A Combined Metabolomic and Proteomic Analysis of Gestational Diabetes Mellitus.

The aim of this pilot study was to apply a novel combined metabolomic and proteomic approach in analysis of gestational diabetes mellitus. The investigation was performed with plasma samples derived from pregnant women with diagnosed gestational diabetes mellitus (n = 18) and a matched control group (n = 13). The mass spectrometry-based analyses allowed to determine 42 free amino acids and low molecular-weight peptide profiles. Different expressions of several peptides and altered amino acid profiles were observed in the analyzed groups. The combination of proteomic and metabolomic data allowed obtaining the model with a high discriminatory power, where amino acids ethanolamine, L-citrulline, L-asparagine, and peptide ions with m/z 1488.59; 4111.89 and 2913.15 had the highest contribution to the model. The sensitivity (94.44%) and specificity (84.62%), as well as the total group membership classification value (90.32%) calculated from the post hoc classification matrix of a joint model were the highest when compared with a single analysis of either amino acid levels or peptide ion intensities. The obtained results indicated a high potential of integration of proteomic and metabolomics analysis regardless the sample size. This promising approach together with clinical evaluation of the subjects can also be used in the study of other diseases.

Sequence variants in COL4A1 and COL4A2 genes in Ecuadorian families with keratoconus.

PURPOSE: Keratoconus (KTCN) is a non-inflammatory, usually bilateral disorder of the eye which results in the conical shape and the progressive thinning of the cornea. Several studies have suggested that genetic factors play a role in the etiology of the disease. Several loci were previously described as possible candidate regions for familial KTCN; however, no causative mutations in any genes have been identified for any of these loci. The purpose of this study was to evaluate role of the collagen genes collagen type IV, alpha-1 (COL4A1) and collagen type IV, alpha-2 (COL4A2) in KTCN in Ecuadorian families. METHODS: COL4A1 and COL4A2 in 15 Ecuadorian KTCN families were examined with polymerase chain reaction amplification, and direct sequencing of all exons, promoter and intron-exon junctions was performed. RESULTS: Screening of COL4A1 and COL4A2 revealed numerous alterations in coding and non-coding regions of both genes. We detected three missense substitutions in COL4A1: c.19G>C (Val7Leu), c.1663A>C (Thr555Pro), and c.4002A>C (Gln1334His). Five non-synonymous variants were identified in COL4A2: c.574G>T (Val192Phe), c.1550G>A (Arg517Lys), c.2048G>C (Gly683Ala), c.2102A>G (Lys701Arg), and c.2152C>T (Pro718Ser). None of the identified sequence variants completely segregated with the affected phenotype. The Gln1334His variant was possibly damaging to protein function and structure. CONCLUSIONS: This is the first mutation screening of COL4A1 and COL4A2 genes in families with KTCN and linkage to a locus close to these genes. Analysis of COL4A1 and COL4A2 revealed no mutations indicating that other genes are involved in KTCN causation in Ecuadorian families.

IGF-1 gene polymorphisms in Polish families with high-grade myopia.

PURPOSE: Recent work has suggested that insulin-like growth factor 1 (IGF-1) gene polymorphisms are genetically linked with high-grade myopia (HM), which is a complex-trait eye disorder in which numerous candidate loci and genes are thought to play a role. We investigated whether the IGF-1 single nucleotide polymorphisms (SNPs) rs6214, rs10860860, and rs2946834 are associated with HM (≤-6.0 diopters [D]) and any myopia (≤-0.5 D) phenotype in Polish families. METHODS: Forty-two multiplex HM Polish families, of whom 127 had HM, participated in the study. All of the family members (n=306) underwent a detailed ophthalmic examination, including axial length measurements. The IGF-1 SNPs rs6214, rs10860860, and rs2946834 were evaluated by PCR-RFLP and direct sequencing methods. Both Family-Based Association Test (FBAT) and family-based Pedigree Disequilibrium Test (PDT) were used to examine the potential association of the IGF-1 SNPs rs6214, rs10860860, and rs2946834 with HM or any myopia. To determine the distribution of the HM-associated SNPs rs6214 and rs10860860, 543 unrelated individuals from the general Polish population were also analyzed. RESULTS: We found no significant association between the IGF-1 SNPs rs6214, rs10860860, and rs2946834 and HM or any myopia phenotype in Polish HM families. In the general Polish population, the minor allele frequencies of the SNPs rs6214 and rs10860860 did not deviate significantly from the distribution reported for European populations (p=0.629). In the FBAT analysis under the dominant model, the haplotype consisted of T allele of rs10860860, with C allele of rs2946834 of IGF-1 was found less frequently transmitted to HM individuals (p=0.0065), pointing to a nonassociated or protective haplotype. CONCLUSIONS: Our results do not support recent studies reporting an association of the SNPs rs6214, rs10860860, and rs2946834 in the IGF-1 gene with HM and any myopia phenotypes. Further replication studies involving other populations are needed to investigate the possible role of IGF-1 as a potential myopia candidate gene.

Deletions and duplications of developmental pathway genes in 5q31 contribute to abnormal phenotypes.

Although copy number changes of 5q31 have been rarely reported, deletions have been associated with some common characteristics, such as short stature, failure to thrive, developmental delay (DD)/intellectual disability (ID), club feet, dislocated hips, and dysmorphic features. We report on three individuals with deletions and two individuals with duplications at 5q31, ranging from 3.6 Mb to 8.1 Mb and 830 kb to 3.4 Mb in size, respectively. All five copy number changes are apparently de novo and involve several genes that are important in developmental pathways, including PITX1, SMAD5, and WNT8A. The individuals with deletions have characteristic features including DD, short stature, club feet, cleft or high palate, dysmorphic features, and skeletal anomalies. Haploinsufficiency of PITX1, a transcription factor important for limb development, is likely the cause for the club feet, skeletal anomalies, and cleft/high palate, while additional genes, including SMAD5 and WNT8A, may also contribute to additional phenotypic features. Two patients with deletions also presented with corneal anomalies. To identify a causative gene for the corneal anomalies, we sequenced candidate genes in a family with apparent autosomal dominant keratoconus with suggestive linkage to 5q31, but no mutations in candidate genes were found. The duplications are smaller than the deletions, and the patients with duplications have nonspecific features. Although development is likely affected by increased dosage of the genes in the region, the developmental disruption appears less severe than that seen with deletion.

Collagen synthesis disruption and downregulation of core elements of TGF-ß, Hippo, and Wnt pathways in keratoconus corneas.

To understand better the factors contributing to keratoconus (KTCN), we performed comprehensive transcriptome profiling of human KTCN corneas for the first time using an RNA-Seq approach. Twenty-five KTCN and 25 non-KTCN corneas were enrolled in this study. After RNA extraction, total RNA libraries were prepared and sequenced. The discovery RNA-Seq analysis (in eight KTCN and eight non-KTCN corneas) was conducted first, after which the replication RNA-Seq experiment was performed on a second set of samples (17 KTCN and 17 non-KTCN corneas). Over 82% of the genes and almost 75% of the transcripts detected as differentially expressed in KTCN and non-KTCN corneas were confirmed in the replication study using another set of samples. We used these differentially expressed genes to generate a network of KTCN-deregulated genes. We found an extensive disruption of collagen synthesis and maturation pathways, as well as downregulation of the core elements of the TGF-ß, Hippo, and Wnt signaling pathways influencing corneal organization. This first comprehensive transcriptome profiling of human KTCN corneas points further to a complex etiology of KTCN.

KTCNlncDB-a first platform to investigate lncRNAs expressed in human keratoconus and non-keratoconus corneas.

Keratoconus (KTCN, OMIM 148300) is a degenerative eye disorder characterized by progressive stromal thinning that leads to a conical shape of the cornea, resulting in optical aberrations and even loss of visual function. The biochemical background of the disease is poorly understood, which motivated us to perform RNA-Seq experiment, aimed at better characterizing the KTCN transcriptome and identification of long non-coding RNAs (lncRNAs) that might be involved in KTCN etiology. The in silico functional studies based on predicted lncRNA:RNA base-pairings led us to recognition of a number of lncRNAs possibly regulating genes with known or plausible links to KTCN. The lncRNA sequences and data regarding their predicted functions in controlling the RNA processing and stability are available for browse, search and download in KTCNlncDB (http://rhesus.amu.edu.pl/KTCNlncDB/), the first online platform devoted to KTCN transcriptome.Database URL: http://rhesus.amu.edu.pl/KTCNlncDB/.

Nonrandom Distribution of miRNAs Genes and Single Nucleotide Variants in Keratoconus Loci.

Despite numerous studies, the causes of both development and progression of keratoconus remain elusive. Previous studies of this disorder focused mainly on one or two genetic factors only. However, in the analysis of such complex diseases all potential factors should be taken into consideration. The purpose of this study was a comprehensive analysis of known keratoconus loci to uncover genetic factors involved in this disease causation in the general population, which could be omitted in the original studies. In this investigation genomic data available in various databases and experimental own data were assessed. The lists of single nucleotide variants and miRNA genes localized in reported keratoconus loci were obtained from Ensembl and miRBase, respectively. The potential impact of nonsynonymous amino acid substitutions on protein structure and function was assessed with PolyPhen-2 and SIFT. For selected protein genes the ranking was made to choose those most promising for keratoconus development. Ranking results were based on topological features in the protein-protein interaction network. High specificity for the populations in which the causative sequence variants have been identified was found. In addition, the possibility of links between previously analyzed keratoconus loci was confirmed including miRNA-gene interactions. Identified number of genes associated with oxidative stress and inflammatory agents corroborated the hypothesis of their effect on the disease etiology. Distribution of the numerous sequences variants within both exons and mature miRNA which forces you to search for a broader look at the determinants of keratoconus. Our findings highlight the complexity of the keratoconus genetics.

Substitution at IL1RN and deletion at SLC4A11 segregating with phenotype in familial keratoconus.

PURPOSE: Keratoconus (KTCN) is a thinning and anterior protrusion of the cornea that results in altered refractive powers and loss of visual acuity. Despite numerous studies, the reasons for development and progression of KTCN remain unknown. Genetic studies have led to identification of several loci linked with KTCN, including a locus in one multigenerational Ecuadorian family. The purpose of this study was to identify sequence variants in candidate genes segregating with the KTCN phenotype in another Ecuadorian family. METHODS: Nonparametric linkage analysis was performed in Ecuadorian family KTCN-019. Candidate genes IL1A, IL1B, IL1RN, and SLC4A11 were selected and examined in this family by direct sequencing of all exons, promoters, and intron-exon junctions. RESULTS: Two novel suggestive loci were identified in 2q13-q14.3 and 20p13-p12.2. Screening of the candidate genes revealed 66 sequence variants, including five novel variants, in both coding and noncoding regions. The substitution c.214+242C > T in the IL1RN gene was observed in all affected individuals and three apparently unaffected family members. The novel deletion of 54 nucleotides in position c.2558+149_2558+203 in SLC4A11 was observed in all patients but one, as well as two healthy individuals and one person with an unknown phenotype. CONCLUSIONS: The analyses of selected genes have led to identification of numerous sequence variants in the examined Ecuadorian family. Both substitution c.214+242C > T in IL1RN and novel deletion c.2558+149_2558+203del54 in SLC4A11 were observed significantly more frequently in family members with KTCN (P = 0.004525 and P = 0.00761, respectively), suggesting involvement of these two genes in KTCN etiology in the studied family.

Novel mutation and three other sequence variants segregating with phenotype at keratoconus 13q32 susceptibility locus.

Keratoconus (KTCN), a non-inflammatory corneal disorder characterized by stromal thinning, represents a major cause of corneal transplantations. Genetic and environmental factors have a role in the etiology of this complex disease. Previously reported linkage analysis revealed that chromosomal region 13q32 is likely to contain causative gene(s) for familial KTCN. Consequently, we have chosen eight positional candidate genes in this region: MBNL1, IPO5, FARP1, RNF113B, STK24, DOCK9, ZIC5 and ZIC2, and sequenced all of them in 51 individuals from Ecuadorian KTCN families and 105 matching controls. The mutation screening identified one mutation and three sequence variants showing 100% segregation under a dominant model with KTCN phenotype in one large Ecuadorian family. These substitutions were found in three different genes: c.2262A>C (p.Gln754His) and c.720+43A>G in DOCK9; c.2377-132A>C in IPO5 and c.1053+29G>C in STK24. PolyPhen analyses predicted that c.2262A>C (Gln754His) is possibly damaging for the protein function and structure. Our results suggest that c.2262A>C (p.Gln754His) mutation in DOCK9 may contribute to the KTCN phenotype in the large KTCN-014 family.

The genetics of keratoconus.

Keratoconus (KTCN) is non-inflammatory thinning and anterior protrusion of the cornea that results in steepening and distortion of the cornea, altered refractive error, and decreased vision. Keratoconus is a complex condition of multifactorial etiology. Both genetic and environmental factors are associated with KTCN. Evidence of genetic etiology includes familial inheritance, discordance between dizygotic twins, and association with other known genetic disorders. Several loci responsible for a familial form of KTCN have been mapped; however, no mutations in any genes have been identified for any of these loci. This article focuses on the genetic aspects. In addition, bioinformatics methods applied in KTCN gene identification process are discussed.