Suppression of the Arabidopsis cinnamoyl-CoA reductase 1-6 intronic T-DNA mutation by epigenetic modification.

Arabidopsis (Arabidopsis thaliana) transfer DNA (T-DNA) insertion collections are popular resources for fundamental plant research. Cinnamoyl-CoA reductase 1 (CCR1) catalyzes an essential step in the biosynthesis of the cell wall polymer lignin. Accordingly, the intronic T-DNA insertion mutant ccr1-6 has reduced lignin levels and shows a stunted growth phenotype. Here, we report restoration of the ccr1-6 mutant phenotype and CCR1 expression levels after a genetic cross with a UDP-glucosyltransferase 72e1 (ugt72e1),-e2,-e3 T-DNA mutant. We discovered that the phenotypic recovery was not dependent on the UGT72E family loss of function but due to an epigenetic phenomenon called trans T-DNA suppression. Via trans T-DNA suppression, the gene function of an intronic T-DNA mutant was restored after the introduction of an additional T-DNA sharing identical sequences, leading to heterochromatinization and splicing out of the T-DNA-containing intron. Consequently, the suppressed ccr1-6 allele was named epiccr1-6. Long-read sequencing revealed that epiccr1-6, not ccr1-6, carries dense cytosine methylation over the full length of the T-DNA. We showed that the SAIL T-DNA in the UGT72E3 locus could trigger the trans T-DNA suppression of the GABI-Kat T-DNA in the CCR1 locus. Furthermore, we scanned the literature for other potential cases of trans T-DNA suppression in Arabidopsis and found that 22% of the publications matching our query report on double or higher-order T-DNA mutants that meet the minimal requirements for trans T-DNA suppression. These combined observations indicate that intronic T-DNA mutants need to be used with caution since methylation of intronic T-DNA might derepress gene expression and can thereby confound results.

Rare variants in IFFO1, DTNB, NLRC3 and SLC22A10 associate with Alzheimer's disease CSF profile of neuronal injury and inflammation.

Alzheimer's disease (AD) biomarkers represent several neurodegenerative processes, such as synaptic dysfunction, neuronal inflammation and injury, as well as amyloid pathology. We performed an exome-wide rare variant analysis of six AD biomarkers (ß-amyloid, total/phosphorylated tau, NfL, YKL-40, and Neurogranin) to discover genes associated with these markers. Genetic and biomarker information was available for 480 participants from two studies: EMIF-AD and ADNI. We applied a principal component (PC) analysis to derive biomarkers combinations, which represent statistically independent biological processes. We then tested whether rare variants in 9576 protein-coding genes associate with these PCs using a Meta-SKAT test. We also tested whether the PCs are intermediary to gene effects on AD symptoms with a SMUT test. One PC loaded on NfL and YKL-40, indicators of neuronal injury and inflammation. Four genes were associated with this PC: IFFO1, DTNB, NLRC3, and SLC22A10. Mediation tests suggest, that these genes also affect dementia symptoms via inflammation/injury. We also observed an association between a PC loading on Neurogranin, a marker for synaptic functioning, with GABBR2 and CASZ1, but no mediation effects. The results suggest that rare variants in IFFO1, DTNB, NLRC3, and SLC22A10 heighten susceptibility to neuronal injury and inflammation, potentially by altering cytoskeleton structure and immune activity disinhibition, resulting in an elevated dementia risk. GABBR2 and CASZ1 were associated with synaptic functioning, but mediation analyses suggest that the effect of these two genes on synaptic functioning is not consequential for AD development.

Whole-exome rare-variant analysis of Alzheimer's disease and related biomarker traits.

INTRODUCTION: Despite increasing evidence of a role of rare genetic variation in the risk of Alzheimer's disease (AD), limited attention has been paid to its contribution to AD-related biomarker traits indicative of AD-relevant pathophysiological processes. METHODS: We performed whole-exome gene-based rare-variant association studies (RVASs) of 17 AD-related traits on whole-exome sequencing (WES) data generated in the European Medical Information Framework for Alzheimer's Disease Multimodal Biomarker Discovery (EMIF-AD MBD) study (n = 450) and whole-genome sequencing (WGS) data from ADNI (n = 808). RESULTS: Mutation screening revealed a novel probably pathogenic mutation (PSEN1 p.Leu232Phe). Gene-based RVAS revealed the exome-wide significant contribution of rare coding variation in RBKS and OR7A10 to cognitive performance and protection against left hippocampal atrophy, respectively. DISCUSSION: The identification of these novel gene-trait associations offers new perspectives into the role of rare coding variation in the distinct pathophysiological processes culminating in AD, which may lead to identification of novel therapeutic and diagnostic targets.

Mitochondrial GpC and CpG DNA Hypermethylation Cause Metabolic Stress-Induced Mitophagy and Cholestophagy.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by a constant accumulation of lipids in the liver. This hepatic lipotoxicity is associated with a dysregulation of the first step in lipid catabolism, known as beta oxidation, which occurs in the mitochondrial matrix. Eventually, this dysregulation will lead to mitochondrial dysfunction. To evaluate the possible involvement of mitochondrial DNA methylation in this lipid metabolic dysfunction, we investigated the functional metabolic effects of mitochondrial overexpression of CpG (MSssI) and GpC (MCviPI) DNA methyltransferases in relation to gene expression and (mito)epigenetic signatures. Overall, the results show that mitochondrial GpC and, to a lesser extent, CpG methylation increase bile acid metabolic gene expression, inducing the onset of cholestasis through mito-nuclear epigenetic reprogramming. Moreover, both increase the expression of metabolic nuclear receptors and thereby induce basal overactivation of mitochondrial respiration. The latter promotes mitochondrial swelling, favoring lipid accumulation and metabolic-stress-induced mitophagy and autophagy stress responses. In conclusion, both mitochondrial GpC and CpG methylation create a metabolically challenging environment that induces mitochondrial dysfunction, which may contribute to the progression of MASLD.

Genomic Analysis of a Strain Collection Containing Multidrug-, Extensively Drug-, Pandrug-, and Carbapenem-Resistant Modern Clinical Isolates of Acinetobacter baumannii.

In this study, we characterize a new collection that comprises multidrug-resistant (MDR), extensively drug-resistant (XDR), pandrug-resistant (PDR), and carbapenem-resistant modern clinical isolates of Acinetobacter baumannii collected from hospitals through national microbiological surveillance in Belgium. Bacterial isolates (n = 43) were subjected to whole-genome sequencing (WGS), combining Illumina (MiSeq) and Nanopore (MinION) technologies, from which high-quality genomes (chromosome and plasmids) were de novo assembled. Antimicrobial susceptibility testing was performed along with genome analyses, which identified intrinsic and acquired resistance determinants along with their genetic environments and vehicles. Furthermore, the bacterial isolates were compared to the most prevalent A. baumannii sequence type 2 (ST2) (Pasteur scheme) genomes available from the BIGSdb database. Of the 43 strains, 40 carried determinants of resistance to carbapenems; blaOXA-23 (n = 29) was the most abundant acquired antimicrobial resistance gene, with 39 isolates encoding at least two different types of OXA enzymes. According to the Pasteur scheme, the majority of the isolates were globally disseminated clones of ST2 (n = 25), while less frequent sequence types included ST636 (n = 6), ST1 (n = 4), ST85 and ST78 (n = 2 each), and ST604, ST215, ST158, and ST10 (n = 1 each). Using the Oxford typing scheme, we identified 22 STs, including two novel types (ST2454 and ST2455). While the majority (26/29) of blaOXA-23 genes were chromosomally carried, all blaOXA-72 genes were plasmid borne. Our results show the presence of high-risk clones of A. baumannii within Belgian health care facilities with frequent occurrences of genes encoding carbapenemases, highlighting the crucial need for constant surveillance.

Structural variants identified by Oxford Nanopore PromethION sequencing of the human genome.

We sequenced the genome of the Yoruban reference individual NA19240 on the long-read sequencing platform Oxford Nanopore PromethION for evaluation and benchmarking of recently published aligners and germline structural variant calling tools, as well as a comparison with the performance of structural variant calling from short-read sequencing data. The structural variant caller Sniffles after NGMLR or minimap2 alignment provides the most accurate results, but additional confidence or sensitivity can be obtained by a combination of multiple variant callers. Sensitive and fast results can be obtained by minimap2 for alignment and a combination of Sniffles and SVIM for variant identification. We describe a scalable workflow for identification, annotation, and characterization of tens of thousands of structural variants from long-read genome sequencing of an individual or population. By discussing the results of this well-characterized reference individual, we provide an approximation of what can be expected in future long-read sequencing studies aiming for structural variant identification.

Methplotlib: analysis of modified nucleotides from nanopore sequencing.

SUMMARY: Modified nucleotides play a crucial role in gene expression regulation. Here, we describe methplotlib, a tool developed for the visualization of modified nucleotides detected from Oxford Nanopore Technologies sequencing platforms, together with additional scripts for statistical analysis of allele-specific modification within-subjects and differential modification frequency across subjects. AVAILABILITY AND IMPLEMENTATION: The methplotlib command-line tool is written in Python3, is compatible with Linux, Mac OS and the MS Windows 10 Subsystem for Linux and released under the MIT license. The source code can be found at https://github.com/wdecoster/methplotlib and can be installed from PyPI and bioconda. Our repository includes test data, and the tool is continuously tested at travis-ci.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Loss of DPP6 in neurodegenerative dementia: a genetic player in the dysfunction of neuronal excitability.

Emerging evidence suggested a converging mechanism in neurodegenerative brain diseases (NBD) involving early neuronal network dysfunctions and alterations in the homeostasis of neuronal firing as culprits of neurodegeneration. In this study, we used paired-end short-read and direct long-read whole genome sequencing to investigate an unresolved autosomal dominant dementia family significantly linked to 7q36. We identified and validated a chromosomal inversion of ca. 4 Mb, segregating on the disease haplotype and disrupting the coding sequence of dipeptidyl-peptidase 6 gene (DPP6). DPP6 resequencing identified significantly more rare variants-nonsense, frameshift, and missense-in early-onset Alzheimer's disease (EOAD, p value = 0.03, OR = 2.21 95% CI 1.05-4.82) and frontotemporal dementia (FTD, p = 0.006, OR = 2.59, 95% CI 1.28-5.49) patient cohorts. DPP6 is a type II transmembrane protein with a highly structured extracellular domain and is mainly expressed in brain, where it binds to the potassium channel Kv4.2 enhancing its expression, regulating its gating properties and controlling the dendritic excitability of hippocampal neurons. Using in vitro modeling, we showed that the missense variants found in patients destabilize DPP6 and reduce its membrane expression (p < 0.001 and p < 0.0001) leading to a loss of protein. Reduced DPP6 and/or Kv4.2 expression was also detected in brain tissue of missense variant carriers. Loss of DPP6 is known to cause neuronal hyperexcitability and behavioral alterations in Dpp6-KO mice. Taken together, the results of our genomic, genetic, expression and modeling analyses, provided direct evidence supporting the involvement of DPP6 loss in dementia. We propose that loss of function variants have a higher penetrance and disease impact, whereas the missense variants have a variable risk contribution to disease that can vary from high to low penetrance. Our findings of DPP6, as novel gene in dementia, strengthen the involvement of neuronal hyperexcitability and alteration in the homeostasis of neuronal firing as a disease mechanism to further investigate.

miRVaS: a tool to predict the impact of genetic variants on miRNAs.

Genetic variants in or near miRNA genes can have profound effects on miRNA expression and targeting. As user-friendly software for the impact prediction of miRNA variants on a large scale is still lacking, we created a tool called miRVaS. miRVaS automates this prediction by annotating the location of the variant relative to functional regions within the miRNA hairpin (seed, mature, loop, hairpin arm, flanks) and by annotating all predicted structural changes within the miRNA due to the variant. In addition, the tool defines the most important region that is predicted to have structural changes and calculates a conservation score that is indicative of the reliability of the structure prediction. The output is presented in a tab-separated file, which enables fast screening, and in an html file, which allows visual comparison between wild-type and variant structures. All separate images are provided for downstream use. Finally, we tested two different approaches on a small test set of published functionally validated genetic variants for their capacity to predict the impact of variants on miRNA expression.

Identification of rare copy number variants in high burden schizophrenia families.

Over the last years, genome-wide studies consistently showed an increased burden of rare copy number variants (CNVs) in schizophrenia patients, supporting the "common disease, rare variant" hypothesis in at least a subset of patients. We hypothesize that in families with a high burden of disease, and thus probably a high genetic load influencing disease susceptibility, rare CNVs might be involved in the etiology of schizophrenia. We performed a genome-wide CNV analysis in the index patients of eight families with multiple schizophrenia affected members, and consecutively performed a detailed family analysis for the most relevant CNVs. One index patient showed a DRD5 containing duplication. A second index patient presented with an NRXN1 containing deletion and two adjacent duplications containing MYT1L and SNTG2. Detailed analysis in the subsequent families showed segregation of the identified CNVs. With this study we show the importance of screening high burden families for rare CNVs, which will not only broaden our knowledge concerning the molecular genetic mechanisms involved in schizophrenia but also allow the use of the obtained genetic data to provide better clinical care to these families in general and to non-symptomatic causal CNV carriers in particular.

Phenotypic Characterization and Heterogeneity among Modern Clinical Isolates of Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic pathogenic bacterium prioritized by WHO and CDC because of its increasing antibiotic resistance. Heterogeneity among strains represents the hallmark of A. baumannii bacteria. We wondered to what extent extensively used strains, so-called reference strains, reflect the dynamic nature and intrinsic heterogeneity of these bacteria. We analyzed multiple phenotypic traits of 43 nonredundant, modern, and multidrug-resistant, extensively drug-resistant, and pandrug-resistant clinical isolates and broadly used strains of A. baumannii. Comparison of these isolates at the genetic and phenotypic levels confirmed a high degree of heterogeneity. Importantly, we observed that a significant portion of modern clinical isolates strongly differs from several historically established strains in the light of colony morphology, cellular density, capsule production, natural transformability, and in vivo virulence. The significant differences between modern clinical isolates of A. baumannii and established strains could hamper the study of A. baumannii, especially concerning its virulence and resistance mechanisms. Hence, we propose a variable collection of modern clinical isolates that are characterized at the genetic and phenotypic levels, covering a wide range of the phenotypic spectrum, with six different macrocolony type groups, from avirulent to hypervirulent phenotypes, and with naturally noncapsulated to hypermucoid strains, with intermediate phenotypes as well. Strain-specific mechanistic observations remain interesting per se, and established "reference" strains have undoubtedly been shown to be very useful to study basic mechanisms of A. baumannii biology. However, any study based on a specific strain of A. baumannii should be compared to modern and clinically relevant isolates. IMPORTANCE Acinetobacter baumannii is a bacterium prioritized by the CDC and WHO because of its increasing antibiotic resistance, leading to treatment failures. The hallmark of this pathogen is the high heterogeneity observed among isolates, due to a very dynamic genome. In this context, we tested if a subset of broadly used isolates, considered "reference" strains, was reflecting the genetic and phenotypic diversity found among currently circulating clinical isolates. We observed that the so-called reference strains do not cover the whole diversity of the modern clinical isolates. While formerly established strains successfully generated a strong base of knowledge in the A. baumannii field and beyond, our study shows that a rational choice of strain, related to a specific biological question, should be taken into consideration. Any data obtained with historically established strains should also be compared to modern and clinically relevant isolates, especially concerning drug screening, resistance, and virulence contexts.

Less cognitive and neurological deficits in schizophrenia patients carrying risk variant in ZNF804A.

BACKGROUND: The rs1344706 single nucleotide polymorphism in the ZNF804A gene is a common variant with strong evidence for association with schizophrenia. Recent studies show an association of rs1344706 with cognitive functioning, and there is some evidence suggesting that the risk allele may increase susceptibility for a subtype of schizophrenia with relatively spared cognition. METHODS: We tested the effect of rs1344706 genotype in 89 schizophrenia patients on 3 basic cognitive domains (working memory, processing speed and attention) shown to be severely impaired in schizophrenia. Also we investigated the effect of rs1344706 on the severity of neurological soft signs, subtle impairments in motor and sensory functions highly frequent in schizophrenia patients. Neurological soft signs and cognitive deficits are central features of schizophrenia and are tightly linked with clinical, social and functional outcome. RESULTS: Our results show an association of higher rs1344706 risk allele load with improved performance on processing speed and with fewer neurological soft signs. CONCLUSIONS: Together with other studies, our findings suggest that ZNF804A is associated with a subtype of schizophrenia with better cognitive and neurological functioning. Discovery of the specific pathways through which ZNF804A is exerting this effect may lead to better prevention, diagnosis and treatment for a specific group of schizophrenia patients.

Rare copy number variants in neuropsychiatric disorders: Specific phenotype or not?

From a number of genome-wide association studies it was shown that de novo and/or rare copy number variants (CNVs) are found at an increased frequency in neuropsychiatric diseases. In this study we examined the prevalence of CNVs in six genomic regions (1q21.1, 2p16.3, 3q29, 15q11.2, 15q13.3, and 16p11.2) previously implicated in neuropsychiatric diseases. Hereto, a cohort of four neuropsychiatric disorders (schizophrenia, bipolar disorder, major depressive disorder, and intellectual disability) and control individuals from three different populations was used in combination with Multilpex Amplicon Quantifiaction (MAQ) assays, capable of high resolution (kb range) and custom-tailored CNV detection. Our results confirm the etiological candidacy of the six selected CNV regions for neuropsychiatric diseases. It is possible that CNVs in these regions can result in disturbed brain development and in this way lead to an increased susceptibility for different neuropsychiatric disorders, dependent on additional genetic and environmental factors. Our results also suggest that the neurodevelopmental component is larger in the etiology of schizophrenia and intellectual disability than in mood disorders. Finally, our data suggest that deletions are in general more pathogenic than duplications. Given the high frequency of the examined CNVs (1-2%) in patients of different neuropsychiatric disorders, screening of large cohorts with an affordable and feasible method like the MAQ assays used in this study is likely to result in important progress in unraveling the genetic factors leading to an increased susceptibility for several psychiatric disorders.

Identification of a CACNA2D4 deletion in late onset bipolar disorder patients and implications for the involvement of voltage-dependent calcium channels in psychiatric disorders.

The GWAS-based association of CACNA1C with bipolar disorder (BPD) is one of the strongest genetic findings to date. CACNA1C belongs to the family of CACN genes encoding voltage-dependent calcium channels (VDCCs). VDCCs are involved in brain circuits and cognitive processes implicated in BPD and schizophrenia (SZ). Recently, it was shown that rare copy number variations (CNVs) are found at an increased frequency in SZ and to a lesser extent also in BPD, suggesting the involvement of CNVs in the causation of these diseases. We hypothesize that CNVs in CACN genes can influence the susceptibility to BPD, SZ, and/or schizoaffective disorder (SZA). A search for CNVs in eight CACN genes in a patient-control sample of European decent was performed. A total of 709 BP patients, 645 SZ patients, 189 SZA patients, and 1,470 control individuals were screened using the Multiplex Amplicon Quantification (MAQ) method. We found a rare, partial deletion of 35.7 kb in CACNA2D4 in two unrelated late onset bipolar I patients and in one control individual. All three deletions shared the same breakpoints removing exons 17-26 of CACNA2D4, comprising part of the CACHE domain. Based on the data we cannot claim causality to BPD of the identified CACNA2D4 deletion but nevertheless this deletion can be important in unraveling the underlying processes leading to psychiatric diseases in general and BPD in particular.

Scarless excision of an insertion sequence restores capsule production and virulence in Acinetobacter baumannii.

We identify a new mechanism mediating capsule production and virulence in the WHO and CDC priority ESKAPE pathogen Acinetobacter baumannii. Non-capsulated and avirulent bacteria can revert into a capsulated and virulent state upon scarless excision of an ISAba13 insertion sequence under stress conditions. Reversion events fully restore capsule production and in vivo virulence. This increases our knowledge about A. baumannii genome dynamics, and the regulation of capsule production, virulence and resistance.

Long-Read Sequencing to Unravel Complex Structural Variants of CEP78 Leading to Cone-Rod Dystrophy and Hearing Loss.

Inactivating variants as well as a missense variant in the centrosomal CEP78 gene have been identified in autosomal recessive cone-rod dystrophy with hearing loss (CRDHL), a rare syndromic inherited retinal disease distinct from Usher syndrome. Apart from this, a complex structural variant (SV) implicating CEP78 has been reported in CRDHL. Here we aimed to expand the genetic architecture of typical CRDHL by the identification of complex SVs of the CEP78 region and characterization of their underlying mechanisms. Approaches used for the identification of the SVs are shallow whole-genome sequencing (sWGS) combined with quantitative polymerase chain reaction (PCR) and long-range PCR, or ExomeDepth analysis on whole-exome sequencing (WES) data. Targeted or whole-genome nanopore long-read sequencing (LRS) was used to delineate breakpoint junctions at the nucleotide level. For all SVs cases, the effect of the SVs on CEP78 expression was assessed using quantitative PCR on patient-derived RNA. Apart from two novel canonical CEP78 splice variants and a frameshifting single-nucleotide variant (SNV), two SVs affecting CEP78 were identified in three unrelated individuals with CRDHL: a heterozygous total gene deletion of 235 kb and a partial gene deletion of 15 kb in a heterozygous and homozygous state, respectively. Assessment of the molecular consequences of the SVs on patient's materials displayed a loss-of-function effect. Delineation and characterization of the 15-kb deletion using targeted LRS revealed the previously described complex CEP78 SV, suggestive of a recurrent genomic rearrangement. A founder haplotype was demonstrated for the latter SV in cases of Belgian and British origin, respectively. The novel 235-kb deletion was delineated using whole-genome LRS. Breakpoint analysis showed microhomology and pointed to a replication-based underlying mechanism. Moreover, data mining of bulk and single-cell human and mouse transcriptional datasets, together with CEP78 immunostaining on human retina, linked the CEP78 expression domain with its phenotypic manifestations. Overall, this study supports that the CEP78 locus is prone to distinct SVs and that SV analysis should be considered in a genetic workup of CRDHL. Finally, it demonstrated the power of sWGS and both targeted and whole-genome LRS in identifying and characterizing complex SVs in patients with ocular diseases.

Oligonucleotide DNA microarray profiling of lung adenocarcinoma revealed significant downregulation and deletions of vasoactive intestinal peptide receptor 1.

The purpose of this study was to find novel gene(s) involved in the development of lung adenocarcinoma (AD). Using DNA microarrays, we identified 31 up-regulated and 8 downregulated genes in 12 AD. Real time PCR was used to measure expression of VIPR1 and SPP1 mRNA and possible losses or gains of genes in 32 AD. We describe significant upregulation of the SPP1 gene, downregulation of VIPR1, and losses of the VIPR1 gene. Our findings complement a proposed VIPR1 tumor suppressor role, in which deletions in the 3p22 chromosome region are an important mechanism leading to loss of the VIPR1 gene.

Darier disease in Slovenia: spectrum of ATP2A2 mutations and relation to patients' phenotypes.

ATP2A2 encodes the sarco/endoplasmic reticulum Ca2+- ATPase (SERCA2) and has been identified as a defective gene in Darier disease (DD). It is an autosomal dominant genodermatosis, which is characterized by loss of adhesion between suprabasal epidermal keratinocytes (acantholysis) and abnormal keratinization (dyskeratosis). We examined 28 Slovenian patients with DD (the cohort of patients represents over 50% of all DD patients in Slovenia) and screened genomic DNA for ATP2A2 mutations and RNA for splice site mutations. We identified 7 different ATP2A2 mutations, 4 of which are novel: A516P, R559G, 544+1del6, and 1762-6del18. We also found two previously described polymorphisms 2741+54 G>A in intron XVIII and 2172 G>A (A724A) in exon 15, with allele frequencies of 64.2% and 11.3%, respectively. The mutations are scattered throughout the gene and affect the actuator, phosphorylation, stalk and transmembrane domains of SERCA2. A P160L mutation in a Slovene patient with severe DD and a history of deafness is another consistent genotype-phenotype correlation. It seems that mutations of the ATP2A2 gene may also play a role in the pathogenesis of deafness, which seems to be a new phenotypic characteristic of DD patients.

Critical length in long-read resequencing.

Long-read sequencing has substantial advantages for structural variant discovery and phasing of variants compared to short-read technologies, but the required and optimal read length has not been assessed. In this work, we used long reads simulated from human genomes and evaluated structural variant discovery and variant phasing using current best practice bioinformatics methods. We determined that optimal discovery of structural variants from human genomes can be obtained with reads of minimally 20 kb. Haplotyping variants across genes only reaches its optimum from reads of 100 kb. These findings are important for the design of future long-read sequencing projects.

Polymorphisms in COL4A3 and COL4A4 genes associated with keratoconus.

PURPOSE: Alterations in collagen type IV, alpha-3 (COL4A3) and collagen type IV, alpha-4 (COL4A4) genes may be responsible for a decrease in collagen types I and III, a feature often detected in keratoconus (KC). To evaluate the significance of alterations in COL4A3 and COL4A4 genes in KC patients, we screened both genes and estimated the significance of polymorphisms in Slovenian patients with KC. METHODS: The study included 104 unrelated patients with KC and 157 healthy blood donors. Diagnosis was established by clinical examination, electronic refractometry, and keratometry. DNA was extracted from blood, and gene exons were amplified by PCR. Non-isotopic high-resolution single-stranded conformation analysis (SSCA) was used to screen COL4A3 and COL4A4 genes, and migration shifts detected by SSCA were subsequently sequenced. For statistical evaluation, control blood donors were chosen according to age, sex, and not having blood relationship. Neither patients nor control blood donors chosen for statistical analysis were in blood relationship. We used Fisher's exact test for statistical evaluation, with p<0.05 considered significant. RESULTS: We detected eight polymorphisms in the COL4A3 gene and six in the COL4A4 gene. Allele differences in D326Y in COL4A3 and M1237V and F1644F in COL4A4 are significantly distinctive of KC patients (Fisher's exact test, p<0.05). When analyzing different genotypes under three models (dominant, recessive, and additive), we established that P141L, D326Y, and G895G in COL4A3 and P482S, M1327V, V1516V, and F1644F in COL4A4 have significant differences in genotype distribution between KC patients and the control group. CONCLUSIONS: This is the first mutational screening of COL4A3 and COL4A4 genes in KC patients to establish the status of these genes and compare them to a control population. Analysis of COL4A3 and COL4A4 revealed no mutations related to KC patients, but specific genotypes of seven previously described polymorphisms are significantly associated with KC under dominant, recessive, or additive models. Differences in the expression of type IV collagen in previously published data about chromosomal instabilities in the regions in which the analyzed genes were mapped and our data indicate a probability that some of the polymorphisms we detected could be related to KC.