at higher abundance in the mineral and potable springs. The present study constitutes an example for standard water spring monitoring integrated with microbial community composition on a regional scale, and provides information which could be useful in the design and application of future water management policies in Trentino.

Microbiome characterization of alpine water springs for human consumption reveals site- and usage-specific microbial signatures

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Optimization of long-range PCR protocol to prepare filaggrin exon 3 libraries for PacBio long-read sequencing

Towards a Long-Read Sequencing Approach for the Molecular Diagnosis of RPGRORF15 Genetic Variants

Microbiome characterization of alpine water springs for human consumption reveals site- and usage-specific microbial signatures