Genetic Variants in Carbohydrate Digestive Enzyme and Transport Genes Associated with Risk of Irritable Bowel Syndrome.

Irritable Bowel Syndrome (IBS) is characterized by abdominal pain and alterations in bowel pattern, such as constipation (IBS-C), diarrhea (IBS-D), or mixed (IBS-M). Since malabsorption of ingested carbohydrates (CHO) can cause abdominal symptoms that closely mimic those of IBS, identifying genetic mutations in CHO digestive enzymes associated with IBS symptoms is critical to ascertain IBS pathophysiology. Through candidate gene association studies, we identify several common variants in TREH, SI, SLC5A1 and SLC2A5 that are associated with IBS symptoms. By investigating rare recessive Mendelian or oligogenic inheritance patterns, we identify case-exclusive rare deleterious variation in known disease genes (SI, LCT, ALDOB, and SLC5A1) as well as candidate disease genes (MGAM and SLC5A2), providing potential evidence of monogenic or oligogenic inheritance in a subset of IBS cases. Finally, our data highlight that moderate to severe IBS-associated gastrointestinal symptoms are often observed in IBS cases carrying one or more of deleterious rare variants.

Sequencing individual genomes with recurrent genomic disorder deletions: an approach to characterize genes for autosomal recessive rare disease traits.

BACKGROUND: In medical genetics, discovery and characterization of disease trait contributory genes and alleles depends on genetic reasoning, study design, and patient ascertainment; we suggest a segmental haploid genetics approach to enhance gene discovery and molecular diagnostics. METHODS: We constructed a genome-wide map for nonallelic homologous recombination (NAHR)-mediated recurrent genomic deletions and used this map to estimate population frequencies of NAHR deletions based on large-scale population cohorts and region-specific studies. We calculated recessive disease carrier burden using high-quality pathogenic or likely pathogenic variants from ClinVar and gnomAD. We developed a NIRD (NAHR deletion Impact to Recessive Disease) score for recessive disorders by quantifying the contribution of NAHR deletion to the overall allele load that enumerated all pairwise combinations of disease-causing alleles; we used a Punnett square approach based on an assumption of random mating. Literature mining was conducted to identify all reported patients with defects in a gene with a high NIRD score; meta-analysis was performed on these patients to estimate the representation of NAHR deletions in recessive traits from contemporary human genomics studies. Retrospective analyses of extant clinical exome sequencing (cES) were performed for novel rare recessive disease trait gene and allele discovery from individuals with NAHR deletions. RESULTS: We present novel genomic insights regarding the genome-wide impact of NAHR recurrent segmental variants on recessive disease burden; we demonstrate the utility of NAHR recurrent deletions to enhance discovery in the challenging context of autosomal recessive (AR) traits and biallelic variation. Computational results demonstrate new mutations mediated by NAHR, involving recurrent deletions at 30 genomic regions, likely drive recessive disease burden for over 74% of loci within these segmental deletions or at least 2% of loci genome-wide. Meta-analyses on 170 literature-reported patients implicate that NAHR deletions are depleted from the ascertained pool of AR trait alleles. Exome reanalysis of personal genomes from subjects harboring recurrent deletions uncovered new disease-contributing variants in genes including COX10, ERCC6, PRRT2, and OTUD7A. CONCLUSIONS: Our results demonstrate that genomic sequencing of personal genomes with NAHR deletions could dramatically improve allele and gene discovery and enhance clinical molecular diagnosis. Moreover, results suggest NAHR events could potentially enable human haploid genetic screens as an approach to experimental inquiry into disease biology.

Detection of low-level parental somatic mosaicism for clinically relevant SNVs and indels identified in a large exome sequencing dataset.

BACKGROUND: Due to the limitations of the current routine diagnostic methods, low-level somatic mosaicism with variant allele fraction (VAF) < 10% is often undetected in clinical settings. To date, only a few studies have attempted to analyze tissue distribution of low-level parental mosaicism in a large clinical exome sequencing (ES) cohort. METHODS: Using a customized bioinformatics pipeline, we analyzed apparent de novo single-nucleotide variants or indels identified in the affected probands in ES trio data at Baylor Genetics clinical laboratories. Clinically relevant variants with VAFs between 30 and 70% in probands and lower than 10% in one parent were studied. DNA samples extracted from saliva, buccal cells, redrawn peripheral blood, urine, hair follicles, and nail, representing all three germ layers, were tested using PCR amplicon next-generation sequencing (amplicon NGS) and droplet digital PCR (ddPCR). RESULTS: In a cohort of 592 clinical ES trios, we found 61 trios, each with one parent suspected of low-level mosaicism. In 21 parents, the variants were validated using amplicon NGS and seven of them by ddPCR in peripheral blood DNA samples. The parental VAFs in blood samples varied between 0.08 and 9%. The distribution of VAFs in additional tissues ranged from 0.03% in hair follicles to 9% in re-drawn peripheral blood. CONCLUSIONS: Our study illustrates the importance of analyzing ES data using sensitive computational and molecular methods for low-level parental somatic mosaicism for clinically relevant variants previously diagnosed in routine clinical diagnostics as apparent de novo.

Clinical characterization of individuals with the distal 1q21.1 microdeletion.

Distal 1q21.1 microdeletions have shown highly variable clinical expressivity and incomplete penetrance, with affected individuals manifesting a broad spectrum of nonspecific features. The goals of this study were to better describe the phenotypic spectrum of patients with distal 1q21.1 microdeletions and to compare the clinical features among affected individuals. We performed a retrospective chart review of 47 individuals with distal 1q21.1 microdeletions tested at a large clinical genetic testing laboratory, with most patients being clinically evaluated in the same children's hospital. Health information such as growth charts, results of imaging studies, developmental history, and progress notes were collected. Statistical analysis was performed using Fisher's exact test to compare clinical features among study subjects. Common features in our cohort include microcephaly (51.2%), seizures (29.8%), developmental delay (74.5%), failure to thrive (FTT) (68.1%), dysmorphic features (63.8%), and a variety of congenital anomalies such as cardiac abnormalities (23.4%) and genitourinary abnormalities (19.1%). Compared to prior literature, we found that seizures, brain anomalies, and FTT were more prevalent among our study cohort. Females were more likely than males to have microcephaly (p = 0.0199) and cardiac abnormalities (p = 0.0018). Based on existing genome-wide clinical testing results, at least a quarter of the cohort had additional genetic findings that may impact the phenotype of the individual. Our study represents the largest cohort of distal 1q21.1 microdeletion carriers available in the literature thus far, and it further illustrates the wide spectrum of clinical manifestations among symptomatic individuals. These results may allow for improved genetic counseling and management of affected individuals. Future studies may help to elucidate the underlying molecular mechanisms impacting the phenotypic variability observed with this microdeletion.

Biases in arginine codon usage correlate with genetic disease risk.

PURPOSE: The persistence of hypermutable CGN (CGG, CGA, CGC, CGU) arginine codons at high frequency suggests the possibility of negative selective pressure at these sites and that arginine codon usage could be a predictive indicator of human disease genes. METHODS: We analyzed arginine codons (CGN, AGG, AGA) from all canonical Ensembl protein coding gene transcripts before comparing the frequency of CGN codons between genes with and without human disease associations and with gnomAD constraint metrics. RESULTS: The frequency of CGN codons among a gene's total arginine codon count was higher in genes linked to syndromic autism spectrum disorder (ASD) compared with genes not associated with ASD. A comparison of genes annotated as dominant or recessive with control genes not matching either classification revealed a progressive increase in CGN codon frequency. Moreover, CGN frequency was positively correlated with a gene's probability of loss-of-function intolerance (pLI) score and negatively correlated with observed-over-expected ratios for both loss-of-function and missense variants. CONCLUSION: Our findings indicate that genes utilizing CGN arginine codons rather than AGG or AGA are more likely to underlie single-gene disorders, particularly for dominant phenotypes, and thus constitute candidate genes for the study of human genetic disease.

Edematous severe acute malnutrition is characterized by hypomethylation of DNA.

Edematous severe acute childhood malnutrition (edematous SAM or ESAM), which includes kwashiorkor, presents with more overt multi-organ dysfunction than non-edematous SAM (NESAM). Reduced concentrations and methyl-flux of methionine in 1-carbon metabolism have been reported in acute, but not recovered, ESAM, suggesting downstream DNA methylation changes could be relevant to differences in SAM pathogenesis. Here, we assess genome-wide DNA methylation in buccal cells of 309 SAM children using the 450 K microarray. Relative to NESAM, ESAM is characterized by multiple significantly hypomethylated loci, which is not observed among SAM-recovered adults. Gene expression and methylation show both positive and negative correlation, suggesting a complex transcriptional response to SAM. Hypomethylated loci link to disorders of nutrition and metabolism, including fatty liver and diabetes, and appear to be influenced by genetic variation. Our epigenetic findings provide a potential molecular link to reported aberrant 1-carbon metabolism in ESAM and support consideration of methyl-group supplementation in ESAM.

Novel parent-of-origin-specific differentially methylated loci on chromosome 16.

BACKGROUND: Congenital malformations associated with maternal uniparental disomy of chromosome 16, upd(16)mat, resemble those observed in newborns with the lethal developmental lung disease, alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Interestingly, ACDMPV-causative deletions, involving FOXF1 or its lung-specific upstream enhancer at 16q24.1, arise almost exclusively on the maternally inherited chromosome 16. Given the phenotypic similarities between upd(16)mat and ACDMPV, together with parental allelic bias in ACDMPV, we hypothesized that there may be unknown imprinted loci mapping to chromosome 16 that become functionally unmasked by chromosomal structural variants. RESULTS: To identify parent-of-origin biased DNA methylation, we performed high-resolution bisulfite sequencing of chromosome 16 on peripheral blood and cultured skin fibroblasts from individuals with maternal or paternal upd(16) as well as lung tissue from patients with ACDMPV-causative 16q24.1 deletions and a normal control. We identified 22 differentially methylated regions (DMRs) with ≥ 5 consecutive CpG methylation sites and varying tissue-specificity, including the known DMRs associated with the established imprinted gene ZNF597 and DMRs supporting maternal methylation of PRR25, thought to be paternally expressed in lymphoblastoid cells. Lastly, we found evidence of paternal methylation on 16q24.1 near LINC01082 mapping to the FOXF1 enhancer. CONCLUSIONS: Using high-resolution bisulfite sequencing to evaluate DNA methylation across chromosome 16, we found evidence for novel candidate imprinted loci on chromosome 16 that would not be evident in array-based assays and could contribute to the birth defects observed in patients with upd(16)mat or in ACDMPV.

Aberrant DNA methylation as a diagnostic biomarker of diabetic embryopathy.

PURPOSE: Maternal diabetes is a known teratogen that can cause a wide spectrum of birth defects, collectively referred to as diabetic embryopathy (DE). However, the pathogenic mechanisms underlying DE remain uncertain and there are no definitive tests to establish the diagnosis. Here, we explore the potential of DNA methylation as a diagnostic biomarker for DE and to inform disease pathogenesis. METHODS: Bisulfite sequencing was used to identify gene regions with differential methylation between DE neonates and healthy infants born with or without prenatal exposure to maternal diabetes, and to investigate the role of allele-specific methylation at implicated sites. RESULTS: We identified a methylation signature consisting of 237 differentially methylated loci that distinguished infants with DE from control infants. These loci were found proximal to genes associated with Mendelian syndromes that overlap the DE phenotype (e.g., CACNA1C, TRIO, ANKRD11) or genes known to influence embryonic development (e.g., BRAX1, RASA3). Further, we identified allele-specific methylation (ASM) at 11 of these loci, within which 61.5% of ASM single-nucleotide variants are known expression quantitative trait loci (eQTLs). CONCLUSIONS: Our study suggests a role for aberrant DNA methylation and cis-sequence variation in the pathogenesis of DE and highlights the diagnostic potential of DNA methylation for teratogenic birth defects.

TRX-LOGOS - a graphical tool to demonstrate DNA information content dependent upon backbone dynamics in addition to base sequence.

BACKGROUND: It is now widely-accepted that DNA sequences defining DNA-protein interactions functionally depend upon local biophysical features of DNA backbone that are important in defining sites of binding interaction in the genome (e.g. DNA shape, charge and intrinsic dynamics). However, these physical features of DNA polymer are not directly apparent when analyzing and viewing Shannon information content calculated at single nucleobases in a traditional sequence logo plot. Thus, sequence logos plots are severely limited in that they convey no explicit information regarding the structural dynamics of DNA backbone, a feature often critical to binding specificity. SOFTWARE AND IMPLEMENTATION: We present TRX-LOGOS, an R software package and Perl wrapper code that interfaces the JASPAR database for computational regulatory genomics. TRX-LOGOS extends the traditional sequence logo plot to include Shannon information content calculated with regard to the dinucleotide-based BI-BII conformation shifts in phosphate linkages on the DNA backbone, thereby adding a visual measure of intrinsic DNA flexibility that can be critical for many DNA-protein interactions. TRX-LOGOS is available as an R graphics module offered at both SourceForge and as a download supplement at this journal. RESULTS: To demonstrate the general utility of TRX logo plots, we first calculated the information content for 416 Saccharomyces cerevisiae transcription factor binding sites functionally confirmed in the Yeastract database and matched to previously published yeast genomic alignments. We discovered that flanking regions contain significantly elevated information content at phosphate linkages than can be observed at nucleobases. We also examined broader transcription factor classifications defined by the JASPAR database, and discovered that many general signatures of transcription factor binding are locally more information rich at the level of DNA backbone dynamics than nucleobase sequence. We used TRX-logos in combination with MEGA 6.0 software for molecular evolutionary genetics analysis to visually compare the human Forkhead box/FOX protein evolution to its binding site evolution. We also compared the DNA binding signatures of human TP53 tumor suppressor determined by two different laboratory methods (SELEX and ChIP-seq). Further analysis of the entire yeast genome, center aligned at the start codon, also revealed a distinct sequence-independent 3 bp periodic pattern in information content, present only in coding region, and perhaps indicative of the non-random organization of the genetic code. CONCLUSION: TRX-LOGOS is useful in any situation in which important information content in DNA can be better visualized at the positions of phosphate linkages (i.e. dinucleotides) where the dynamic properties of the DNA backbone functions to facilitate DNA-protein interaction.

Synonymous codon bias and functional constraint on GC3-related DNA backbone dynamics in the prokaryotic nucleoid.

While mRNA stability has been demonstrated to control rates of translation, generating both global and local synonymous codon biases in many unicellular organisms, this explanation cannot adequately explain why codon bias strongly tracks neighboring intergene GC content; suggesting that structural dynamics of DNA might also influence codon choice. Because minor groove width is highly governed by 3-base periodicity in GC, the existence of triplet-based codons might imply a functional role for the optimization of local DNA molecular dynamics via GC content at synonymous sites (≈GC3). We confirm a strong association between GC3-related intrinsic DNA flexibility and codon bias across 24 different prokaryotic multiple whole-genome alignments. We develop a novel test of natural selection targeting synonymous sites and demonstrate that GC3-related DNA backbone dynamics have been subject to moderate selective pressure, perhaps contributing to our observation that many genes possess extreme DNA backbone dynamics for their given protein space. This dual function of codons may impose universal functional constraints affecting the evolution of synonymous and non-synonymous sites. We propose that synonymous sites may have evolved as an 'accessory' during an early expansion of a primordial genetic code, allowing for multiplexed protein coding and structural dynamic information within the same molecular context.