Genetic variation among 82 pharmacogenes: The PGRNseq data from the eMERGE network.

Genetic variation can affect drug response in multiple ways, although it remains unclear how rare genetic variants affect drug response. The electronic Medical Records and Genomics (eMERGE) Network, collaborating with the Pharmacogenomics Research Network, began eMERGE-PGx, a targeted sequencing study to assess genetic variation in 82 pharmacogenes critical for implementation of "precision medicine." The February 2015 eMERGE-PGx data release includes sequence-derived data from ∼5,000 clinical subjects. We present the variant frequency spectrum categorized by variant type, ancestry, and predicted function. We found 95.12% of genes have variants with a scaled Combined Annotation-Dependent Depletion score above 20, and 96.19% of all samples had one or more Clinical Pharmacogenetics Implementation Consortium Level A actionable variants. These data highlight the distribution and scope of genetic variation in relevant pharmacogenes, identifying challenges associated with implementing clinical sequencing for drug treatment at a broader level, underscoring the importance for multifaceted research in the execution of precision medicine.

A genome-wide association study identifies variants in KCNIP4 associated with ACE inhibitor-induced cough.

The most common side effect of angiotensin-converting enzyme inhibitor (ACEi) drugs is cough. We conducted a genome-wide association study (GWAS) of ACEi-induced cough among 7080 subjects of diverse ancestries in the Electronic Medical Records and Genomics (eMERGE) network. Cases were subjects diagnosed with ACEi-induced cough. Controls were subjects with at least 6 months of ACEi use and no cough. A GWAS (1595 cases and 5485 controls) identified associations on chromosome 4 in an intron of KCNIP4. The strongest association was at rs145489027 (minor allele frequency=0.33, odds ratio (OR)=1.3 (95% confidence interval (CI): 1.2-1.4), P=1.0 × 10(-8)). Replication for six single-nucleotide polymorphisms (SNPs) in KCNIP4 was tested in a second eMERGE population (n=926) and in the Genetics of Diabetes Audit and Research in Tayside, Scotland (GoDARTS) cohort (n=4309). Replication was observed at rs7675300 (OR=1.32 (1.01-1.70), P=0.04) in eMERGE and at rs16870989 and rs1495509 (OR=1.15 (1.01-1.30), P=0.03 for both) in GoDARTS. The combined association at rs1495509 was significant (OR=1.23 (1.15-1.32), P=1.9 × 10(-9)). These results indicate that SNPs in KCNIP4 may modulate ACEi-induced cough risk.

A polymorphism in HLA-G modifies statin benefit in asthma.

Several reports have shown that statin treatment benefits patients with asthma; however, inconsistent effects have been observed. The mir-152 family (148a, 148b and 152) has been implicated in asthma. These microRNAs suppress HLA-G expression, and rs1063320, a common SNP in the HLA-G 3'UTR that is associated with asthma risk, modulates miRNA binding. We report that statins upregulate mir-148b and 152, and affect HLA-G expression in an rs1063320-dependent fashion. In addition, we found that individuals who carried the G minor allele of rs1063320 had reduced asthma-related exacerbations (emergency department visits, hospitalizations or oral steroid use) compared with non-carriers (P=0.03) in statin users ascertained in the Personalized Medicine Research Project at the Marshfield Clinic (n=421). These findings support the hypothesis that rs1063320 modifies the effect of statin benefit in asthma, and thus may contribute to variation in statin efficacy for the management of this disease.

Design and anticipated outcomes of the eMERGE-PGx project: a multicenter pilot for preemptive pharmacogenomics in electronic health record systems.

We describe here the design and initial implementation of the eMERGE-PGx project. eMERGE-PGx, a partnership of the Electronic Medical Records and Genomics Network and the Pharmacogenomics Research Network, has three objectives: (i) to deploy PGRNseq, a next-generation sequencing platform assessing sequence variation in 84 proposed pharmacogenes, in nearly 9,000 patients likely to be prescribed drugs of interest in a 1- to 3-year time frame across several clinical sites; (ii) to integrate well-established clinically validated pharmacogenetic genotypes into the electronic health record with associated clinical decision support and to assess process and clinical outcomes of implementation; and (iii) to develop a repository of pharmacogenetic variants of unknown significance linked to a repository of electronic health record-based clinical phenotype data for ongoing pharmacogenomics discovery. We describe site-specific project implementation and anticipated products, including genetic variant and phenotype data repositories, novel variant association studies, clinical decision support modules, clinical and process outcomes, approaches to managing incidental findings, and patient and clinician education methods.

A PheWAS approach in studying HLA-DRB1*1501.

HLA-DRB1 codes for a major histocompatibility complex class II cell surface receptor. Genetic variants in and around this gene have been linked to numerous autoimmune diseases. Most notably, an association between HLA-DRB1*1501 haplotype and multiple sclerosis (MS) has been defined. Utilizing electronic health records and 4235 individuals within Marshfield Clinic's Personalized Medicine Research Project, a reverse genetic screen coined phenome-wide association study (PheWAS) tested association of rs3135388 genotype (tagging HLA-DRB1*1501) with 4841 phenotypes. As expected, HLA-DRB1*1501 was associated with MS (International Classification of Disease version 9-CM (ICD9) 340, P=0.023), whereas the strongest association was with alcohol-induced cirrhosis of the liver (ICD9 571.2, P=0.00011). HLA-DRB1*1501 also demonstrated association with erythematous conditions (ICD9 695, P=0.0054) and benign neoplasms of the respiratory and intrathoracic organs (ICD9 212, P=0.042), replicating previous findings. This study not only builds on the feasibility/utility of the PheWAS approach, represents the first external validation of a PheWAS, but may also demonstrate the complex etiologies associated with the HLA-DRB1*1501 loci.

The oral-systemic personalized medicine model at Marshfield Clinic.

Periodontal disease and diabetes, two diseases that have achieved epidemic status, share a bidirectional relationship driven by micro-inflammatory processes. The present review frames the current understanding of the pathological processes that appear to link these diseases and advances the hypothesis that reversal of the epidemic is possible through application of interdisciplinary intervention and advancement of oral-systemic personalized medicine. An overview of how Marshfield Clinic's unique clinical, informatics and bio-repository resources and infrastructures are being aligned to advance oral-systemic personalized medicine is presented as an interventional model with the potential to reverse the epidemic trends seen for these two chronic diseases over the past several decades. The overall vision is to engineer a transformational shift in paradigm from 'personalized medicine' to 'personalized health'.

Mutations in the human orthologue of the mouse underwhite gene (uw) underlie a new form of oculocutaneous albinism, OCA4.

Oculocutaneous albinism (OCA) affects approximately 1/20,000 people worldwide. All forms of OCA exhibit generalized hypopigmentation. Reduced pigmentation during eye development results in misrouting of the optic nerves, nystagmus, alternating strabismus, and reduced visual acuity. Loss of pigmentation in the skin leads to an increased risk for skin cancer. Two common forms and one infrequent form of OCA have been described. OCA1 (MIM 203100) is associated with mutations of the TYR gene encoding tyrosinase (the rate-limiting enzyme in the production of melanin pigment) and accounts for approximately 40% of OCA worldwide. OCA2 (MIM 203200), the most common form of OCA, is associated with mutations of the P gene and accounts for approximately 50% of OCA worldwide. OCA3 (MIM 203290), a rare form of OCA and also known as "rufous/red albinism," is associated with mutations in TYRP1 (encoding tyrosinase-related protein 1). Analysis of the TYR and P genes in patients with OCA suggests that other genes may be associated with OCA. We have identified the mouse underwhite gene (uw) and its human orthologue, which underlies a new form of human OCA, termed "OCA4." The encoded protein, MATP (for "membrane-associated transporter protein") is predicted to span the membrane 12 times and likely functions as a transporter.

The mouse p (pink-eyed dilution) and human P genes, oculocutaneous albinism type 2 (OCA2), and melanosomal pH.

Recessive mutations of the mouse p (pink-eyed dilution) gene lead to hypopigmentation of the eyes, skin, and fur. Mice lacking a functional p protein have pink eyes and light gray fur (if non-agouti) or cream-colored fur (if agouti). The human orthologue is the P protein. Humans lacking a functional P protein have oculocutaneous albinism type 2 (OCA2). Melanocytes from p-deficient mice or OCA2 individuals contain small, minimally pigmented melanosomes. The mouse and human proteins are predicted to have 12 membrane spanning domains and possess significant sequence homology to a number of membrane transport proteins, some of which are involved in the transport of anions. The p protein has been localized to the melanosome membrane. Recently, it has been shown that melanosomes from p protein-deficient melanocytes have an abnormal pH. Melanosomes in cultured melanocytes derived from wild-type mice are typically acidic, whereas melanosomes from p protein-deficient mice are non-acidic. Melanosomes and related endosome-derived organelles (i.e., lysosomes) are thought to have an adenosine triphosphate (ATP)-driven proton pump that helps to generate an acidic lumen. To compensate for the charge of these protons, anions must also be transported to the lumen of the melanosome. In light of these observations, a model of p protein function is presented in which the p protein, together with the ATP-driven proton pump, regulates the pH of the melanosome.

Cloning, characterization and chromosome mapping of the human SOX6 gene.

The Sox gene family encodes an important group of transcription factors harboring the conserved high-mobility group (HMG) box originally identified in the mouse and human testis determining gene Sry. We have cloned and sequenced SOX6, a member of the human Sox gene family. SOX6 cDNAs isolated from a human myoblast cDNA library show 94.3% amino acid identity to mouse Sox6 throughout the gene, and 100% identity in the critical HMG box and coiled-coil domains. The human SOX6 gene was localized to chromosome 11p15.2-11p15.3 in a region of shared synteny with distal mouse chromosome 7. An analysis of the genomic structure of the human SOX6 gene revealed 16 exons. We identified three SOX6 cDNAs that are generated by alternative splicing. Northern blot analysis revealed that SOX6 is expressed in a wide variety of tissues, most abundantly in skeletal muscle, suggesting an important role for SOX6 in muscle. Mice homozygous for a null mutation of Sox6 (p(100H)) die suddenly within the first 2 weeks after birth, most likely from cardiac conduction defects (Hagiwara et al., 2000). Thus, there is a possibility that human SOX6 is similarly involved in an, as yet, unidentified human cardiac disorder.

The underwhite (uw) locus acts autonomously and reduces the production of melanin.

The mouse has provided several significant models for hypopigmentation disorders, including the major forms of albinism. Mutations at the mouse underwhite locus confer one of the most severe hypopigmentation phenotypes, similar to mutations at the pink-eyed dilution locus that is a model for type 2 oculocutaneous albinism. A melanocyte cell line established from underwhite mutant mice failed to pigment under conditions that support pigment production in wild-type melanocytes and melanoblasts from underwhite skin graft transplants failed to produce melanin in normal skin, demonstrating that the action of the gene encoded by the underwhite locus is intrinsic to melanocytes. Mice with mutations at the underwhite locus and either the pink-eyed dilution locus or the melanocortin receptor 1 locus exhibited more severe hypopigmentation than either mutation alone, suggesting that the actions of these genes are independent. These results demonstrate that the underwhite locus is a major determinant of mammalian pigmentation.

Aberrant pH of melanosomes in pink-eyed dilution (p) mutant melanocytes.

In past studies, we cloned the mouse p gene and its human homolog P, which is associated with oculocutaneous albinism type 2. Both mouse and human genes are expressed in melanocytes and encode proteins predicted to have 12 membrane-spanning domains with structural homology to known ion transporters. We have also demonstrated that the p protein is localized to the melanosomal membrane and does not function as a tyrosine transporter. In this study, immunohistochemistry and confocal microscopy were used to show that the p protein plays an important role in the generation or maintenance of melanosomal pH. Melanosomes (and their precursor compartments) were defined by antiserum directed against the melanosomal marker tyrosinase related protein 1. Acidic vesicles were identified by 3-(2, 4-dinitroanilino)-3'-amino-N-methyldipropylamine incorporation, visualized with anti-dinitrophenol. In C57BL/6+/+ (wild-type) melanocytes, 94.2% of vesicles demonstrated colocalization of tyrosinase related protein 1 and 3-(2, 4-dinitroanilino)-3'-amino-N-methyldipropylamine, indicating that almost all melanosomes or their precursors were acidic. By contrast, only 7%-8% of the staining vesicles in p mutant cell lines (pJ/pJ and pcp/p6H) showed colocalization of tyrosinase related protein 1 and 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine. Thus, without a functional p protein, most melanosomes and their precursors are not acidic. As mammalian tyrosinase activity in situ is apparently dependent on low pH, we postulate that in the absence of a low pH environment brought about by ionic transport mediated by the p protein, tyrosinase activity is severely impaired, leading to the minimal production of melanin that is characteristic of p mutants. Additionally (or alternatively), an abnormal pH may also impair the assembly of the normal melanogenic complex.

Sox6 is a candidate gene for p100H myopathy, heart block, and sudden neonatal death.

The mouse p locus encodes a gene that functions in normal pigmentation. We have characterized a radiation-induced mutant allele of the mouse p locus that is associated with a failure-to-thrive syndrome, in addition to diminished pigmentation. Mice homozygous for this mutant allele, p(100H), show delayed growth and die within 2 wk after birth. We have discovered that the mutant mice develop progressive atrioventricular heart block and significant ultrastructural changes in both cardiac and skeletal muscle cells. These observations are common characteristics described in human myopathies. The karyotype of p(100H) chromosomes indicated that the mutation is associated with a chromosome 7 inversion. We demonstrate here that the p(100H) chromosomal inversion disrupts both the p gene and the Sox6 gene. Normal Sox6 gene expression has been examined by Northern blot analysis and was found most abundantly expressed in skeletal muscle in adult mouse tissues, suggesting an involvement of Sox6 in muscle maintenance. The p(100H) mutant is thus a useful animal model in the elucidation of myopathies at the molecular level.

The pink-eyed dilution locus controls the biogenesis of melanosomes and levels of melanosomal proteins in the eye.

The pink-eyed dilution (p) locus is known to control the quantity of melanin pigment made within melanocytes and retinal pigment epithelium (RPE) in the eye. We have examined the effects of several mutant allele combinations at the murine p locus on the number and morphology of melanosomes in choroidal melanocytes and RPE cells as well as on the levels of four proteins known to be present within melanosomes: tyrosinase, tyrosinase-related proteins 1 and 2 (TRP-1 and TRP-2) and lysosome-associated membrane protein-1 (LAMP-1). By electron microscopy, we observed a modest diminution in the size and number of choroidal melanosomes in pbs/pJ mice but a more dramatic decrease in the RPE in comparison with wild-type P/P mice. By contrast, a drastic reduction in melanosome size and number was present in the choroid and RPE of pun/pun and p6H/pcp mice, and in the RPE of p6H/pcp mice, melanosomes were essentially undetectable. In wild-type mice, levels of tyrosinase, TRP-1 and TRP-2 were high at birth and showed a second peak of expression at 10-14 days of age, declining to undetectable levels by 42 days. All three mutant allele combinations reduced the levels of these melanosomal proteins with the relative severity of effects being p6H/pcp>pun/pun>pbs/pJ. In the null p6H/pcp mice, levels of these proteins were extremely low at birth, no postnatal peak was observed, and levels declined to undetectable by 14 days. Levels of LAMP-1 in wild-type mice rose initially and then declined whereas in the mutant mice, levels decreased gradually from birth. Higher levels of LAMP-1 were observed in each of the mutants than in the wild-type mice at 21 days of age. Our results demonstrate that mutations at the p locus affect the size, number, shape and contents of melanosomes, implicating the p gene product in the normal biogenesis of this organelle.

A new allelic series for the underwhite gene on mouse chromosome 15.

A new allelic series at the underwhite gene is described. Three of the alleles in the series--uw, uwd, and Uwdbr--arose as spontaneous mutations on different genetic backgrounds at The Jackson Laboratory. We report here the visible phenotypes and dominance hierarchy of these alleles, all of which are defined by a reduction of pigmentation in both eye and coat color. Electron microscopic analysis of retinal epithelium suggests that the primary defect is in the melanosome. The degree of severity of melanosome anomalies in the retina correlates with the degree of hypopigmentation in the coat. The perturbed gene and its gene product are unknown. We show that the uw locus is genetically distinct from Myo10, a suggested candidate gene for this mutation.

A very large protein with diverse functional motifs is deficient in rjs (runty, jerky, sterile) mice.

Three radiation-induced alleles of the mouse p locus, p6H, p25H, and pbs, cause defects in growth, coordination, fertility, and maternal behavior in addition to p gene-related hypopigmentation. These alleles are associated with disruption of the p gene plus an adjacent gene involved in the disorders listed. We have identified this adjacent gene, previously named rjs (runty jerky sterile), by positional cloning. The rjs cDNA is very large, covering 15,264 nucleotides. The predicted rjs-encoded protein (4,836 amino acids) contains several sequence motifs, including three RCC1 repeats, a structural motif in common with cytochrome b5, and a HECT domain in common with E6-AP ubiquitin ligase. On the basis of sequence homology and conserved synteny, the rjs gene is the single mouse homolog of a previously described five- or six-member human gene family. This family is represented by at least two genes, HSC7541 and KIAA0393, from human chromosome 15q11-q13. HSC7541 and KIAA0393 lie close to, or within, a region commonly deleted in most Prader-Willi syndrome patients. Previous work has suggested that the multiple phenotypes in rjs mice might be due to a common neuroendocrine defect. In addition to this proposed mode of action, alternative functions of the rjs gene are evaluated in light of its known protein homologies.

Identification of a novel transcript produced by the gene responsible for the Hermansky-Pudlak syndrome in Puerto Rico.

Hermansky-Pudlak Syndrome (HPS) is a rare, autosomal recessive disorder that is characterized by oculocutaneous albinism, a predisposition to mild bleeding caused by storage-pool deficient platelets, and a ceroid storage disorder. A gene responsible for HPS in Puerto Rico maps to chromosome 10q2 and isolation of the gene has been reported. We have now identified a variant HPS cDNA that contains the same 5' sequence as the published HPS gene and a unique 3' sequence. Analysis of genomic DNA suggests that the two cDNA are derived from alternative transcripts of a single gene; two polyadenylated transcripts were found in normal human melanocytes, human bone marrow cells, human melanoma cells, lymphoblastoid cell lines, and megakaryocytic leukemia cells by reverse transcriptase polymerase chain reaction and northern analysis. The splicing exhibited by this gene is identical to the splicing found to produce two alternative transcripts of the Chediak-Higashi Syndrome gene, another pigment disorder exhibiting platelet storage pool deficiency. These studies show that the HPS gene on chromosome 10 is complex and may have more than one biologically active transcript.

Mutations of the human P gene associated with Type II oculocutaneous albinism (OCA2). Mutations in brief no. 205. Online.

Mutations in the human P gene lead to oculocutaneous albinism type 2 (OCA2, MIM #203200), the most common type of albinism in humans. The P gene encodes a 110 kDa protein that is associated with melonosomal membranes and contains 12 potential membrane spanning domains. The specific function of the P protein is currently unknown. We report 7 new mutations in the P gene associated with OCA2. This includes 6 missense mutations (S86R, C112F, A368V, T592I, A724P and A787V) and one frameshift mutation (1047del7). We also report 8 polymorphisms including one amino acid substitution, D/A257. We and others have found many polymorphisms of the P gene in the coding region, several of which result in amino acid substitutions, making molecular diagnosis problematic. In contrast to this is the tyrosinase gene associated with OCA1, with a limited number of polymorphic variations in the coding region. There is also no apparent clustering of P gene missense mutations in contrast to the clustering observed by the tyrosinase gene missense mutations that define functional domains of the protein. Further mutational analysis is needed to help define the critical functional domains of the P protein and to allow a definitive diagnosis of OCA2.

Expression of PACE4 in chemically induced carcinomas is associated with spindle cell tumor conversion and increased invasive ability.

Gene expression changes associated with the conversion of squamous cell carcinoma (SCC) to a more advanced malignant spindle cell carcinoma (SPCC) were determined by differential display. Using an animal model of human SCC progression, we provide evidence of increased PACE4 expression in SPCC cell lines and primary tumors induced by chemical carcinogenesis protocols, thus implicating this proprotein convertase in the process of tumor progression. Exogenous overexpression of PACE4 cDNA in mouse SCC cells of low invasive ability resulted in enhanced tumor cell invasiveness that was absent in parental or mock-transfected SCC cells. In addition, the PACE4-transfected cells acquired the ability to process prostromelysin 3 into its active enzyme form. Taken together, these results show that up-regulation of PACE4 expression is associated with SCC conversion to SPCC and suggests that activation of essential PACE4 substrates, such as the metalloproteinase stromelysin 3, is required for tumor cell invasion.

Oculocutaneous albinism in an isolated Tonga community in Zimbabwe.

Oculocutaneous albinism (OCA) is a recessively inherited genetic condition prevalent throughout sub-Saharan Africa. We now describe a cluster of tyrosinase positive OCA (OCA2) cases belonging to the Tonga ethnic group living in the Zambezi valley of northern Zimbabwe. The prevalence in this region was 1 in 1000, which is four times higher than that for the country as a whole. The gene frequency for OCA2 in this population was calculated as 0.0316, with a carrier rate of 1 in 16. Molecular analysis showed that all five affected subjects from two independent families examined were found to be homozygous for an interstitial 2.7 kb deletion mutation commonly found in OCA2 subjects in Africa. An obligate carrier from another family was heterozygous for this deletion allele. Affected subjects in this isolated community suffered health, social, and economic problems.