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# The Precise Structures and Stereochemistry of Trihydroxy-linoleates Esterified in Human and Porcine Epidermis and Their Significance in Skin Barrier Function : IMPLICATION OF AN EPOXIDE HYDROLASE IN THE TRANSFORMATIONS OF LINOLEATE *

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### Abstract

Creation of an intact skin water barrier, a prerequisite for life on dry land, requires the lipoxygenase-catalyzed oxidation of the essential fatty acid linoleate, which is esterified to the ω-hydroxyl of an epidermis-specific ceramide. Oxidation of the linoleate moiety by lipoxygenases is proposed to facilitate enzymatic cleavage of the ester bond, releasing free ω-hydroxyceramide for covalent binding to protein, thus forming the corneocyte lipid envelope, a key component of the epidermal barrier. Herein, we report the transformations of esterified linoleate proceed beyond the initial steps of oxidation and epoxyalcohol synthesis catalyzed by the consecutive actions of 12 R-LOX and epidermal LOX3. The major end product in human and porcine epidermis is a trihydroxy derivative, formed with a specificity that implicates participation of an epoxide hydrolase in converting epoxyalcohol to triol. Of the 16 possible triols arising from hydrolysis of 9,10-epoxy-13-hydroxy-octadecenoates, using LC-MS and chiral analyses, we identify and quantify specifically 9 R,10 S,13 R-trihydroxy-11 E-octadecenoate as the single major triol esterified in porcine epidermis and the same isomer with lesser amounts of its 10 R diastereomer in human epidermis. The 9 R,10 S,13 R-triol is formed by S N2 hydrolysis of the 9 R,10 R-epoxy-13 R-hydroxy-octadecenoate product of the LOX enzymes, a reaction specificity characteristic of epoxide hydrolase. The high polarity of triol over the primary linoleate products enhances the concept that the oxidations disrupt corneocyte membrane lipids, promoting release of free ω-hydroxyceramide for covalent binding to protein and sealing of the waterproof barrier.

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### Abnormal maternal behaviour and growth retardation associated with loss of the imprinted gene Mest.

(1998)
Mest (also known as Peg1), an imprinted gene expressed only from the paternal allele during development, was disrupted by gene targeting in embryonic stem (ES) cells. The targeted mutation is imprinted and reversibly silenced by passage through the female germ line. Paternal transmission activates the targeted allele and causes embryonic growth retardation associated with reduced postnatal survival rates in mutant progeny. More significantly, Mest-deficient females show abnormal maternal behaviour and impaired placentophagia, a distinctive mammalian behaviour. Our results provide evidence for the involvement of an imprinted gene in the control of adult behaviour.
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### Targeted disruption of soluble epoxide hydrolase reveals a role in blood pressure regulation.

(2000)
Renal microsomal cytochrome P-450 monooxygenase-dependent metabolism of arachidonic acid generates a series of regioisomeric epoxyeicosatrienoic acids that can be further metabolized by soluble epoxide hydrolase to the corresponding dihydroxyeicosatrienoic acids. Evidence exists that these metabolites affect renal function and, in particular, blood pressure regulation. To examine this possibility, blood pressure and renal arachidonic acid metabolism were examined in mice with a targeted disruption of the soluble epoxide hydrolase gene. Systolic blood pressure of male soluble epoxide hydrolase-null mice was lower compared with wild-type mice in both the absence and presence of dietary salt loading. Both female soluble epoxide hydrolase-null and wild-type female mice also had significantly lower systolic blood pressure than male wild-type mice. Renal formation of epoxyeicosatrienoic and dihydroxyeicosatrienoic acids was markedly lower for soluble epoxide hydrolase-null versus wild-type mice of both sexes. Although disruption of soluble epoxide hydrolase in female mice had minimal effects on blood pressure, deletion of this gene feminized male mice by lowering systolic blood pressure and altering arachidonic acid metabolism. These data provide the first direct evidence for a role for soluble epoxide hydrolase in blood pressure regulation and identify this enzyme as a novel and attractive target for therapeutic intervention in hypertension.
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### Prediction of Human Disease Genes by Human-Mouse Conserved Coexpression Analysis

(2008)
Introduction In the last two decades, positional cloning has been remarkably successful in the identification of genes involved in human disorders. More recently, our ability to map genetic disease loci has strikingly increased due to the availability of the entire genome sequence. Nevertheless, once a disease locus has been mapped, the identification of the mutation responsible for the phenotype still represents a very demanding task, because the mapped region may typically contain hundreds of candidates [1]. Accordingly, many phenotypes mapped on the genome by linkage analysis are not yet associated to any validated disease gene (850 OMIM entries for phenotypes with unknown molecular basis had at least one associated disease locus on July 2nd, 2007). Therefore, the definition of strategies that can pinpoint the most likely targets to be sequenced in patients is of critical importance [1]. Many different strategies have been proposed to prioritize genes located in critical map intervals. Some of the methods so far developed rely on the observation that disease genes tend to share common global properties, which can be deduced directly by absolute and comparative sequence analysis [2]. However, most of the available prioritization strategies are based on the widely accepted idea that genes and proteins of living organisms deploy their functions as part of sophisticated functional modules, based on a complex series of physical, metabolic and regulatory interactions [3],[4]. Although this principle has been extensively used even in the pre-genome era to identify the critical players of many different biological phenomena, the present availability of genome-scale information on gene function, protein-protein interactions and gene expression in different experimental models allows unprecedented opportunities for approaching the prioritization problem with greater efficiency. In theory, the use of functional gene annotations would represent the most straightforward approach for candidate prioritization. However, although this strategy may be very useful in selected cases [5],[6], at the present stage it has clear limitations, either because it overlooks non-annotated genes [6],[7] or because it is not evident how the annotated functions of the candidates relate to the disease phenotype. Therefore, computational methods less biased toward already consolidated knowledge, may have strong advantages [1]. In particular, protein-protein interaction maps and gene coexpression data from microarray experiments represent extremely rich sources of potentially relevant information. Recently, the direct integration of a very heterogeneous human interactome with a text mining-based map of phenotype similarity has allowed the prediction of high confidence candidates within large disease-associated loci [8]. Although this approach is highly efficient, it is clearly not exhaustive because very close functional relationships between genes and proteins are possible in the absence of direct molecular binding. In addition, the protein-protein interaction space is currently under-sampled and many genuine biological interactions have not yet been identified in experiments. Conversely, high-throughput experiments are known to result in a large fraction of false positives. The consistently low overlap of protein-protein interactions between large-scale experiments, even when the same proteins are considered, is testament to these problems [9]. Finally, many of the known protein-protein interactions have been ascertained through low-throughput experiments and are thus strongly biased towards better-studied proteins [10]. Since genes involved in the same functions tend to show very similar expression profiles, coexpression analysis could be a very powerful approach for inferring functional relationships, which may correlate with similar disease phenotypes. Accordingly, global analyses have shown that genes highly coexpressed across microarray experiments display very similar functional annotation [11]. However, with notable exceptions [12], so far the coexpression criterion has not been employed very successfully for the prediction of genetic disease candidates and has been used to this purpose only in combination with other independent evidence [5],[13]. The noisy nature of high-throughput gene expression datasets may represent one of the possible explanations for this shortcoming. Moreover, even when the coexpression of two genes is reproducibly observed under a high number of experimental conditions, this does not necessarily imply that the genes are functionally related. For instance, extensive meta analysis of microarray data across different species has revealed that neighboring genes are more likely to be coexpressed than genes encoded in distant genomic regions, even if they are not functionally related in any obvious manner [14],[15]. Phylogenetic conservation has been previously proposed as a very strong criterion to identify functionally relevant coexpression links among genes [16],[17]. Indeed, significant coexpression of two or more orthologous genes is very likely due to selective advantage, strongly suggesting a functional relation. Therefore, conserved coexpression could be a much stronger criterion than single species coexpression to relate genes involved in similar disease phenotypes. In this report we show that conserved coexpression and phenome analysis can be effectively integrated to produce accurate predictions of human disease genes. Using this approach we were able to select a small number of strong candidates for 81 human diseases, corresponding to a wide spectrum of different phenotypes. Materials and Methods Figure 1 schematically illustrates our approach. 10.1371/journal.pcbi.1000043.g001 Figure 1 Identification of candidate disease genes by means of conserved co-expression clusters. A locus with positional candidates (green bar) associated with a disease of unknown molecular basis (yellow) is screened for one or more candidate genes (green sphere) that appear in a conserved coexpression cluster (purple spheres) together with at least two other genes known to be involved in similar phenotypes (yellow spheres), as defined by the MimMiner similarity score. Generation of Conserved Coexpression Networks We have generated two human-mouse conserved coexpression networks (CCN), based on cDNA and oligonucleotide microarray platforms, respectively. In the first case, the starting data were log-transformed values of human and mouse ratiometric experiments, downloaded from the Stanford Microarray Database (SMD) [18] (4129 experiments for 102296 EST probes for human and 467 experiments for 80595 EST probes for mouse). The resulting network will be referred to as ‘Stanford’ in the following (Text S1). In the second case (‘Affy’, Text S2), the network was based on previously described series of normal tissue Affymetrix microarray experiments from human [19] (353 experiments corresponding to 65 different tissues for 46241 probe-sets associated to a known gene) and mouse [20] (122 experiments corresponding to 61 tissues for 19692 probe-sets). In the human case, the Affymetrix experiments corresponding to the same tissue were averaged to compensate for the different number of replicates available for the various tissues. In both cases, we used the same procedure to generate a final CCN. In particular, we first generated single species gene coexpression networks (SCN) and then integrated them on the basis of human-mouse orthology, as detailed below. SCNs were generated by first calculating the Pearson correlation coefficients of every row in the expression matrix (cDNA probe or Affymetrix probe-set) with all other rows. A directed edge was established from row r1 to row r2 if r2 fell within the top 1% rows in terms of correlation with r1. The threshold was first chosen on the basis of a previous study, showing that such 1% interval is most significantly enriched in terms of functionally relevant coexpression [16]. Moreover, we confirmed that using a more stringent 0.5% threshold results in strongly reduced sensitivity (data not shown). These directed networks where then converted into undirected SCNs by mapping the rows to the corresponding Entrez Gene identifiers [21]: an edge is established between two Entrez gene IDs G1 and G2 if there is at least one edge from a row assigned to G1 to a row assigned to G2 and vice versa. The correspondence between probe-sets and Entrez gene IDs for the SMD data was established using Unigene (build 190 and 152 for human and mouse, respectively). For Affymetrix data we used the annotation files provided by Affymetrix for each platform. Finally, CCNs were built from SCNs by mapping every Entrez Gene identifier to the corresponding Homologene cluster (build 55) and retaining only the cases in which a one-to-one correspondence could be established between the human and mouse Entrez gene IDs appearing in the SCNs. Phenotype Correlation Genetic disease phenotypes described in OMIM where correlated on the basis of MimMiner [22]. MimMiner assigns a similarity score to all pairs of OMIM phenotype records, based on the text mining analysis of their phenotype descriptions [22]. Two phenotypes were defined to be similar if their score was at least 0.4, because biologically meaningful relationships were mostly detected in phenotype pairs with a similarity score equal or greater than this value [22]. About 1% of all possible pairs of phenotypes included in MimMiner pass this threshold. Global Properties of the CCNs The analysis of the CCNs was based on the construction of coexpression clusters, defined as a given gene (the center of the cluster) plus its nearest neighbors in the conserved coexpression network, thus obtaining one cluster for each gene. The prevalence of genes joining functionally related genes in the CCNs was tested by analyzing the prevalence of Gene Ontology terms within coexpression clusters, compared to the same prevalence in randomized coexpression clusters. We counted the number of coexpression clusters for which at least one Gene Ontology term was significantly overrepresented (P-value less than 10−4 with exact Fisher test), and compared this number with the same number averaged over 100 randomized CCNs. This was done separately for the Affy and Stanford networks, and the results are shown in Figure 2A. 10.1371/journal.pcbi.1000043.g002 Figure 2 Comparison of the Affy and Stanford networks with functional, physical interaction, and disease-related information. (A) Prevalence of functionally related coexpression clusters (see Materials and Methods). (B) Number of edges of the CCN joining proteins previously shown to physically interact by different techniques, as deduced by the HPRD database. (C) Number of edges of the indicated networks connecting genes involved in Mendelian phenotypes sharing a MimMiner score of 0.4 or higher. In each case, the results for the actual CCNs are compared to the results averaged on 100 randomized CCNs, with error bars representing the standard deviation of the latter. The overlap between CCNs and protein interaction networks was evaluated by downloading the list of known interactions between human proteins from HPRD [23],[24]. To take into account the different experimental methods on which the HPRD interactions are based and their varying degree of reliability, we analyzed separately in-vivo, in vitro and yeast double hybrid interactions. In each case, separately for the Affy and Stanford networks, we compared the overlap between the CCNs and the protein interaction network to the same overlap averaged over 100 randomized CCNs. The results are shown in Figure 2B. Finally to verify whether the CCNs were enriched in edges joining genes causing similar phenotypes, we constructed a network of human genes in which an edge was placed between every pair of genes known to be involved in the same disease or in diseases with MimMiner similarity score at least equal to 0.4. The mapping between OMIM phenotypes and genes known to cause them was obtained from Ensembl, version 45 [25]. We then evaluated the overlap between this network and the CCNs, again compared to the same overlap averaged over 100 randomized CCNs (Figure 2C). Identification of Candidate Genes in Disease Loci The lists of genes contained in OMIM loci of unknown molecular basis were obtained from Ensembl, version 45 [25]. To identify likely candidates for a given disease-associated genomic locus, we first extracted from the networks disease-relevant conserved coexpression clusters. A cluster was considered relevant to a given disease d if it contained at least two genes experimentally known to cause phenotypes similar to d. These clusters will be called ‘disease clusters’ in the following. The genes of the disease clusters, which are also contained in the map interval of the locus associated to d, were retained for scoring as described below. Scoring and Estimation of the False Discovery Rate The size of the coexpression clusters and of the loci are very heterogeneous: coexpression clusters contain between 2 and 186 genes while loci associated to OMIM diseases with unknown molecular basis vary between 3 and 2153 genes. Therefore the genes selected in the previous step were assigned a probabilistic score based on the null hypothesis in which the coexpression clusters are random sets of genes. The score is essentially the probability that the two events leading to the identification of a candidate gene for a disease occur by chance. The two events are (1) the presence in a coexpression cluster of at least 2 genes known to be involved in phenotypes similar to the disease in question and (2) the presence in the same coexpression cluster of a gene located within the locus relevant to the disease. First, we computed a P-value p1 for associating a cluster to the given disease by chance. This P-value is given by the cumulative hypergeometric distribution considering: the number RDC of genes linked to similar phenotypes that have been found in the cluster (at least 2 to associate the cluster with the disease); the number Rall of genes in the network that are linked to similar phenotypes; the number GDC of genes in the cluster; and the total number of genes Gall in the network: Second, we computed the P-value p2 of the overlap between the disease cluster and the genetic locus associated to the disease. This P-value is given by the cumulative hypergeometric distribution considering: the number LDC of genes in the locus that are also present in the given disease cluster; the number Lall of genes in the locus that are present in the network; and GDC and Gall are defined as above: In the null hypothesis, associating a cluster with a disease and finding a gene in the cluster that belongs to the appropriate orphan locus are independent events. Thus, the total score for a predicted candidate is given by the product p1 • p2. When a candidate is found in more than one disease cluster, we consider only the lowest (best) score. The cutoff on such scores was determined by estimating the false discovery rate (FDR) for each possible cutoff using 100 randomized CCNs per dataset: The false discovery rate is defined as the ratio between the average number of predictions made using randomized CCNs and the number of predictions made using the real CCN, with the same cutoff. The cutoffs thus obtained for a 10% FDR were 4.49·10−6 for the Affy and 2.67·10−6 for the Stanford network, respectively. Precision and Leave-One-Out Procedure To estimate the precision of our procedure we used a leave-one-out strategy: For every gene experimentally associated to an OMIM phenotype we constructed artificial loci centered on the disease gene, and removed all associations between this particular phenotype and all the genes known to cause it, so as to simulate a phenotype with unknown molecular basis. The association between phenotypes similar to the one under examination and the corresponding genes was instead retained. In order to take into account the variability of locus sizes we constructed artificial loci of various sizes, by taking the disease gene plus the N closest genes on each side of the chromosome (according to their start position on the chromosome). The artificial loci thus contained up to 2N+1 genes, but could contain fewer genes when the disease gene was close to one of the chromosome ends. In the following discussion, such artificial loci will be denoted as N20, N50, … N500 for locus sizes up to 41, 101 … 1001 genes, respectively. This range of locus sizes was chosen based on the observed size distribution of orphan loci: OMIM loci for diseases with unknown molecular basis contain an average number of about 273 genes (median: 180 genes). We considered the disease gene as correctly identified if it was selected as a candidate by our method with the same score threshold that we used for the orphan loci. The precision is defined as the ratio between the number of cases with correctly identified disease genes and the number of cases with at least one selected candidate, that is, the fraction of cases with selected candidates in which the disease gene was among the candidate list. Results Generation and Global Properties of Two Different Human-Mouse Conserved Coexpression Networks Conserved coexpression has been previously reported to be an efficient criterion to identify functionally related genes [16],[17]. Therefore, to discover new relationships between human genes with a high potential relevance for disease phenotypes, we produced the two human-mouse gene coexpression networks described above, covering different platforms and experimental conditions. In particular, the Stanford network (supporting file S1) was generated from data based on cDNA platforms, corresponding mostly to experiments performed on tumor cell lines. In contrast, the Affy network (Text S2) was derived from normal tissue data, generated on Affymetrix platforms in two independent studies [19],[20]. The Stanford network has 8512 nodes (genes) and 56397 edges, with an average connectivity of 13.2 edges per node. The Affy network is composed of 12766 nodes and 155403 edges, with an average connectivity of 24.3 edges per node. Both networks contain a large connected component of 2305 and 4122 genes, respectively, with some other small connected components containing only a few nodes. As expected from previous studies on gene coexpression networks [26], the two networks are topologically similar to other biological networks, characterized by the existence of a few highly connected nodes (hubs), but they show a connectivity distribution more similar to an exponential law than to a scale-free one (data not shown). More importantly, if compared with 100 random permutations, both networks show a strong prevalence of edges between genes that are annotated to the same Gene Ontology (GO) keyword (Figure 2A). This confirms that human-mouse conserved coexpression is a valuable criterion to identify functionally related genes. Accordingly, both networks show a highly significant overlap with protein-protein interactions reported in the Human Protein Reference Database (HPRD) [23],[24] (Figure 2B). Since many genes, such as those involved in basic cellular functions, should be coexpressed regardless of the particular experimental situation, we would expect the two networks to have many common links. Indeed, they share 2305 edges, between the 7332 common nodes, which represents a striking overlap (the randomized Affy networks had on average 88.4 edges in common with the Stanford network, with a standard deviation of 8.7). On the other hand, the large number of specific links that characterize the two networks indicates that they provide highly complementary information. Finally, to evaluate the capability of conserved coexpression to link genes involved in similar disease phenotypes, we measured in both networks the prevalence of links between genes associated to phenotypes with similar descriptions [22]. Interestingly, both networks showed a strong enrichment, if compared with the average number obtained from the randomized networks (Figure 2C). We concluded that the two networks represent complementary resources that could efficiently predict disease-relevant relationships among human genes. Integration of Coexpression and Phenotypic Information to Identify Likely Candidate Genes in Disease Loci The high prevalence of links between genes involved in similar disease phenotypes, observed in both networks, suggests that they could provide valuable information to identify likely candidates in mapped disease loci. Therefore, we devised an algorithm that integrates our CCNs with phenotype and mapping information to predict candidate disease genes in large genomic regions (Figure 1). As detailed in Materials and Methods, the procedure is based on the extraction from the network of the disease clusters, which we consider to be associated to a given disease since they contain at least two genes involved in similar phenotypes. The genes that are present in both the OMIM phenotype loci and the corresponding disease clusters are considered as candidates and assigned a score based on the size of the locus and of the disease cluster. Randomized runs allowed us to select a score threshold corresponding to a 10% FDR. To evaluate how our procedure could perform on the loci characterized by unknown molecular basis, we applied a leave-one-out strategy to all the Ensembl genes associated to at least one OMIM disease ID, by constructing artificial loci of variable size around each gene. We then measured the fraction of artificial loci for which we obtained at least one candidate (Figure 3A), the average number of candidates found for these loci (Figure 3B) and the precision (Figure 3C), defined as explained in Materials and Methods. Of the 1762 disease genes contained in OMIM, we could analyze 1426, whose associated phenotypes are present in the MimMiner similarity matrix. 10.1371/journal.pcbi.1000043.g003 Figure 3 Performance of the identification of candidate disease genes as determined by a leave-one-out strategy. Artificial loci of different sizes were constructed around known disease genes as explained in the text. (A) Fraction of the artificial loci for which it was possible to identify at least one candidate gene, as a function of the locus size. (B) Average number of candidates in the loci for which at least one candidate was identified, as a function of the locus size. (C) Precision as a function of the locus size. Precision is determined as the ratio between the number of loci whose candidate list contained the starting disease gene and the number of loci with candidates. Filled triangles indicate the results obtained with the Affy network, while empty boxes refer to the Stanford network. The precision obtained obviously decreases when the size of the artificial loci is increased. However, it is interesting to notice that, while for the smallest artificial loci the precision was excellent (about 68% for both networks), even with the largest artificial loci it was still remarkable (37.5% for the Affy and 29.1% for the Stanford networks, respectively). For N90 (artificial loci with a maximum of 181 genes, very close to the median size of 180 genes for OMIM phenotype loci with unknown molecular basis), for example, the leave-one-out validation yielded at least one candidate for 47.8% (Affy) and 12.7% (Stanford) of the disease loci (Figure 3A). In these cases, an average of 3.67 (Affy) and 2.17 (Stanford) candidates were returned (Figure 3B), that contained the true disease gene in 49.3% (Affy) and 43.6% (Stanford) of the cases (Figure 3C). That is, for both networks, when candidates were returned, the very short candidate lists contained the disease-causing gene with a probability of over 40%. We next determined how our method performs compared to other existing approaches. This comparison was based on the enrichment in correctly identified disease genes (by leave-one-out) with respect to randomized networks. In our case the fold enrichment is defined as the ratio between number of disease genes correctly recalled in the leave-one out procedure and the same number averaged on 100 randomized CCNs. For this evaluation we used the N90 artificial loci (see Materials and Methods), which are closest in size to the actual orphan loci (median size 180). Fold enrichment values were computed for several published methods by Lage et al [8] (see their Supplementary material). It must be noticed that the exact definition of the fold enrichment depends on the method under consideration, thus comparison must be taken with caution. However, Table 1 shows that our methods compares favorably with many previously published ones: when considering that, as discussed above, the use of gene expression data allows a less biased analysis compared to most other methods, we conclude that our approach provides a significant and original contribution to the identification of disease genes. 10.1371/journal.pcbi.1000043.t001 Table 1 Fold enrichment of different methods for candidate gene prioritization. Method Data Source Fold Enrichment Lage et al. [8] PPI 23.1a Perez-Iratxeta et al. [35] FA 19.4a Conserved coexpression (Affy network) GE 14.4 Freudenberg and Propping [36] FA 13.3a Conserved coexpression (Stanford network) GE 10.0 Oti et al. [10] PPI 10.0a Adie et al. [37] SBF 5.6a Turner et al. [6] FA 5.2a Franke et al. [5] FA, GE, PPI 3.6a PPI, protein-protein interaction; FA, functional annotation; GE, gene expression; SBF, sequence-based features (e.g., sequence length). a Values taken from the supplementary material of Lage et al. [8]. Prediction of Candidates for Orphan Disease Loci The above results indicate that conserved coexpression can be efficiently combined with phenotype correlation data to provide high confidence candidates within genetic disease loci. Therefore, we applied our procedure to 850 OMIM phenotype entries with at least one mapped disease locus but unknown molecular basis. In Table 2 we provide the list of all the 321 candidates (gene-locus pairs) obtained with 10% FDR. We obtained predictions for 81 loci, 67 of which where only from the Affy network, 5 only from the Stanford and 9 from both. Interestingly, in 4 of the latter cases, the list of candidates from the two networks contained at least one common gene (Table 2). 10.1371/journal.pcbi.1000043.t002 Table 2 List of the candidate genes found for 81 OMIM loci with unknown molecular basis. OMIM ID Disease and Locus Locus Size HUGO Ensembl ID Status Net. Disease Cluster Size P-Value 119540 Cleft palate, isolated; 2q32 75 COL3A1 ENSG00000168542 1*(i) S 22 1.16E-06 COL5A2 ENSG00000204262 1*(i) S 22 1.16E-06 121210 Febrile convulsions, familial, 1; 8q13-q21 188 C8orf46 ENSG00000169085 1 A 143 4.12E-10 EFCBP1 ENSG00000123119 1 A 143 4.12E-10 RALYL ENSG00000184672 1 A 143 4.12E-10 STMN2 ENSG00000104435 1 A 143 4.12E-10 GDAP1 ENSG00000104381 1 A 139 1.79E-09 C8orf34 ENSG00000165084 1 A 110 2.95E-08 STAU2 ENSG00000040341 1 A 173 3.08E-08 130080 Ehlers-Danlos syndrome, type VIII; 12p13 277 A2M ENSG00000175899 1 S 50 4.79E-11 VWF ENSG00000110799 1 A 99 9.97E-10 C1S ENSG00000182326 1 S 87 1.25E-09 MFAP5 ENSG00000197614 1 A 111 1.90E-09 EMP1 ENSG00000134531 1 A 72 2.37E-09 CD163 ENSG00000177575 1 A 44 3.34E-09 TNFRSF1A ENSG00000067182 1 A 61 4.34E-09 TSPAN9 ENSG00000011105 1 S 112 5.28E-09 CSDA ENSG00000060138 1 A 55 9.95E-09 LTBR ENSG00000111321 1 A 73 1.70E-06 CD9 ENSG00000010278 1 S;A 49 2.34E-06 142700 Acetabular dysplasia; 13q22 36 KLF5 ENSG00000102554 1 A 54 1.18E-06 145410 Hypertelorism with esophageal abnormality and hypospadias; 22q11.2 271 KLHL22 ENSG00000185214 1 A 15 1.84E-07 MAPK1 ENSG00000100030 1 A 15 1.84E-07 MICAL3 ENSG00000099972 1 A 15 1.84E-07 154275 Malignant hyperthermia, susceptibility to, 2; 17q11.2-q24 881 CACNB1 ENSG00000067191 3$(ii) A 37 1.06E-06 CACNG1 ENSG00000108878 3$(ii) A 37 1.06E-06 SCN4A ENSG00000007314 1 A 37 1.06E-06 156232 Mesomelic dysplasia, Kantaputra type; 2q24-q32 296 HOXD10 ENSG00000128710 2 A 13 1.80E-07 HOXD8 ENSG00000175879 1 A 13 1.80E-07 GRB14 ENSG00000115290 1 A 63 3.14E-07 HOXD11 ENSG00000128713 1 A 63 3.14E-07 HOXD3 ENSG00000128652 1 A 63 3.14E-07 156600 Microcoria, congenital; 13q31-q32 99 DCT ENSG00000080166 1 A 18 4.06E-08 SLITRK6 ENSG00000184564 1 A 18 4.06E-08 162820 Neutrophil chemotactic response; 7q22-qter 712 EPHB6 ENSG00000106123 1 A 46 6.04E-07 EPHA1 ENSG00000146904 1 A 54 1.08E-06 163000 Nevi flammei, familial multiple; 5q13-q22 327 AGGF1 ENSG00000164252 2*(iii) A 77 3.72E-08 RASA1 ENSG00000145715 2#(iii) A 77 3.72E-08 164210 Hemifacial microsomia; 14q32 414 BTBD7 ENSG00000011114 1 S 109 2.15E-07 DICER1 ENSG00000100697 1 S 109 2.15E-07 EIF5 ENSG00000100664 1 S 109 2.15E-07 JAG2 ENSG00000184916 1 S 109 2.15E-07 PAPOLA ENSG00000090060 1 S 109 2.15E-07 PPP2R5C ENSG00000078304 1 S 109 2.15E-07 VRK1 ENSG00000100749 1 S 109 2.15E-07 177720 Pseudohyperkalemia, familial, 1, due to red cell leak; 16q23-q24 170 CA5A ENSG00000174990 1 A 11 1.56E-07 180020 Retinal cone dystrophy 1; 6q25-q26 133 PLEKHG1 ENSG00000120278 1 A 18 1.10E-09 SYTL3 ENSG00000164674 1 A 18 1.10E-09 181430 Scapuloperoneal myopathy; 12q15-q23.1 215 MYF6 ENSG00000111046 3 A 104 8.24E-09 PHLDA1 ENSG00000139289 1 A 134 3.50E-08 LUM ENSG00000139329 1 A 90 3.66E-07 183600 Split-hand/foot malformation 1; 2q31 126 HOXD10 ENSG00000128710 1 A 27 8.16E-10 HOXD11 ENSG00000128713 1 A 27 8.16E-10 HOXD3 ENSG00000128652 1 A 27 8.16E-10 HOXD8 ENSG00000175879 1 A 27 8.16E-10 HOXD9 ENSG00000128709 1 A 27 8.16E-10 HOXD13 ENSG00000128714 2 A 28 2.67E-07 185000 Stomatocytosis I; 9q34.1 135 LCN2 ENSG00000148346 1 A 35 3.34E-07 203650 Alopecia-mental retardation syndrome 1; 3q26.3-q27.3 150 ABCC5 ENSG00000114770 1 S 23 6.51E-06 LIPH ENSG00000163898 2 S 23 6.51E-06 213200 Spinocerebellar ataxia, autosomal recessive 2; 9q34-qter 290 CACNA1B ENSG00000148408 1 A 31 2.00E-06 GRIN1 ENSG00000176884 1 A 50 3.38E-06 213600 Basal ganglia calcification, idiopathic, 1; 14q 1215 GPHN ENSG00000171723 1 A 61 2.11E-06 214900 Cholestasis-lymphedema syndrome; 15q 1074 CYP1A2 ENSG00000140505 1 A 73 2.66E-06 LIPC ENSG00000166035 1 A 73 2.66E-06 218400 Craniometaphyseal dysplasia, autosomal recessive; 6q21-q22 215 GJA1 ENSG00000152661 1 S 65 8.67E-07 225000 Rosselli-gulienetti syndrome; 11q23-q24 314 BACE1 ENSG00000186318 1 S 65 4.88E-07 CRYAB ENSG00000109846 1 S 65 4.88E-07 TAGLN ENSG00000149591 1 S 65 4.88E-07 255160 Myopathy, hyaline body, autosomal recessive; 3p22.2-p21.32 84 CMYA1 ENSG00000168334 1 A 119 4.01E-18 HHATL ENSG00000010282 1 A 119 4.01E-18 SCN5A ENSG00000183873 1 A 83 1.74E-09 259450 Bruck syndrome 1; 17p12 37 MYOCD ENSG00000141052 1 A 67 3.43E-06 PMP22 ENSG00000109099 1 A 68 3.64E-06 268700 Saccharopinuria; 7q31.3 39 AASS ENSG00000008311 2#(iv) A 141 9.48E-15 SLC13A1 ENSG00000081800 1*(iv) A 88 1.80E-09 TSPAN12 ENSG00000106025 1*(iv) A 56 3.35E-07 300046 Mental retardation, X-linked 23; Xq23-q24 116 ACSL4 ENSG00000068366 2 A 36 2.38E-06 WDR44 ENSG00000131725 1 A 36 2.38E-06 300148 Mental retardation, epileptic seizures, hypogonadism and hypogenitalism, microcephaly, and obesity; Xp22.13-p21.1 117 PDHA1 ENSG00000131828 1 A 40 1.03E-09 300195 Alport syndrome, mental retardation, midface hypoplasia, and elliptocytosis; Xq22.3 40 COL4A5 ENSG00000188153 2#(v) A 20 3.85E-06 300324 Mental retardation, X-linked 53; Xq22.2-q26 327 ACSL4 ENSG00000068366 2 A 19 1.41E-06 ARHGEF6 ENSG00000129675 2 A 19 1.41E-06 300489 Spinal muscular atrophy, distal, x-linked recessive; Xq13.1-q21 182 ITGB1BP2 ENSG00000147166 1 A 60 2.41E-06 SH3BGRL ENSG00000131171 1 A 26 3.16E-06 309610 Prieto X-linked mental retardation syndrome; Xp11-q21 481 ATRX ENSG00000085224 2 S 24 6.25E-08 HUWE1 ENSG00000086758 1 S 24 6.25E-08 OGT ENSG00000147162 1 S 24 6.25E-08 USP9X ENSG00000124486 1 S 24 6.25E-08 MAGED1 ENSG00000179222 1 A 21 1.13E-07 MAGED2 ENSG00000102316 1 A 21 1.13E-07 DIAPH2 ENSG00000147202 1 S 17 2.28E-06 CRSP2 ENSG00000180182 1 S 107 6.20E-06 RNF12 ENSG00000131263 1 S 107 6.20E-06 UTX ENSG00000147050 1 S 107 6.20E-06 310440 Myopathy, X-linked, with excessive autophagy; Xq28 151 SLC6A8 ENSG00000130821 1 A 76 8.25E-11 SRPK3 ENSG00000184343 1 A 62 1.12E-09 IL9R ENSG00000124334 1 A 79 6.05E-09 DNASE1L1 ENSG00000013563 1 A 29 6.40E-08 BGN ENSG00000182492 1 S 112 7.32E-08 311510 Parkinsonism, early-onset, with mental retardation; Xq28 151 BCAP31 ENSG00000185825 1 S 26 3.48E-07 IRAK1 ENSG00000184216 1 S 26 3.48E-07 SSR4 ENSG00000180879 1 S 26 3.48E-07 RAB39B ENSG00000155961 1 A 66 2.02E-06 314580 Wieacker syndrome; Xq13-q21 182 ITGB1BP2 ENSG00000147166 1 A 87 5.02E-08 PHKA1 ENSG00000067177 1 A 87 5.02E-08 MAGEE1 ENSG00000198934 1 A 144 1.89E-07 APOOL ENSG00000155008 1 A 54 2.97E-06 600131 Epilepsy, childhood absence, 1; 8q24 247 NIBP ENSG00000167632 1 A 139 1.38E-07 LYNX1 ENSG00000180155 1 A 173 4.55E-07 BAI1 ENSG00000181790 1 A 88 8.59E-07 600175 Spinal muscular atrophy, distal, congenital nonprogressive; 12q23-q24 445 HSPB8 ENSG00000152137 2 A 108 1.88E-07 MYBPC1 ENSG00000196091 1 A 108 1.88E-07 MYL2 ENSG00000111245 1 A 99 2.33E-06 600593 Craniosynostosis, Adelaide type; 4p16 160 MSX1 ENSG00000163132 1 A 28 6.29E-07 600624 Cone-rod dystrophy 1; 18q21.1-q21.3 148 SERPINB2 ENSG00000197632 1 A 63 3.75E-06 SERPINB4 ENSG00000057149 1 A 63 3.75E-06 SERPINB5 ENSG00000206075 1 A 63 3.75E-06 SERPINB7 ENSG00000166396 1 A 63 3.75E-06 600792 Deafness, neurosensory, autosomal recessive 5; 14q12 43 COCH ENSG00000100473 2 A 31 1.36E-06 FOXG1B ENSG00000176165 1 A 31 1.36E-06 600964 Refsum disease with increased pipecolic acidemia; 10pter-p11.2 312 PHYH ENSG00000107537 2 A 46 3.04E-06 600977 Cone-rod dystrophy 5; 17p13-p12 319 RCVRN ENSG00000109047 1*( vi) A 12 1.04E-06 PITPNM3 ENSG00000091622 1*( vi) A 10 3.62E-06 601202 Cataract, anterior polar, 2; 17p13 282 RCVRN ENSG00000109047 1 A 12 3.53E-07 601251 Retinal cone dystrophy 2; 17p 494 RCVRN ENSG00000109047 1*(vii) A 12 4.20E-07 PITPNM3 ENSG00000091622 1*(vii) A 10 2.65E-06 601362 Digeorge syndrome/velocardiofacial syndrome spectrum of malformation 2; 10p14-p13 71 GATA3 ENSG00000107485 2 A 29 1.75E-07 601676 Acute insulin response; 1p31 156 ANGPTL3 ENSG00000132855 1 A 46 4.21E-08 CTH ENSG00000116761 1 A 46 4.21E-08 CRYZ ENSG00000116791 1 A 37 1.84E-07 601764 Convulsions, benign familial infantile, 1; 19q 1002 ATP1A3 ENSG00000105409 1 A 100 2.71E-08 FXYD7 ENSG00000142290 1 A 100 2.71E-08 SCN1B ENSG00000105711 2$(viii) A 100 2.71E-08 APLP1 ENSG00000105290 1 A 82 6.25E-08 CA11 ENSG00000063180 1 A 82 6.25E-08 TTYH1 ENSG00000167614 1 A 82 6.25E-08 LRRC4B ENSG00000131409 1 A 125 4.11E-07 MAG ENSG00000105695 1 A 125 4.11E-07 TMEM145 ENSG00000167619 1 A 125 4.11E-07 ZNF536 ENSG00000198597 1 A 125 4.11E-07 ZNF8 ENSG00000083842 1 A 173 7.17E-07 CPT1C ENSG00000169169 1 A 115 8.94E-07 CADM4 ENSG00000105767 1 A 73 9.65E-07 LIN7B ENSG00000104863 1 A 139 1.05E-06 PLD3 ENSG00000105223 1 A 139 1.05E-06 SPTBN4 ENSG00000160460 1 A 139 1.05E-06 GRIK5 ENSG00000105737 1 A 88 3.73E-06 601846 Vacuolar neuromyopathy; 19p13.3 238 ITGB1BP3 ENSG00000077009 1 A 119 2.44E-14 NRTN ENSG00000171119 1 A 108 2.89E-13 GNG7 ENSG00000176533 1 A 144 2.41E-10 PRTN3 ENSG00000196415 1 A 113 1.20E-09 MKNK2 ENSG00000099875 1 A 45 2.27E-07 TRIP10 ENSG00000125733 1 A 45 2.27E-07 602067 Cardiomyopathy, dilated, 1f; 6q23 74 AHI1 ENSG00000135541 1 A 144 3.32E-14 EYA4 ENSG00000112319 1 A 134 9.27E-13 TCF21 ENSG00000118526 1 A 33 3.69E-12 HEBP2 ENSG00000051620 1 A 72 2.44E-06 603165 Dermatitis, atopic; 1q21 334 ANXA9 ENSG00000143412 1 A 72 2.24E-09 ECM1 ENSG00000143369 1 A 72 2.24E-09 FLG ENSG00000143631 2 A 72 2.24E-09 LOR ENSG00000203782 1 A 72 2.24E-09 S100A14 ENSG00000189334 1 A 72 2.24E-09 SPRR1B ENSG00000169469 1 A 72 2.24E-09 603204 Epilepsy, nocturnal frontal lobe, type 2; 15q24 97 LINGO1 ENSG00000169783 1 A 68 2.45E-08 SCAMP5 ENSG00000198794 1 A 51 9.65E-08 603511 Muscular dystrophy, limb-girdle, type 1d; 7q 1069 ASB10 ENSG00000146926 1 A 72 8.28E-09 ASB15 ENSG00000146809 1 A 72 8.28E-09 FLNC ENSG00000128591 3(ix) A 72 8.28E-09 PPP1R3A ENSG00000154415 1 A 72 8.28E-09 PDK4 ENSG00000004799 1 A 120 8.73E-09 MYLC2PL ENSG00000106436 1 A 119 9.20E-08 CD36 ENSG00000135218 1 A 144 1.13E-07 EXOC4 ENSG00000131558 1 A 144 1.13E-07 EPHA1 ENSG00000146904 1 A 26 2.89E-07 FOXP2 ENSG00000128573 1 A 26 2.89E-07 603786 Stargardt disease 4; 4p 361 APBB2 ENSG00000163697 1 S 77 2.34E-06 FAM114A1 ENSG00000197712 1 S 77 2.34E-06 UGDH ENSG00000109814 1 S 77 2.34E-06 604288 Cardiomyopathy, dilated, 1h; 2q14-q22 278 ACVR2A ENSG00000121989 1 A 134 2.24E-08 HNMT ENSG00000150540 1 A 134 2.24E-08 604364 Epilepsy, partial, with variable foci; 22q11-q12 474 CACNG2 ENSG00000166862 1 A 73 1.18E-07 SEZ6L ENSG00000100095 1 A 73 1.18E-07 SLC25A18 ENSG00000182902 1 A 73 1.18E-07 GANZ ENSG00000128266 1 A 51 8.91E-07 C22orf25 ENSG00000183597 1 A 39 1.31E-06 604454 Welander distal myopathy; wdm; 2p13 111 HK2 ENSG00000159399 1 A 107 1.01E-13 ANTXR1 ENSG00000169604 1 S;A 98 1.75E-06 ANXA4 ENSG00000196975 1 A 73 3.04E-06 604499 Hyperlipidemia, combined, 2; 11p 637 F2 ENSG00000180210 1 A 103 1.02E-06 HPX ENSG00000110169 1 A 103 1.02E-06 SAA4 ENSG00000148965 1 A 103 1.02E-06 604781 Ichthyosis, nonlamellar and nonerythrodermic, congenital, autosomal recessive; 19p13.2-p13.1 434 ABHD9 ENSG00000105131 1 A 57 4.18E-14 CASP14 ENSG00000105141 1 A 57 4.18E-14 CYP4F22 ENSG00000171954 1 A 57 4.18E-14 KIAA1543 ENSG00000076826 1 A 33 5.44E-10 604801 Muscular dystrophy, congenital, 1b; 1q42 194 ACTA1 ENSG00000143632 2 A 119 1.66E-16 CABC1 ENSG00000163050 1 A 119 1.66E-16 NID1 ENSG00000116962 1 S 112 4.04E-08 C1orf198 ENSG00000119280 1 S 68 6.50E-07 ABCB10 ENSG00000135776 1 S 98 6.70E-07 605021 Myoclonic epilepsy, infantile; 16p13 366 A2BP1 ENSG00000078328 1 A 100 3.94E-08 CASKIN1 ENSG00000167971 1 A 100 3.94E-08 MAPK8IP3 ENSG00000138834 1 A 100 3.94E-08 SYNGR3 ENSG00000127561 1 A 139 8.45E-07 FLYWCH1 ENSG00000059122 1 A 173 1.72E-06 605285 Neuropathy, hereditary motor and sensory, russe type; 10q23.2 26 SNCG ENSG00000173267 1 A 32 5.27E-09 605480 Systemic lupus erythematosus, susceptibility to; 4p16-p15.2 239 CRMP1 ENSG00000072832 1 A 51 1.21E-06 KCNIP4 ENSG00000185774 1 A 51 1.21E-06 LGI2 ENSG00000153012 1 A 51 1.21E-06 NSG1 ENSG00000168824 1 A 52 1.32E-06 PPP2R2C ENSG00000074211 1 A 52 1.32E-06 605582 Cardiomyopathy, dilated, 1k; 6q12-q16 209 ME1 ENSG00000065833 3$(x) A 76 9.60E-08 TPBG ENSG00000146242 1 A 134 4.83E-07 605642 Thyroid carcinoma, papillary, with papillary renal neoplasia; 1q21 334 MRPL9 ENSG00000143436 1 A 9 1.17E-06 PRKAB2 ENSG00000131791 1 A 58 2.97E-06 605711 Multiple mitochondrial dysfunctions syndrome; 2p14-p13 142 MCEE ENSG00000124370 1 A 54 1.28E-06 605751 Convulsions, benign familial infantile, 2; 16p12-q12 407 GNAO1 ENSG00000087258 1 A 173 4.17E-08 MT3 ENSG00000087250 1 A 173 4.17E-08 SEZ6L2 ENSG00000174938 1 A 84 2.67E-07 ITFG1 ENSG00000129636 1 A 159 1.14E-06 USP31 ENSG00000103404 1 A 159 1.14E-06 CE110 ENSG00000103540 1 A 157 2.84E-06 PRKCB1 ENSG00000166501 1 A 157 2.84E-06 605809 Myasthenia, familial infantile, 1; 17p13 282 hCG_1776018 ENSG00000188265 1 A 32 1.86E-06 CHRNB1 ENSG00000170175 2£(xi) A 68 3.01E-06 ENO3 ENSG00000108515 1 A 68 3.01E-06 MYH1 ENSG00000109061 1 A 68 3.01E-06 606070 Myopathy, distal 2; 5q 1116 MYOT ENSG00000120729 2 A 120 2.33E-09 THBS4 ENSG00000113296 1 A 120 2.33E-09 DBN1 ENSG00000113758 1 S 45 3.10E-08 LOX ENSG00000113083 1 S 45 3.10E-08 LOC493869 ENSG00000164294 1 S 45 3.10E-08 SLIT3 ENSG00000184347 1 S 45 3.10E-08 SPARC ENSG00000113140 1 S 45 3.10E-08 HSPB3 ENSG00000169271 1 A 114 5.19E-08 MEF2C ENSG00000081189 1 A 114 5.19E-08 AFAP1L1 ENSG00000157510 1 A 144 6.16E-08 EDIL3 ENSG00000164176 1 A 144 6.16E-08 FGF1 ENSG00000113578 1 A 144 6.16E-08 GABRG2 ENSG00000113327 1 A 144 6.16E-08 SCAMP1 ENSG00000085365 1 A 144 6.16E-08 CTNNA1 ENSG00000044115 1 S 84 7.06E-08 EGR1 ENSG00000120738 1 S 84 7.06E-08 IL6ST ENSG00000134352 1 S 84 7.06E-08 NR2F1 ENSG00000175745 1 S 84 7.06E-08 PCDHGA12 ENSG00000081853 1 S 84 7.06E-08 PPIC ENSG00000168938 1 S 84 7.06E-08 VCAN ENSG00000038427 1 S 84 7.06E-08 GABRP ENSG00000094755 1 A 62 9.02E-08 SPINK5 ENSG00000133710 1 A 120 9.28E-08 SYNPO ENSG00000171992 1 S 65 4.99E-07 EFNA5 ENSG00000184349 1 S 75 5.90E-07 PDGFRB ENSG00000113721 1 S 75 5.90E-07 PAM ENSG00000145730 1 S 112 1.27E-06 REEP5 ENSG00000129625 1 S 112 1.27E-06 LAEVR ENSG00000172901 1 A 44 1.35E-06 MAP1B ENSG00000131711 1 S 98 2.10E-06 P4HA2 ENSG00000072682 1 S 98 2.10E-06 DPYSL3 ENSG00000113657 1 S 90 2.34E-06 NR3C1 ENSG00000113580 1 S 64 2.39E-06 GFM2 ENSG00000164347 1 A 68 2.53E-06 U384 ENSG00000048162 1 A 68 2.53E-06 CKMT2 ENSG00000131730 1 A 68 3.90E-06 606257 Stature quantitative trait locus 3; 12p11.2-q14 548 KRT1 ENSG00000167768 1 A 72 1.32E-06 KRT2 ENSG00000172867 1 A 72 1.32E-06 KRT71 ENSG00000139648 1 A 72 1.32E-06 KRT8 ENSG00000170421 1 A 72 1.32E-06 PP11 ENSG00000111405 1 A 72 1.32E-06 606483 Charcot-Marie-Tooth disease, dominant intermediate A; 10q24.1-q25.1 179 SFRP5 ENSG00000120057 1 A 69 1.61E-06 606545 Ichthyosis, lamellar, 5; 17p13.2-p13.1 210 ALOX12B ENSG00000179477 2 A 58 1.78E-17 ALOXE3 ENSG00000179148 2 A 58 1.78E-17 GGT6 ENSG00000167741 1 A 58 1.78E-17 ENO3 ENSG00000108515 1 A 71 1.65E-16 SOX15 ENSG00000129194 1 A 41 3.05E-10 MYH2 ENSG00000125414 1 A 65 1.35E-09 CLDN7 ENSG00000181885 1 A 186 5.49E-07 606708 Split-hand/foot malformation 5; 2q31 126 HOXD10 ENSG00000128710 1 A 13 4.55E-07 HOXD8 ENSG00000175879 1 A 13 4.55E-07 HOXD11 ENSG00000128713 1 A 63 1.67E-06 HOXD3 ENSG00000128652 1 A 63 1.67E-06 HOXD13 ENSG00000128714 2$(xii) A 28 2.08E-06 606744 Seckel syndrome 2; 18p11.31-q11.2 169 VAPA ENSG00000101558 1 A 36 3.68E-06 607086 Aortic aneurysm, familial thoracic 1; 11q23.3-q24 250 TAGLN ENSG00000149591 1 A 92 8.74E-07 MCAM ENSG00000076706 1 A 138 1.83E-06 607088 Spinal muscular atrophy, distal, autosomal recessive, 3; 11q13 354 ACTN3 ENSG00000204633 1 A 108 2.28E-10 PYGM ENSG00000068976 1 A 108 2.28E-10 EHBP1L1 ENSG00000173442 1 A 87 5.07E-08 LRP16 ENSG00000133315 1 A 144 5.11E-08 P2RY2 ENSG00000175591 1 A 120 1.87E-07 CCND1 ENSG00000110092 1 S 98 3.99E-07 SERPINH1 ENSG00000149257 1 S;A 98 3.99E-07 PLCB3 ENSG00000149782 1 A 52 1.03E-06 PDE2A ENSG00000186642 1 S 65 1.72E-06 FOLR2 ENSG00000165457 1 A 85 1.82E-06 UCP3 ENSG00000175564 1 A 85 1.82E-06 CTTN ENSG00000085733 1 S 79 1.84E-06 CD248 ENSG00000174807 1 A 72 2.05E-06 DGAT2 ENSG00000062282 1 A 75 2.55E-06 ARHGEF17 ENSG00000110237 1 S 84 2.68E-06 607221 Epilepsy, partial, with pericentral spikes; 4p15 86 KCNIP4 ENSG00000185774 1 A 143 1.65E-07 LGI2 ENSG00000153012 1 A 51 2.31E-07 607936 Exfoliative ichthyosis, autosomal recessive, ichthyosis bullosa of siemens-like; 12q13 355 KRT1 ENSG00000167768 2 A 57 6.70E-20 KRT2 ENSG00000172867 2 A 57 6.70E-20 KRT8 ENSG00000170421 1 A 57 6.70E-20 KRT84 ENSG00000161849 1 A 57 6.70E-20 SDR-O ENSG00000170426 1 A 57 6.70E-20 KRT71 ENSG00000139648 1 A 52 1.55E-19 PP11 ENSG00000111405 1 A 61 1.06E-18 VDR ENSG00000111424 1 A 61 1.06E-18 KRT4 ENSG00000170477 1 A 71 1.97E-15 KRT76 ENSG00000185069 1 A 67 2.51E-13 RARG ENSG00000172819 1 A 76 2.38E-11 GLS2 ENSG00000135423 1 A 33 4.03E-09 SLC38A4 ENSG00000139209 1 A 33 4.03E-09 HOXC13 ENSG00000123364 1 A 117 4.15E-09 KRT82 ENSG00000161850 1 A 41 8.03E-07 608096 Epilepsy, familial temporal lobe; 12q22-q23.3 163 ANKS1B ENSG00000185046 1 A 143 1.04E-06 608224 Deafness, autosomal dominant nonsyndromic sensorineural 41; 12q24.32-qter 71 POLE ENSG00000177084 1 A 19 1.00E-06 ULK1 ENSG00000177169 1 A 19 1.00E-06 608318 Coronary heart disease, susceptibility to, 4; 14q32 414 SERPINA10 ENSG00000140093 1 A 9 1.25E-06 608423 Muscular dystrophy, limb-girdle, type 1f; 7q32.1-q32.2 71 FAM40B ENSG00000128578 1 A 113 4.50E-11 FLNC ENSG00000128591 2(ix) S;A 112 3.44E-08 LEP ENSG00000174697 1 A 24 1.45E-06 608762 Epilepsy, idiopathic generalized, susceptibility to, 3; 9q32-q33 161 GOLGA1 ENSG00000136935 1 A 173 4.49E-06 608816 Myoclonic epilepsy, juvenile, 3; 6p21 395 PACSIN1 ENSG00000124507 1 A 173 1.01E-07 The column ‘status’ reviews the current knowledge about the association of the candidate with the disease. In particular, 1 = gene not previously associated with the disease; 2 = gene involved in mendelian phenotype sharing a MimMiner similarity score of 0.4 or higher with the phenotypic description of the locus; 3 = gene previously considered as a candidate for clinical similarity, but with a MimMiner similarity score to the locus lower than 0.4. Moreover, genes annotated with “#” represent the actual disease gene, because mutations have been found in patients; genes annotated with “£” have been excluded by refining the map interval; genes annotated with “*” could be excluded because mutations have been found in a different gene of the same locus; genes annotated with “$” are at the moment excluded because they have been screened but no mutations considered to be relevant have been found. The above statements are supported by the indicated references: i = [38]; ii = [39]; iii = [40]; iv = [41]; v = [42]; vi = [43]; vii = [44]; viii = [45]; ix = [46]; x = [47]; xi = [48]; xii = [49]. The column “Net.” indicates the networks from which the candidate was predicted: A = Affy; S = Stanford. Notably, for three OMIM phenotypes (163000, familial multiple nevi flammei; 268700, saccharopinuria; 300195; AMMECR1) our predictions include the actual disease genes that, although not yet correctly annotated in OMIM, have been found to be mutated in patients (see Table 2). For 22 loci, at least one of the candidates obtained from either network was already known to be involved in phenotypes similar to those described for the locus. These genes represent the most obvious candidates and our results should be considered as further, independent evidence for their possible involvement in the disease. However, it must be noted that some of them were previously excluded, either by the direct identification of crossovers or by the negative results of mutation screenings. Nevertheless, since mutations have most likely been searched only within the annotated exons, we think that the decision to definitively rule out the involvement of such candidates should be taken cautiously. Moreover, even silent exonic mutations, although often considered innocuous polymorphisms, can have severe effects on proteins by disrupting splicing patterns [27],[28]. In most cases only few candidates are given for a locus, thus providing extremely focused working hypotheses for the identification of the actual disease genes, which in many cases are made even stronger by the available sequence or functional information. For instance, one of the two candidates provided for the OMIM phenotype entry 607221 (partial epilepsy with pericentral spikes, located on 4p15) corresponds to KCNIP4 (Figure 4). This protein has been show to specifically modulate the activity of Kv4 A-type potassium channels [29], which are well known regulators of membrane excitability [30] and have been recently involved in epilepsy [31]. Another interesting example is given by the 605285 phenotype entry (hereditary motor and sensory neuropathy, Russe type, mapped to 10q23.2, a locus comprising only 26 candidate genes). The only prediction for this locus is gamma-synuclein (SNCG), which is a very strong candidate both for the low P-value and for the known role of synucleins in neurodegenerative disorders [32]. 10.1371/journal.pcbi.1000043.g004 Figure 4 Example of identification of candidate disease genes. Identification of KCNIP4 (green) in the genomic locus 4p15 (containing 86 genes; orange), associated with partial epilepsy with pericentral spikes (OMIM ID 607221). KCNIP4 was found in a disease-related cluster composed of LOC399947 and its nearest neighbors (red and purple spheres, respectively) that contains 5 other genes known to be involved in similar phenotypes (yellow spheres). A second candidate for this disease was found in another disease-associated cluster (not shown; see Table 2). Even when the number of candidates for a particular locus is substantially higher, our results may provide a strong restriction of the experimental search field, which can be further narrowed by additional evidences. For instance, the phenotype with OMIM ID 130080 (Ehlers-Danlos syndrome, type VIII), is mapped to 12p13, containing 277 genes. In this case, the Affy and Stanford networks provide 8 and 4 candidates, respectively. Interestingly, the candidate with the lowest associated P-value is the Alpha-2-macroglobulin precursor (A2M), whose absence was previously reported in a patient with Ehlers-Danlos syndrome [33]. A second interesting protein for this locus is CD9 that is the only candidate provided by both networks and that is known to regulate collagen matrix organization by interacting with Beta1 integrin [34]. In general, given the highly stringent criteria that we adopted and considering that the starting data underlying the two networks are completely independent, we propose that the 4 common candidates (Table 2) should be considered as those having the highest priority for experimental validation. Since a recent study has identified high confidence candidate genes by integrating protein-protein interaction with phenotypic information [8], we evaluated the number of common predictions, and found that a candidate is proposed by both approaches for 7 loci. In 5 cases the candidate proposed by Lage et al. did not overlap with our predictions. The only, remarkable exception was Filamin C (FLNC), which was found as a candidate on chromosome 7q for both the 608423 (limb-girdle muscular dystrophy type 1F) and the 603511 (limb-girdle muscular dystrophy type 1D) OMIM phenotype entries. Interestingly, the FLNC gene has been previously implicated in myopathy, but mutations have not been reported in families mapping to the above loci. Thus, our results can be considered as further supporting evidence, pointing to the actual involvement of this gene in limb-girdle muscular dystrophy. Discussion In the present report we have shown that the integration of massive gene expression data with phenotype similarity maps can allow to efficiently identify high-probability candidates for many orphan human genetic disease loci, even when these comprise hundreds of genes. The comparison of the two different networks clearly showed that the Affy network is characterized by a much better signal/noise ratio than the Stanford network. Although this may be partially due to the different source material (normal tissues in the first case, mostly tumor cell lines in the second case) we think that this could also depend on the higher technical standards reached by oligonucleotide-based platforms. Nevertheless, it is also important to notice that the analysis of the Stanford network allowed the prediction of many candidates that were not obtained from the Affy network, indicating that different datasets can result in complementary predictions. Interestingly, in both cases the gene coexpression criterion proved to be much more effective than it could be expected from previous work, where it has been mostly used in combination with other high-throughput information sources. We think that these results strongly underscore two critical points. The first is the importance of using a restrictive filter to select biologically relevant coexpression links, such as our phylogenetic filter selecting links which are under selective pressure and therefore more likely to imply functional relationships. The second is the usefulness of systematic phenotype analysis methods, which may capture disease similarities that could easily escape human operator-based approaches. Although very significant under the statistical point of view, the overlapping between conserved-coexpression links and physical protein-protein interaction data appeared to be rather limited in absolute terms, strengthening the idea that these criteria may cover partially overlapping subsets of the functional interaction space. Our results strongly suggest that, in most of the cases, requiring the concordance of coexpression data and protein-protein interaction data may worsen, instead of improving, the performances of both methods. Therefore, we envisage the independent use of both types of evidence to predict functional relationships and candidate disease genes. However, in the limited cases for which these approaches provide convergent results, they can be used as strong additive evidence. In conclusion, we propose that our method and our list of candidates will provide a useful support for the identification of new disease-relevant genes. Supporting Information Text S1 Stanford human-mouse conserved coexpression network. Each row contains a link between two Entrez-Gene identifiers. (0.66 MB TXT) Click here for additional data file. Text S2 Affy human-mouse conserved coexpression network. Each row contains a link between two Entrez-Gene identifiers. (1.89 MB TXT) Click here for additional data file.
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### Author and article information

###### Journal
J Biol Chem
J. Biol. Chem
jbc
jbc
JBC
The Journal of Biological Chemistry
American Society for Biochemistry and Molecular Biology (11200 Rockville Pike, Suite 302, Rockville, MD 20852-3110, U.S.A. )
0021-9258
1083-351X
8 July 2016
5 May 2016
5 May 2016
: 291
: 28
: 14540-14554
###### Affiliations
From the Departments of []Pharmacology and
[]Biochemistry and the Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and
the [§ ]Systems Immunity Research Institute, School of Medicine, Cardiff University, Cardiff CF14 4XN, Wales, United Kingdom
###### Author notes
[3 ] To whom correspondence should be addressed: Dept. of Pharmacology, Vanderbilt University School of Medicine, 23rd Ave. at Pierce, Nashville, TN 37232-6602. Tel.: 615-343-4495; E-mail: alan.brash@ 123456vanderbilt.edu .
[1]

Both authors contributed equally to this work.

[2]

A Marie Curie Outgoing Fellow.

###### Article
M115.711267
10.1074/jbc.M115.711267
4938176
27151221
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Author's Choice—Final version free via Creative Commons CC-BY license.

###### Funding
Funded by: National Institute of Arthritis and Musculoskeletal and Skin Diseases http://dx.doi.org/10.13039/100000069
Award ID: AR051968
Funded by: National Cancer Institute http://dx.doi.org/10.13039/100000054
Award ID: P30 CA068485
Funded by: Medical Research Council http://dx.doi.org/10.13039/501100000265
Award ID: MR/M011445/1
###### Categories
Lipids

Biochemistry