Gut Microbiome-How Does Two-Month Consumption of Fiber-Enriched Rolls Change Microbiome in Patients Suffering from MASLD?

BACKGROUND: The occurrence of metabolic dysfunction-associated steatotic liver disease (MASLD) is a growing global problem which commonly affects patients with co-existing diseases/conditions, such as type 2 diabetes and dyslipidemia. The effective treatment of MASLD is still limited; however, diet plays a significant role in its management. There are multiple beneficial properties of dietary fiber, including its ability to modify the gut microbiome. Therefore, the aim of this study was to determine the effect of the consumption of fiber-enriched rolls on the gut microbiome and microbial metabolites in patients suffering from MASLD. METHODS: The participants were recruited according to the inclusion criteria and were required to consume fiber-enriched rolls containing either 6 g or 12 g of fiber. There were three assessment timepoints, when the anthropometric and laboratory parameters were measured, and 16s on nanopore sequencing of the fecal microbiome was conducted. RESULTS: Firmicutes and Bacteroidetes were the most abundant phyla in the patients living with MASLD. It was demonstrated that the amount of short-chain fatty acids (SCFAs) changed after the consumption of fiber-enriched rolls; however, this was strongly associated with both the timepoint and the type of SCFAs-acetate and butyrate. Additionally, the high-fiber diet was related to the increase in phyla diversity (p = 0.006571). CONCLUSIONS: Overall, the introduction of an appropriate amount of fiber to the diet seems to be promising for patients suffering from MASLD due to its ability to create an improvement in gut microbiome-related aspects.

DNA enrichment and tagmentation method for species-level identification and strain-level differentiation using ON-rep-seq.

Despite the massive developments within culture-independent methods for detection of microorganisms during the last decade, culture-based methods remain a cornerstone in microbiology. Yet, the problem of rapid, accurate and inexpensive identification of bacterial isolates down to species/strain level remains unresolved. We have developed a new method for bacterial DNA enrichment and tagmentation allowing fast (<24 h) and cost-effective species level identification and strain level differentiation using the MinION portable sequencing platform (ON-rep-seq). DNA library preparation for 96 isolates takes less than 5 h and ensures highly reproducible distribution of reads that can be used to generate strain level specific read length counts profiles (LCp). We have developed a pipeline that by correcting reads error within peaks of LCp generates a set of high quality (>99%) consensus reads. Whereas, the information from high quality reads is used to retrieve species level taxonomy, comparison of LCp allows for strain level differentiation.

Truncated Robust Principal Component Analysis and Noise Reduction for Single Cell RNA Sequencing Data.

The development of single cell RNA sequencing (scRNA-seq) has enabled innovative approaches to investigating mRNA abundances. In our study, we are interested in extracting the systematic patterns of scRNA-seq data in an unsupervised manner; thus, we have developed two extensions of robust principal component analysis (RPCA). First, we present a truncated version of RPCA (tRPCA), which is much faster and memory efficient. Second, we introduce a noise reduction in tRPCA with L 2 regularization. Unlike RPCA that only considers a low-rank L and sparse S matrices, the proposed method can also extract a noise E matrix inherent in modern genomic data. We demonstrate its usefulness by applying our methods on the peripheral blood mononuclear cell scRNA-seq data. Particularly, the clustering of a low-rank L matrix showcases better classification of unlabeled single cells. Overall, the proposed variants are well suited for high-dimensional and noisy data that are routinely generated in genomics.

Mapping of breakpoints in balanced chromosomal translocations by shallow whole-genome sequencing points to EFNA5, BAHD1 and PPP2R5E as novel candidates for genes causing human Mendelian disorders.

BACKGROUND: Mapping the breakpoints in de novo balanced chromosomal translocations (BCT) in symptomatic individuals provides a unique opportunity to identify in an unbiased way the likely causative genetic defect and thus find novel human disease candidate genes. Our aim was to fine-map breakpoints of de novo BCTs in a case series of nine patients. METHODS: Shallow whole-genome mate pair sequencing (SGMPS) together with long-range PCR and Sanger sequencing. In one case (BCT disrupting BAHD1 and RET) cDNA analysis was used to verify expression of a fusion transcript in cultured fibroblasts. RESULTS: In all nine probands 11 disrupted genes were found, that is, EFNA5, EBF3, LARGE, PPP2R5E, TXNDC5, ZNF423, NIPBL, BAHD1, RET, TRPS1 and SLC4A10. Five subjects had translocations that disrupted genes with so far unknown (EFNA5, BAHD1, PPP2R5E, TXNDC5) or poorly delineated impact on the phenotype (SLC4A10, two previous reports of BCT disrupting the gene). The four genes with no previous disease associations (EFNA5, BAHD1, PPP2R5E, TXNDC5), when compared with all human genes by a bootstrap test, had significantly higher pLI (p<0.017) and DOMINO (p<0.02) scores indicating enrichment in genes likely to be intolerant to single copy damage. Inspection of individual pLI and DOMINO scores, and local topologically associating domain structure suggested that EFNA5, BAHD1 and PPP2R5E were particularly good candidates for novel disease loci. The pathomechanism for BAHD1 may involve deregulation of expression due to fusion with RET promoter. CONCLUSION: SGMPS in symptomatic carriers of BCTs is a powerful approach to delineate novel human gene-disease associations.

Novel variants identified with next-generation sequencing in Polish patients with cone-rod dystrophy.

Purpose: The aim of this study was to identify the molecular genetic basis of cone-rod dystrophy in 18 unrelated families of Polish origin. Cone-rod dystrophy is one of the inherited retinal dystrophies, which constitute a highly heterogeneous group of disorders characterized by progressive dysfunction of photoreceptors and retinal pigment epithelium (RPE) cells. Methods: The study group was composed of four groups of patients representing different Mendelian inheritance of the disease: autosomal dominant (AD), autosomal recessive (AR), X-linked recessive (XL), and autosomal recessive or X-linked recessive (AR/XL). The combined molecular strategy included Sanger sequencing of the RPGR-ORF15 gene (three families with XL and three families with the AR/XL mode of inheritance), mutation-specific microarray analysis of the ABCA4 gene (five families with the AR mode of inheritance and two families with the AR/XL mode of inheritance), targeted next-generation sequencing (NGS) of inherited retinal disease-associated (IRD) genes (seven families with the AD mode of inheritance and five families with the AR mode of inheritance), and whole exome sequencing, performed in select families who had been mutation-negative in the analysis with the targeted NGS panel (one family with the AD mode of inheritance, one family with the AR mode of inheritance, and two families with the AR/XL mode of inheritance). Results: Based on this combined strategy, we managed to identify potentially causative variants in seven out of 18 families with CRD. Five of these variants are novel: c.3142_3143dupAA, p.(Glu1049Argfs*41) in the RPGR-ORF15 gene, two variants: c.1612delT, p.(Trp538Glyfs*15) and c.2389dupG, p.(Ile798Hisfs*20) in the PROM1 gene in one family, c.592A>C, p.(Ser198Arg) in the PRPH2 gene and the variant c.1691A>G, p.(Asp564Gly) in the ATF6 gene that we have already reported to be pathogenic. NGS on the IRD panel allowed the molecular basis of CRD to be identified in four out of 14 families with a total detection rate of 38%. WES allowed identification of the molecular genetic basis of CRD in one family. Conclusions: This is the first report on the spectrum of disease genes and pathogenic variants causing CRD in the Polish population. The study presents five novel variants identified in four genes and therefore, broadens the spectrum of probable pathogenic variants associated with CRD.

Phenotypic consequences of gene disruption by a balanced de novo translocation involving SLC6A1 and NAA15.

Mapping of de novo balanced chromosomal translocations (BCTs) in patients with sporadic poorly characterized disease(s) is an unbiased method of finding candidate gene(s) responsible for the observed symptoms. We present a paediatric patient suffering from epilepsy, developmental delay (DD) and atrial septal defect IIº (ASD) requiring surgery. Karyotyping indicated an apparently balanced de novo reciprocal translocation 46,XX,t(3;4)(p25.3;q31.1), whereas aCGH did not reveal any copy number changes. Using shallow mate-pair whole genome sequencing and direct Sanger sequencing of breakpoint regions we found that translocation disrupted SLC6A1 and NAA15 genes. Our results confirm two previous reports indicating that loss of function of a single allele of SLC6A1 causes epilepsy. In addition, we extend existing evidence that disruption of NAA15 is associated with DD and with congenital heart defects.

The association between 38 previously reported polymorphisms and psoriasis in a Polish population: High predicative accuracy of a genetic risk score combining 16 loci.

OBJECTIVES: To confirm the association of previously discovered psoriasis (Ps) risk loci with the disease in a Polish population and to create predictive models based on the combination of these single nucleotide polymorphisms (SNPs). MATERIAL AND METHODS: Thirty-eight SNPs were genotyped in 480 Ps patients and 490 controls. Alleles distributions were compared between patients and controls, as well as between different Ps sub-phenotypes. The genetic risk score (GRS) was calculated to assess the cumulative risk conferred by multiple loci. RESULTS: We confirmed associations of several loci with Ps: HLA-C, REL, IL12B, TRIM39/RPP21, POU5F1, MICA. The analysis of ROC curves showed that GRS combining 16 SNPs at least nominally (uncorrected P<0.05) associated with Ps (GRS-N) had significantly better discriminative power than GRS combining SNPs associated with Ps after the Bonferroni correction (AUC 0.776 vs. 0.750, P = 1 x 10-4) or HLA-C (AUC 0.776 vs. 0.694, P<1 x 10-5). On the other hand, adding additional SNPs to the model did not improve its discriminatory ability (AUC 0.782 for GRS combining all SNPs, P>0.05). In order to assess the total risk conferred by GRS-N, we calculated ORs according to GRS-N quartile - the Ps OR for top vs. bottom GRS-N quartiles was 12.29 (P<1 x 10-6). The analysis of different Ps sub-phenotypes showed an association of GRS-N with age of onset and family history of Ps. CONCLUSIONS: We confirmed the association of Ps with several previously identified genetic risk factors in a Polish population. We found that a GRS combining 16 SNPs at least nominally associated with Ps had a significantly better discriminatory ability than HLA-C or GRS combining SNPs associated with Ps after the Bonferroni correction. In contrast, adding additional SNPs to GRS did not increase significantly the discriminative power.

Gene Expression Profile of the Clinically Aggressive Micropapillary Variant of Bladder Cancer.

BACKGROUND: Progression of conventional urothelial carcinoma of the bladder to a tumor with unique microscopic features referred to as micropapillary carcinoma is coupled with aggressive clinical behavior signified by a high propensity for metastasis to regional lymph nodes and distant organs resulting in shorter survival. OBJECTIVE: To analyze the expression profile of micropapillary cancer and define its molecular features relevant to clinical behavior. DESIGN, SETTING, AND PARTICIPANTS: We retrospectively identified 43 patients with micropapillary bladder cancers and a reference set of 89 patients with conventional urothelial carcinomas and performed whole-genome expression messenger RNA profiling. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The tumors were segregated into distinct groups according to hierarchical clustering analyses. They were also classified according to luminal, p53-like, and basal categories using a previously described algorithm. We applied Ingenuity Pathway Analysis software (Qiagen, Redwood City, CA, USA) and gene set enrichment analysis for pathway analyses. Cox proportional hazards models and Kaplan-Meier methods were used to assess the relationship between survival and molecular subtypes. The expression profile of micropapillary cancer was validated for selected markers by immunohistochemistry on parallel tissue microarrays. RESULTS AND LIMITATIONS: We show that the striking features of micropapillary cancer are downregulation of miR-296 and activation of chromatin-remodeling complex RUVBL1. In contrast to conventional urothelial carcinomas that based on their expression can be equally divided into luminal and basal subtypes, micropapillary cancer is almost exclusively luminal, displaying enrichment of active peroxisome proliferator-activated receptor γ and suppression of p63 target genes. As with conventional luminal urothelial carcinomas, a subset of micropapillary cancers exhibit activation of wild-type p53 downstream genes and represent the most aggressive molecular subtype of the disease with the shortest survival. The involvement of miR-296 and RUVBL1 in the development of micropapillary bladder cancer was identified by the analyses of correlative associations of genome expression profiles and requires mechanistic validation. CONCLUSIONS: Micropapillary cancer evolves through the luminal pathway and is characterized by the activation of miR-296 and RUVBL1 target genes. PATIENT SUMMARY: Our observations have important implications for prognosis and for possible future development of more effective therapies for micropapillary bladder cancer.

Spaced seeds improve k-mer-based metagenomic classification.

MOTIVATION: Metagenomics is a powerful approach to study genetic content of environmental samples, which has been strongly promoted by next-generation sequencing technologies. To cope with massive data involved in modern metagenomic projects, recent tools rely on the analysis of k-mers shared between the read to be classified and sampled reference genomes. RESULTS: Within this general framework, we show that spaced seeds provide a significant improvement of classification accuracy, as opposed to traditional contiguous k-mers. We support this thesis through a series of different computational experiments, including simulations of large-scale metagenomic projects.Availability and implementation, Supplementary information: Scripts and programs used in this study, as well as supplementary material, are available from http://github.com/gregorykucherov/spaced-seeds-for-metagenomics. CONTACT: gregory.kucherov@univ-mlv.fr.

Application of array comparative genomic hybridization in 256 patients with developmental delay or intellectual disability.

We used whole-genome exon-targeted oligonucleotide array comparative genomic hybridization (array CGH) in a cohort of 256 patients with developmental delay (DD)/intellectual disability (ID) with or without dysmorphic features, additional neurodevelopmental abnormalities, and/or congenital malformations. In 69 patients, we identified 84 non-polymorphic copy-number variants, among which 41 are known to be clinically relevant, including two recently described deletions, 4q21.21q21.22 and 17q24.2. Chromosomal microarray analysis revealed also 15 potentially pathogenic changes, including three rare deletions, 5q35.3, 10q21.3, and 13q12.11. Additionally, we found 28 copy-number variants of unknown clinical significance. Our results further support the notion that copy-number variants significantly contribute to the genetic etiology of DD/ID and emphasize the efficacy of the detection of novel candidate genes for neurodevelopmental disorders by whole-genome array CGH.

Multiple samples aCGH analysis for rare CNVs detection.

BACKGROUND: DNA copy number variations (CNV) constitute an important source of genetic variability. The standard method used for CNV detection is array comparative genomic hybridization (aCGH). RESULTS: We propose a novel multiple sample aCGH analysis methodology aiming in rare CNVs detection. In contrast to the majority of previous approaches, which deal with cancer datasets, we focus on constitutional genomic abnormalities identified in a diverse spectrum of diseases in human. Our method is tested on exon targeted aCGH array of 366 patients affected with developmental delay/intellectual disability, epilepsy, or autism. The proposed algorithms can be applied as a post-processing filtering to any given segmentation method. CONCLUSIONS: Thanks to the additional information obtained from multiple samples, we could efficiently detect significant segments corresponding to rare CNVs responsible for pathogenic changes. The robust statistical framework applied in our method enables to eliminate the influence of widespread technical artifact termed 'waves'.

Functional performance of aCGH design for clinical cytogenetics.

Array-comparative genomic hybridization (aCGH) technology enables rapid, high-resolution analysis of genomic rearrangements. With the use of it, genome copy number changes and rearrangement breakpoints can be detected and analyzed at resolutions down to a few kilobases. An exon array CGH approach proposed recently accurately measures copy-number changes of individual exons in the human genome. The crucial and highly non-trivial starting task is the design of an array, i.e. the choice of appropriate (multi)set of oligos. The success of the whole high-level analysis depends on the quality of the design. Also, the comparison of several alternative designs of array CGH constitutes an important step in development of new diagnostic chip. In this paper, we deal with these two often neglected issues. We propose a new approach to measure the quality of array CGH designs. Our measures reflect the robustness of rearrangements detection to the noise (mostly experimental measurement error). The method is parametrized by the segmentation algorithm used to identify aberrations. We implemented the efficient Monte Carlo method for testing noise robustness within DNAcopy procedure. Developed framework has been applied to evaluation of functional quality of several optimized array designs.

Application of custom-designed oligonucleotide array CGH in 145 patients with autistic spectrum disorders.

Autism spectrum disorders (ASDs) are a heterogeneous group of neurodevelopmental disorders, including childhood autism, atypical autism, and Asperger syndrome, with an estimated prevalence of 1.0-2.5% in the general population. ASDs have a complex multifactorial etiology, with genetic causes being recognized in only 10-20% of cases. Recently, copy-number variants (CNVs) have been shown to contribute to over 10% of ASD cases. We have applied a custom-designed oligonucleotide array comparative genomic hybridization with an exonic coverage of over 1700 genes, including 221 genes known to cause autism and autism candidate genes, in a cohort of 145 patients with ASDs. The patients were classified according to ICD-10 standards and the Childhood Autism Rating Scale protocol into three groups consisting of 45 individuals with and 69 individuals without developmental delay/intellectual disability (DD/ID), and 31 patients, in whom DD/ID could not be excluded. In 12 patients, we have identified 16 copy-number changes, eight (5.5%) of which likely contribute to ASDs. In addition to known recurrent CNVs such as deletions 15q11.2 (BP1-BP2) and 3q13.31 (including DRD3 and ZBTB20), and duplications 15q13.3 and 16p13.11, our analysis revealed two novel genes clinically relevant for ASDs: ARHGAP24 (4q21.23q21.3) and SLC16A7 (12q14.1). Our results further confirm the diagnostic importance of array CGH in detection of CNVs in patients with ASDs and demonstrate that CNVs are an important cause of ASDs as a heterogeneous condition with a variety of contributory genes.

Application of array comparative genomic hybridization in 102 patients with epilepsy and additional neurodevelopmental disorders.

Copy-number variants (CNVs) collectively represent an important cause of neurodevelopmental disorders such as developmental delay (DD)/intellectual disability (ID), autism, and epilepsy. In contrast to DD/ID, for which the application of microarray techniques enables detection of pathogenic CNVs in -10-20% of patients, there are only few studies of the role of CNVs in epilepsy and genetic etiology in the vast majority of cases remains unknown. We have applied whole-genome exon-targeted oligonucleotide array comparative genomic hybridization (array CGH) to a cohort of 102 patients with various types of epilepsy with or without additional neurodevelopmental abnormalities. Chromosomal microarray analysis revealed 24 non-polymorphic CNVs in 23 patients, among which 10 CNVs are known to be clinically relevant. Two rare deletions in 2q24.1q24.3, including KCNJ3 and 9q21.13 are novel pathogenic genetic loci and 12 CNVs are of unknown clinical significance. Our results further support the notion that rare CNVs can cause different types of epilepsy, emphasize the efficiency of detecting novel candidate genes by whole-genome array CGH, and suggest that the clinical application of array CGH should be extended to patients with unexplained epilepsies.

Assessment of the role of copy-number variants in 150 patients with congenital heart defects.

BACKGROUND: Congenital heart defects are the most common group of major birth anomalies and one of the leading causes of infant deaths. Mendelian and chromosomal syndromes account for about 20% of congenital heart defects and in some cases are associated with other malformations, intellectual disability, and/or dysmorphic features. The remarkable conservation of genetic pathways regulating heart development in animals suggests that genetic factors can be responsible for a significantly higher percentage of cases. THE AIM: Assessment of the role of CNVs in the etiology of congenital heart defects using microarray studies. MATERIAL AND METHODS: Genome-wide array comparative genomic hybridization, targeting genes known to play an important role in heart development or responsible for abnormal cardiac phenotype was used in the study on 150 patients. In addition, we have used multiplex ligation-dependent probe amplification specific for chromosome 22q11.2 region. RESULTS: We have identified 21 copy-number variants, including 13 known causative recurrent rearrangements (12 deletions 22q11.2 and one deletion 7q11.23), three potentially pathogenic duplications (5q14.2, 15q13.3, and 22q11.2), and five variants likely benign for cardiac anomalies. We suggest that abnormal copy-number of the ARRDC3 and KLF13 genes can be responsible for heart defects. CONCLUSIONS: Our study demonstrates that array comparative genomic hybridization enables detection of clinically significant chromosomal imbalances in patients with congenital heart defects.

Detection of clinically relevant exonic copy-number changes by array CGH.

Array comparative genomic hybridization (aCGH) is a powerful tool for the molecular elucidation and diagnosis of disorders resulting from genomic copy-number variation (CNV). However, intragenic deletions or duplications--those including genomic intervals of a size smaller than a gene--have remained beyond the detection limit of most clinical aCGH analyses. Increasing array probe number improves genomic resolution, although higher cost may limit implementation, and enhanced detection of benign CNV can confound clinical interpretation. We designed an array with exonic coverage of selected disease and candidate genes and used it clinically to identify losses or gains throughout the genome involving at least one exon and as small as several hundred base pairs in size. In some patients, the detected copy-number change occurs within a gene known to be causative of the observed clinical phenotype, demonstrating the ability of this array to detect clinically relevant CNVs with subkilobase resolution. In summary, we demonstrate the utility of a custom-designed, exon-targeted oligonucleotide array to detect intragenic copy-number changes in patients with various clinical phenotypes.