Author Correction: Prioritizing multiple therapeutic targets in parallel using automated DNA-encoded library screening.

This corrects the article DOI: 10.1038/ncomms16081.

Prioritizing multiple therapeutic targets in parallel using automated DNA-encoded library screening.

The identification and prioritization of chemically tractable therapeutic targets is a significant challenge in the discovery of new medicines. We have developed a novel method that rapidly screens multiple proteins in parallel using DNA-encoded library technology (ELT). Initial efforts were focused on the efficient discovery of antibacterial leads against 119 targets from Acinetobacter baumannii and Staphylococcus aureus. The success of this effort led to the hypothesis that the relative number of ELT binders alone could be used to assess the ligandability of large sets of proteins. This concept was further explored by screening 42 targets from Mycobacterium tuberculosis. Active chemical series for six targets from our initial effort as well as three chemotypes for DHFR from M. tuberculosis are reported. The findings demonstrate that parallel ELT selections can be used to assess ligandability and highlight opportunities for successful lead and tool discovery.

Genomic structure and organization of the high grade Myopia-2 locus (MYP2) critical region: mutation screening of 9 positional candidate genes.

PURPOSE: Myopia is a common complex eye disorder, with implications for blindness due to increased risk of retinal detachment, chorioretinal degeneration, premature cataracts, and glaucoma. A genomic interval of 2.2 centiMorgans (cM) was defined on chromosome band 18p11.31 using 7 families diagnosed with autosomal dominant high myopia and was designated the MYP2 locus. To characterize this region, we analyzed 9 known candidate genes localized to within the 2.2 cM interval by direct sequencing. METHODS: Using public databases, a physical map of the MYP2 interval was compiled. Gene expression studies in ocular tissues using complementary DNA library screens, microarray experiments, reverse transcription techniques, and expression data identified in external databases aided in prioritizing gene selection for screening. Coding regions, intron-exon boundaries and untranslated exons of all known genes [Clusterin-like 1 (CLUL1), elastin microfibril interfacer 2 (EMILIN2), lipin 2 (LPIN2), myomesin 1 (MYOM1), myosin regulatory light chain 3 (MRCL3), myosin regulatory light chain 2 (MRLC2), transforming growth beta-induced factor (TGIFbeta), large Drosophila homolog associated protein 1 (DLGAP1), and zinc finger protein 161 homolog (ZFP161)] were sequenced using genomic DNA samples from 9 affected and 6 unaffected MYP2 pedigree members, and from 5 external controls (4 unaffected and 1 affected). Gene sequence changes were compared to known variants from public single nucleotide polymorphism (SNP) databases. RESULTS: In total, 103 polymorphisms were found by direct sequencing; 10 were missense, 14 were silent, 26 were not translated, 49 were intronic, 1 insertion, and 3 were homozygous deletions. Twenty-seven polymorphisms were novel. Novel SNPs were submitted to the public database; observed frequencies were submitted for known SNPs. No sequence alterations segregated with the disease phenotype. CONCLUSIONS: Mutation analysis of 9 encoded positional candidate genes on MYP2 loci did not identify sequence alterations associated with the disease phenotype. Further studies of MYP2 candidate genes, including analysis of putative genes predicted in silico, are underway.

Sequence variants in the transforming growth beta-induced factor (TGIF) gene are not associated with high myopia.

PURPOSE: High myopia is a common complex-trait eye disorder, with implications for blindness due to increased risk of retinal detachment, macular degeneration, premature cataracts, and glaucoma. Mapping studies have identified at least four loci for nonsyndromic autosomal dominant high myopia at 18p11.31, 12q22-q23, 17q21-q23, and 7q36. The smallest haplotyped interval for these loci is that of the MYP2 locus on 18p11.31. Recently, the transforming growth beta-induced factor (TGIF) gene was reported to be a candidate gene for MYP2-associated high myopia in single-nucleotide polymorphism studies. The purpose of this study was to determine whether DNA sequence variants in the human TGIF gene are causally related to MYP2-associated high myopia. METHODS: The protein coding regions and intron-exon boundaries of the human TGIF gene were sequenced using genomic DNA samples from MYP2 individuals (affected, unaffected) and external control subjects. The TGIF model used was the April 20, 2003, human genome National Center for Biotechnology Information (NCBI) build 33, which has 10 exons and encodes eight transcript variants. Polymorphic sequence changes were compared to those in the previous report. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to validate TGIF gene expression in ocular tissues. RESULTS: A total of 21 polymorphisms of TGIF were found by direct sequencing: 3 were missense, 2 were silent, 10 were not translated, 4 were intronic, and 2 were homozygous deletions. The 3 missense allelic variants were localized to exon 10 at positions 236C-->T(Pro-->Leu), 244C-->T(Pro-->Ser), and 245C-->T(Pro-->Leu). Silent mutations were observed in exon 10 at positions 177A-->G, 333C-->T. Ten polymorphisms were novel. No sequence alterations were exclusively associated with the affected disease phenotype. RT-PCR results confirmed expression of TGIF in RNA samples derived from human sclera, cornea, optic nerve, and retina. CONCLUSIONS: TGIF is a known candidate gene for MYP2-associated high myopia, based on its mapped location within the MYP2 interval. Mutation analysis of the encoded TGIF gene for MYP2 autosomal dominant high myopia did not identify sequence alterations associated with the disease phenotype. Further studies of MYP2 candidate genes are needed to determine the gene that causes of this potentially blinding disorder.

Exclusion of lumican and fibromodulin as candidate genes in MYP3 linked high grade myopia.

PURPOSE: The proteoglycans lumican and fibromodulin regulate collagen fibril assembly and show expression in ocular tissues. A recent mouse knockout study implicates lumican and fibromodulin as functional candidate genes for high myopia. Lumican maps within the chromosome 12q21-q23 autosomal dominant high grade myopia-3 (MYP3) interval, and fibromodulin maps to chromosome 1q32. We screened individuals for lumican and fibromodulin sequence alterations from the original MYP3 family, and from a second high grade myopia pedigree that showed suggestive linkage to both the MYP3 interval and to chromosome 1q32. METHODS: A total of 10 affected (average spherical refractive error was -16.13 D) and 5 unaffected individuals from the 2 families were screened by direct DNA sequencing. Six primer pairs spanning intron-exon boundaries and coding regions were designed for the 3-exon 1804 base pair (bp) lumican gene. Two primer pairs for the 2-exon 2863 bp fibromodulin gene were designed. Polymerase chain reaction products were sequenced and analyzed using standard fluorescent methods. Sequences were quality scored and aligned for polymorphic analysis. RESULTS: Direct DNA sequencing of lumican amplicons yielded the expected sequence with no evidence of polymorphism or pathologic mutation. Sequencing of fibromodulin amplicons revealed 6 polymorphisms, 1 of which was novel. One polymorphism was a silent mutation, and five were in the 3' untranslated region. No polymorphism segregated with high myopia. CONCLUSIONS: Although null and double null Lum and Fmod mouse models have been developed for high myopia, our human cohort did not show affected status association with these genes. Sequencing of the human lumican and fibromodulin genes has excluded them as candidate genes for MYP3 associated high grade myopia.

Microarray analysis of gene expression in human donor sclera.

PURPOSE: To develop gene expression profiles of human sclera to allow for the identification of novel, uncharacterized genes in this tissue-type, and to identify candidate genes for scleral disorders. METHODS: Total RNA was isolated from 6 donor sources of human sclerae, and reverse transcribed into cDNA using a T7-(dT) 24 primer. The resulting cDNA was in vitro transcribed to produce biotin-labeled cRNA, fragmented, and mixed with hybridization controls before a 16 h hybridization step with oligonucleotide probes on 6 Affymetrix U95A chips. The chips were scanned twice at 570 nM and the data collected using GeneChip software. Array analyses were carried out with Microarray Suite, version 5.0 (Affymetrix), using the expression analysis algorithm to run an absolute analysis after cell intensities were computed. All arrays were scaled to the same target intensity using all probe sets. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to validate the microarray results. RESULTS: There were 3,751 genes with "present" calls assigned independently to all six human scleral samples. These genes could be clustered into 4 major categories; transcription (10%), metabolism (8.8%), cell growth and proliferation (5.4%), and extracellular matrix (2%). Many extracellular matrix proteins, such as collagens 6A3 and 10A1, thrombospondins 2 and 4, and dystroglycan have not previously been shown to be expressed in sclera. RT-PCR results confirmed scleral expression in 7 extracellular matrix genes examined. CONCLUSIONS: This study demonstrated the utility of gene microarray technology in identifying global patterns of scleral gene expression, and provides an extended list of genes expressed in human sclera. Identification of genes expressed in sclera contributes to our understanding of scleral biology, and potentially provides positional candidate genes for scleral disorders such as high myopia.

X-linked high myopia associated with cone dysfunction.

OBJECTIVE: Bornholm eye disease (BED) consists of X-linked high myopia, high cylinder, optic nerve hypoplasia, reduced electroretinographic flicker with abnormal photopic responses, and deuteranopia. The disease maps to chromosome Xq28 and is the first designated high-grade myopia locus (MYP1). We studied a second family from Minnesota with a similar X-linked phenotype, also of Danish descent. All affected males had protanopia instead of deuteranopia. METHODS: X chromosome genotyping, fine-point mapping, and haplotype analysis of the DNA from 22 Minnesota family individuals (8 affected males and 5 carrier females) and 6 members of the original family with BED were performed. Haplotype comparisons and mutation screening of the red-green cone pigment gene array were performed on DNA from both kindreds. RESULTS: Significant maximum logarithm of odds scores of 3.38 and 3.11 at theta = 0.0 were obtained with polymorphic microsatellite markers DXS8106 and DXYS154, respectively, in the Minnesota family. Haplotype analysis defined an interval of 34.4 cM at chromosome Xq27.3-Xq28. Affected males had a red-green pigment hybrid gene consistent with protanopia. We genotyped Xq27-28 polymorphic markers of the family with BED, and narrowed the critical interval to 6.8 cM. The haplotypes of the affected individuals were different from those of the Minnesota pedigree. Bornholm eye disease-affected individuals showed the presence of a green-red hybrid gene consistent with deuteranopia. CONCLUSIONS: Because of the close geographic origin of the 2 families, we expected affected individuals to have the same haplotype in the vicinity of the same mutation. Mapping studies, however, suggested independent mutations of the same gene. The red-green and green-red hybrid genes are common X-linked color vision defects, and thus are unrelated to the high myopia and other eye abnormalities in these 2 families. CLINICAL RELEVANCE: X-linked high myopia with possible cone dysfunction has been mapped to chromosome Xq28 with intervals of 34.4 and 6.8 centimorgan for 2 families of Danish origin.