We report the design and synthesis of novel FTPA-triazole compounds as potent inhibitors of isoprenylcysteine carboxyl methyltransferase (Icmt), through a focus on thioether and isoprenoid mimetics. These mimetics were coupled utilizing a copper-assisted cycloaddition to assemble the potential inhibitors. Using the resulting triazole from the coupling as an isoprenyl mimetic resulted in the biphenyl substituted FTPA triazole 10n. This lipid-modified analog is a potent inhibitor of Icmt (IC50 = 0.8 ± 0.1 μM; calculated Ki = 0.4 μM).

Human protein isoprenylcysteine carboxyl methyltransferase (hIcmt) is the enzyme responsible for the α-carboxyl methylation of the C-termimal isoprenylated cysteine of CaaX proteins, including Ras proteins. This specific posttranslational methylation event has been shown to be important for cellular transformation by oncogenic Ras isoforms. This finding led to interest in hIcmt inhibitors as potential anti-cancer agents. Previous analog studies based on N-acetyl-S-farnesylcysteine identified two prenylcysteine-based low micromolar inhibitors (1a and 1b) of hIcmt, each bearing a phenoxyphenyl amide modification. In this study, a focused library of analogs of 1a and 1b was synthesized and screened versus hIcmt, delineating structural features important for inhibition. Kinetic characterization of the most potent analogs 1a and 1b established that both inhibitors exhibited mixed-mode inhibition and that the competitive component predominated. Using the Cheng – Prusoff method, the Ki values were determined from the IC50 values. Analog 1a has a KIC of 1.4 ± 0.2 μM and a KIU of 4.8 ± 0.5 μM while 1b has a KIC of 0.5 ± 0.07 μM and a KIU of 1.9 ± 0.2 μM. Cellular evaluation of 1b revealed that it alters the subcellular localization of GFP-KRas, and also inhibits both Ras activation and Erk phosphorylation in Jurkat cells.

Genetic variants responsible for susceptibility to obesity and its comorbidities among Hispanic children have not been identified. The VIVA LA FAMILIA Study was designed to genetically map childhood obesity and associated biological processes in the Hispanic population. A genome-wide association study (GWAS) entailed genotyping 1.1 million single nucleotide polymorphisms (SNPs) using the Illumina Infinium technology in 815 children. Measured genotype analysis was performed between genetic markers and obesity-related traits i.e., anthropometry, body composition, growth, metabolites, hormones, inflammation, diet, energy expenditure, substrate utilization and physical activity. Identified genome-wide significant loci: 1) corroborated genes implicated in other studies (MTNR1B, ZNF259/APOA5, XPA/FOXE1 (TTF-2), DARC, CCR3, ABO); 2) localized novel genes in plausible biological pathways (PCSK2, ARHGAP11A, CHRNA3); and 3) revealed novel genes with unknown function in obesity pathogenesis (MATK, COL4A1). Salient findings include a nonsynonymous SNP (rs1056513) in INADL (p = 1.2E-07) for weight; an intronic variant in MTNR1B associated with fasting glucose (p = 3.7E-08); variants in the APOA5-ZNF259 region associated with triglycerides (p = 2.5-4.8E-08); an intronic variant in PCSK2 associated with total antioxidants (p = 7.6E-08); a block of 23 SNPs in XPA/FOXE1 (TTF-2) associated with serum TSH (p = 5.5E-08 to 1.0E-09); a nonsynonymous SNP (p = 1.3E-21), an intronic SNP (p = 3.6E-13) in DARC identified for MCP-1; an intronic variant in ARHGAP11A associated with sleep duration (p = 5.0E-08); and, after adjusting for body weight, variants in MATK for total energy expenditure (p = 2.7E-08) and in CHRNA3 for sleeping energy expenditure (p = 6.0E-08). Unprecedented phenotyping and high-density SNP genotyping enabled localization of novel genetic loci associated with the pathophysiology of childhood obesity.

Osteogenesis imperfecta (OI) is a spectrum of genetic disorders characterized by bone fragility. It is caused by dominant mutations affecting the synthesis and/or structure of type I procollagen or by recessively inherited mutations in genes responsible for the post-translational processing/trafficking of type I procollagen. Recessive OI type VI is unique among OI types in that it is characterized by an increased amount of unmineralized osteoid, thereby suggesting a distinct disease mechanism. In a large consanguineous family with OI type VI, we performed homozygosity mapping and next-generation sequencing of the candidate gene region to isolate and identify the causative gene. We describe loss of function mutations in serpin peptidase inhibitor, clade F, member 1 (SERPINF1) in two affected members of this family and in an additional unrelated patient with OI type VI. SERPINF1 encodes pigment epithelium-derived factor. Hence, loss of pigment epithelium-derived factor function constitutes a novel mechanism for OI and demonstrates its involvement in bone mineralization.

Background

Somatostatin receptor type 5 (SSTR5) P335L is a hypofunctional single nucleotide polymorphism of SSTR5 with implications in tumor diagnostics and therapy. The purpose of this study is to determine whether a SSTR5 P335L-specific monoclonal antibody (mAb) could sufficiently differentiate pancreatic neuroendocrine tumor (PNT) patients with different SSTR5 genotypes.

Methods

Cellular proliferation rate, SSTR5 mRNA level and SSTR5 protein level were measured by performing MTS assay, qRT-PCR and western blotting and immunohistochemistry, respectively. SSTR5 genotype was determined with the TaqMan SNP Genotyping assay.

Results

1) SSTR5 analogue RPL-1980 inhibited cellular proliferation of CAPAN-1 cells more significantly than that of PANC-1 cells. 2) Only PANC-1 (TT) cells, but not CAPAN-1 (CC) cells expressed SSTR5 P335L. 3) In 29 Caucasian PNT patients, 38% had TT genotype for SSTR5 P335L, 24% had CC genotype for WT SSTR5, and 38% had CT genotype for both SSTR5 P335L and WT SSTR5. 4) Immunohistochemistry using SSTR5 P335L mAb detected immunostaining signals only from the PNT specimens with TT and CT genotypes, but not those with CC genotypes.

Conclusions

A SSTR5 P335L mAb that specifically recognizes SSTR5 P335L, but not WT SSTR5, could differentiate PNT patients with different SSTR5 genotypes, thus providing a potential tool for clinical diagnosis of PNT.

Many genomes have been sequenced to high-quality draft status using Sanger capillary electrophoresis and/or newer short-read sequence data and whole genome assembly techniques. However, even the best draft genomes contain gaps and other imperfections due to limitations in the input data and the techniques used to build draft assemblies. Sequencing biases, repetitive genomic features, genomic polymorphism, and other complicating factors all come together to make some regions difficult or impossible to assemble. Traditionally, draft genomes were upgraded to “phase 3 finished” status using time-consuming and expensive Sanger-based manual finishing processes. For more facile assembly and automated finishing of draft genomes, we present here an automated approach to finishing using long-reads from the Pacific Biosciences RS (PacBio) platform. Our algorithm and associated software tool, PBJelly, (publicly available at https://sourceforge.net/projects/pb-jelly/) automates the finishing process using long sequence reads in a reference-guided assembly process. PBJelly also provides “lift-over” co-ordinate tables to easily port existing annotations to the upgraded assembly. Using PBJelly and long PacBio reads, we upgraded the draft genome sequences of a simulated Drosophila melanogaster, the version 2 draft Drosophila pseudoobscura, an assembly of the Assemblathon 2.0 budgerigar dataset, and a preliminary assembly of the Sooty mangabey. With 24× mapped coverage of PacBio long-reads, we addressed 99% of gaps and were able to close 69% and improve 12% of all gaps in D. pseudoobscura. With 4× mapped coverage of PacBio long-reads we saw reads address 63% of gaps in our budgerigar assembly, of which 32% were closed and 63% improved. With 6.8× mapped coverage of mangabey PacBio long-reads we addressed 97% of gaps and closed 66% of addressed gaps and improved 19%. The accuracy of gap closure was validated by comparison to Sanger sequencing on gaps from the original D. pseudoobscura draft assembly and shown to be dependent on initial reference quality.

BACKGROUND AND AIMS

The intestinal microbiomes of healthy children and pediatric patients with irritable bowel syndrome (IBS) are not well defined. Studies in adults have indicated that the gastrointestinal microbiota could be involved in IBS.

METHODS

We analyzed 71 samples from 22 children with IBS (pediatric Rome III criteria) and 22 healthy children, ages 7–12 years, by 16S rRNA gene sequencing, with an average of 54,287 reads/stool sample (average 454 read length = 503 bases). Data were analyzed using phylogenetic-based clustering (Unifrac), or an operational taxonomic unit (OTU) approach using a supervised machine learning tool (randomForest). Most samples were also hybridized to a microarray that can detect 8,741 bacterial taxa (16S rRNA PhyloChip).

RESULTS

Microbiomes associated with pediatric IBS were characterized by a significantly greater percentage of the class Gammaproteobacteria (0.07% vs 0.89% of total bacteria; P <.05); one prominent component of this group was Haemophilus parainfluenzae. Differences highlighted by 454 sequencing were confirmed by high-resolution PhyloChip analysis. Using supervised learning techniques, we were able to classify different subtypes of IBS with a success rate of 98.5%, using limited sets of discriminant bacterial species. A novel Ruminococcus-like microbe was associated with IBS, indicating the potential utility of microbe discovery for gastrointestinal disorders. A greater frequency of pain correlated with an increased abundance of several bacterial taxa from the genus Alistipes.

CONCLUSIONS

Using16S metagenomics by Phylochip DNA hybridization and deep 454 pyrosequencing, we associated specific microbiome signatures with pediatric IBS. These findings indicate the important association between gastrointestinal microbes and IBS in children; these approaches might be used in diagnosis of functional bowel disorders in pediatric patients.

Purpose

To characterize tumor growth and metastatic potential in head and neck squamous cell carcinoma (HNSCC) cell lines in an orthotopic murine model of oral tongue cancer, and to correlate TP53 mutation status with these findings.

Experimental Design

Cells from each of 48 HNSCC cell lines were orthotopically injected into the oral tongues of nude mice. Tumor volume, cervical lymph node metastasis, and mouse survival were recorded. Direct sequencing of the TP53 gene and western blot analysis for the p53 protein after induction with 5-fluorouracil was performed. Cell lines were categorized as either mutant TP53 or wild-type TP53, and lines with TP53 mutation were further categorized on the basis of type of mutation (disruptive or non-disruptive), and level of p53 protein expression. The behavior of tumors in these different groups was compared.

Results

The 48 HNSCC cell lines showed a wide range of behavior from highly aggressive and metastatic to no tumor formation. Mice injected with cells harboring disruptive TP53 mutations had faster tumor growth, greater incidence of cervical lymph node metastasis, and shorter survival than mice injected with cells lacking these mutations.

Conclusions

HNSCC cell lines display a wide spectrum of behavior in an orthotopic model of oral cancer. Cell lines with disruptive TP53 mutations are more aggressive in this system, corroborating clinical reports that have linked these mutations to poor patient outcome.

The human X and Y chromosomes evolved from an ordinary pair of autosomes during the past 200–300 million years1–3. Due to genetic decay, the human MSY (male-specific region of Y chromosome) retains only three percent of the ancestral autosomes’ genes4,5. This evolutionary decay was driven by a series of five “stratification” events. Each event suppressed X-Y crossing over within a chromosome segment or “stratum”, incorporated that segment into the MSY, and subjected its genes to the erosive forces that attend the absence of crossing over2,6. The last of these events occurred 30 million years ago (mya), or 5 million years before the human and Old World monkey (OWM) lineages diverged. Although speculation abounds regarding ongoing decay and looming extinction of the human Y chromosome7–10, remarkably little is known about how many MSY genes were lost in the human lineage in the 25 million years that have followed its separation from the OWM lineage. To explore this question, we sequenced the MSY of the rhesus macaque, an OWM, and compared it to the human MSY. We discovered that, during the last 25 million years, MSY gene loss in the human lineage was limited to the youngest stratum (stratum 5), which comprises three percent of the human MSY. Within the older strata, which collectively comprise the bulk of the human MSY, gene loss evidently ceased more than 25 mya. Likewise, the rhesus MSY has not lost any older genes (from strata 1–4) during the past 25 million years, despite major structural differences from the human MSY. The rhesus MSY is simpler, with few amplified gene families or palindromes that might enable intrachromosomal recombination and repair. We present an empirical reconstruction of human MSY evolution in which each stratum transitioned from rapid, exponential loss of ancestral genes to strict conservation through purifying selection.

Genome sequencing studies indicate that all humans carry many genetic variants predicted to cause loss of function (LoF) of protein-coding genes, suggesting unexpected redundancy in the human genome. Here we apply stringent filters to 2,951 putative LoF variants obtained from 185 human genomes to determine their true prevalence and properties. We estimate that human genomes typically contain ~100 genuine LoF variants with ~20 genes completely inactivated. We identify rare and likely deleterious LoF alleles, including 26 known and 21 predicted severe disease-causing variants, as well as common LoF variants in non-essential genes. We describe functional and evolutionary differences between LoF-tolerant and recessive disease genes, and a method for using these differences to prioritize candidate genes found in clinical sequencing studies.

Pancreatic and duodenal homeobox-1 (PDX-1) is a transcription factor that regulates insulin expression and islet maintenance in the adult pancreas. Our recent studies demonstrate that PDX-1 is an oncogene for pancreatic cancer and is overexpressed in pancreatic cancer. The purpose of this study was to demonstrate that PDX-1 is a therapeutic target for both hormonal symptoms and tumor volume in mouse models of pancreatic cancer, insulinoma and islet neoplasia. Immunohistochemistry of human pancreatic and islet neoplasia specimens revealed marked PDX-1 overexpression, suggesting PDX-1 as a “drugable” target within these diseases. To do so, a novel RNA interference effector platform, bifunctional shRNAPDX-1, was developed and studied in mouse and human cell lines as well as in mouse models of pancreatic cancer, insulinoma and islet neoplasia. Systemic delivery of bi-shRNAhumanPDX-1 lipoplexes resulted in marked reduction of tumor volume and improved survival in a human pancreatic cancer xenograft mouse model. bi-shRNAmousePDX-1 lipoplexes prevented death from hyperinsulinemia and hypoglycemia in an insulinoma mouse model. shRNAmousePDX-1 lipoplexes reversed hyperinsulinemia and hypoglycemia in an immune-competent mouse model of islet neoplasia. PDX-1 was overexpressed in pancreatic neuroendocrine tumors and nesidioblastosis. These data demonstrate that PDX-1 RNAi therapy controls hormonal symptoms and tumor volume in mouse models of pancreatic cancer, insulinoma and islet neoplasia, therefore, PDX-1 is a potential therapeutic target for these pancreatic diseases.

We sequenced 8 melanoma exomes to identify novel somatic mutations in metastatic melanoma. Focusing on the MAP3K family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9. Structural modelling predicts that mutations in the kinase domain may affect the activity and regulation of MAP3K5/9 protein kinases. The position of the mutations and loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely inactivating. In vitro kinase assay shows reduction in kinase activity in MAP3K5 I780F and MAP3K9 W333X mutants. Overexpression of MAP3K5 or MAP3K9 mutant in HEK293T cells reduces phosphorylation of downstream MAP kinases. Attenuation of MAP3K9 function in melanoma cells using siRNA leads to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.

Background

Enrichment of loci by DNA hybridization-capture, followed by high-throughput sequencing, is an important tool in modern genetics. Currently, the most common targets for enrichment are the protein coding exons represented by the consensus coding DNA sequence (CCDS). The CCDS, however, excludes many actual or computationally predicted coding exons present in other databases, such as RefSeq and Vega, and non-coding functional elements such as untranslated and regulatory regions. The number of variants per base pair (variant density) and our ability to interrogate regions outside of the CCDS regions is consequently less well understood.

Results

We examine capture sequence data from outside of the CCDS regions and find that extremes of GC content that are present in different subregions of the genome can reduce the local capture sequence coverage to less than 50% relative to the CCDS. This effect is due to biases inherent in both the Illumina and SOLiD sequencing platforms that are exacerbated by the capture process. Interestingly, for two subregion types, microRNA and predicted exons, the capture process yields higher than expected coverage when compared to whole genome sequencing. Lastly, we examine the variation present in non-CCDS regions and find that predicted exons, as well as exonic regions specific to RefSeq and Vega, show much higher variant densities than the CCDS.

Conclusions

We show that regions outside of the CCDS perform less efficiently in capture sequence experiments. Further, we show that the variant density in computationally predicted exons is more than 2.5-times higher than that observed in the CCDS.

Isoprenylcysteine carboxylmethyltransferase (Icmt) is an integral membrane protein localized to the endoplasmic reticulum of eukaryotic cells that catalyzes the post-translational α-carboxylmethylesterification of CaaX motif proteins, including the oncoprotein Ras. Prior to methylation, these protein substrates all contain an isoprenylcysteine residue at the C-terminus. In this study, we developed a variety of substrates and inhibitors of Icmt that vary in the isoprene moiety in order to gain information about the nature of the lipophilic substrate binding site. These isoprenoid-modified analogs of the minimal Icmt substrate N-acetyl-S-farnesyl cysteine (AFC) were synthesized from newly prepared farnesol analogs. Using both yeast and human Icmt enzymes, these compounds were found to vary widely in their ability to act as substrates, supporting the isoprenoid moiety as a key substrate recognition element for Icmt. Compound 3 is a competitive inhibitor of overexpressed yeast Icmt (KI = 17.1 ± 1.7 μM). Compound 4 shows a mix of competitive and uncompetitive inhibition for both the yeast and the human Icmt proteins (yeast KIC = 35.4 ± 3.4 μM, KIU = 614.4 ± 148 μM, human KIC = 119.3 ± 18.1 μM, KIU = 377.2 ± 42.5 μM). These data further suggest that differences in substrate specificity exist between the human and yeast enzymes. Biological studies suggest that inhibition of Icmt results in Ras mislocalization and loss of cellular transformation ability, making Icmt an attractive and novel anti-cancer target. Further elaboration of the lead compounds synthesized and assayed here may lead to clinically useful higher potency inhibitors.

Through the use of a 1,2-metalate rearrangement, six 7-substituted farnesol analogs were generated in a concise manner. This new synthetic route allowed us to quickly prepare several diverse farnesyl diphosphate analogs with interesting biological activities against mammalian protein-farnesyl transferase.

N-acetyl-S-farnesyl-L-cysteine (AFC) is the minimal substrate for the enzyme isoprenylcysteine carboxyl methyltransferase (Icmt). A series of amide-modified farnesylcysteine analogs were synthesized and screened against human Icmt. From a 23-member library of compounds, six inhibitors were identified and evaluated further. The adamantyl derivative 7c was the most potent inhibitor with an IC50 of 12.4 μM.

Human isoprenylcysteine carboxyl methyltransferase (hIcmt) is a promising anticancer target as it is important for the post-translational modification of oncogenic Ras proteins. We herein report the synthesis and biochemical activity of 41 farnesyl-cysteine based analogs versus hIcmt. We have demonstrated that the amide linkage of a hIcmt substrate can be replaced by a sulfonamide bond to achieve hIcmt inhibition. The most potent sulfonamide-modified farnesylcysteine analog was 6ag with an IC50 of 8.8±0.5 µM for hIcmt.

Isoprenoids constitute an important class of biomolecules that participate in many different cellular processes. Most available detection methods only allow the identification of one or two specific non-sterol isoprenoid intermediates following radioactive or fluorescent labeling. We here report a rapid, non-radioactive and sensitive procedure for the simultaneous detection and quantification of the 8 main non-sterol intermediates of the isoprenoid biosynthesis pathway by means of tandem mass spectrometry. Intermediates were analyzed by HPLC-MS/MS in the multiple reaction monitoring mode using a silica-based C18 HPLC column. For quantification, their stable-isotope-labeled analogues were used as internal standards. HepG2 cells were used to validate the method. Mevalonate, phosphomevalonate and the 6 subsequent isoprenoid-pyrophosphates were readily determined with detection limits ranging from 0.03 to 1.0 μmol/L. The intra- and interassay variations for HepG2 cell homogenates supplemented with isoprenoid intermediates were 3.6–10.9% and 4.4–11.9%, respectively. Under normal culturing conditions, isoprenoid intermediates in HepG2 cells were below detection limits. However, incubation of the cells with pamidronate, an inhibitor of farnesyl pyrophosphate synthase, resulted in increased levels of MVA, IPP/DMAPP and GPP. This method will be suitable to measure profiles of isoprenoid intermediates in cells with compromised isoprenoid biosynthesis, and to determine the specificity of potential inhibitors of the pathway.

N-Acetyl-S-farnesyl cysteine (AFC) is the minimal synthetic substrate for the enzyme Icmt, which methylates prenylated proteins. The desthio-AFC isostere 2 has been synthesized in racemic form. This analog was not an Icmt substrate, but instead was a weak inhibitor with an IC50 of ~325 μM

Background

Somatostatin (SST) inhibits cell proliferation and negatively regulates the release of growth hormones via specific receptors (SSTR). Genetic variation in SSTR has been associated with risk of human cancers but has never been investigated in pancreatic cancer.

Methods

In this retrospective study, we sequenced the SSTR5 gene in paired tumor and blood samples from 33 pancreatic adenocarcinoma patients using the Sanger method. We analyzed 3 single nucleotide polymorphisms (SNPs) in samples from 863 patients with pancreatic ductal adenocarcinoma and 876 healthy controls using the TaqMan method. The associations between gene polymorphisms and pancreatic cancer risk and survival were analyzed by multivariate logistic regression and Cox proportional hazard models, respectively.

Results

We identified no somatic mutations but 3 nonsynonymous SSTR5 SNPs (P109S, L48M, and P335L) in pancreatic tumors. The SSTR5 P109S variant allele was associated with a 1.62-fold increased risk of pancreatic cancer (95% confidence interval [CI]: 1.08–2.43, P = 0.019). Furthermore, the SSTR5 L48M AC variant and smoking had a joint effect on pancreatic cancer risk (pinteraction = 0.035). The odds ratios (95% CIs) were 0.58 (0.34–0.97), 1.49 (1.18–1.89), and 2.27 (1.35–3.83) for the variant genotype alone, smoking alone, and both factors, respectively, compared with no factors. Finally, SSTR5 P335L CC and P109S CC combined were associated with lower overall survival durations in patients with resectable disease.

Conclusion

Our data suggest that SSTR5 genetic variants play a role in pancreatic cancer development and progression.

Background

Enterococci are among the leading causes of hospital-acquired infections in the United States and Europe, with Enterococcus faecalis and Enterococcus faecium being the two most common species isolated from enterococcal infections. In the last decade, the proportion of enterococcal infections caused by E. faecium has steadily increased compared to other Enterococcus species. Although the underlying mechanism for the gradual replacement of E. faecalis by E. faecium in the hospital environment is not yet understood, many studies using genotyping and phylogenetic analysis have shown the emergence of a globally dispersed polyclonal subcluster of E. faecium strains in clinical environments. Systematic study of the molecular epidemiology and pathogenesis of E. faecium has been hindered by the lack of closed, complete E. faecium genomes that can be used as references.

Results

In this study, we report the complete genome sequence of the E. faecium strain TX16, also known as DO, which belongs to multilocus sequence type (ST) 18, and was the first E. faecium strain ever sequenced. Whole genome comparison of the TX16 genome with 21 E. faecium draft genomes confirmed that most clinical, outbreak, and hospital-associated (HA) strains (including STs 16, 17, 18, and 78), in addition to strains of non-hospital origin, group in the same clade (referred to as the HA clade) and are evolutionally considerably more closely related to each other by phylogenetic and gene content similarity analyses than to isolates in the community-associated (CA) clade with approximately a 3–4% average nucleotide sequence difference between the two clades at the core genome level. Our study also revealed that many genomic loci in the TX16 genome are unique to the HA clade. 380 ORFs in TX16 are HA-clade specific and antibiotic resistance genes are enriched in HA-clade strains. Mobile elements such as IS16 and transposons were also found almost exclusively in HA strains, as previously reported.

Conclusions

Our findings along with other studies show that HA clonal lineages harbor specific genetic elements as well as sequence differences in the core genome which may confer selection advantages over the more heterogeneous CA E. faecium isolates. Which of these differences are important for the success of specific E. faecium lineages in the hospital environment remain(s) to be determined.

Ion channel mutations are an important cause of rare Mendelian disorders affecting brain, heart, and other tissues. We performed parallel exome sequencing of 237 channel genes in a well characterized human sample, comparing variant profiles of unaffected individuals to those with the most common neuronal excitability disorder, sporadic idiopathic epilepsy. Rare missense variation in known Mendelian disease genes is prevalent in both groups at similar complexity, revealing that even deleterious ion channel mutations confer uncertain risk to an individual depending on the other variants with which they are combined. Our findings indicate that variant discovery via large scale sequencing efforts is only a first step in illuminating the complex allelic architecture underlying personal disease risk. We propose that in silico modeling of channel variation in realistic cell and network models will be crucial to future strategies assessing mutation profile pathogenicity and drug response in individuals with a broad spectrum of excitability disorders.