Validation of a cost-efficient multi-purpose SNP panel for disease based research

Background

Here we present convergent methodologies using theoretical calculations, empirical assessment on in-house and publicly available datasets as well as in silico simulations, that validate a panel of SNPs for a variety of necessary tasks in human genetics disease research before resources are committed to larger-scale genotyping studies on those samples. While large-scale well-funded human genetic studies routinely have up to a million SNP genotypes, samples in a human genetics laboratory that are not yet part of such studies may be productively utilized in pilot projects or as part of targeted follow-up work though such smaller scale applications require at least some genome-wide genotype data for quality control purposes such as DNA “barcoding” to detect swaps or contamination issues, determining familial relationships between samples and correcting biases due to population effects such as population stratification in pilot studies.

Principal Findings

Empirical performance in classification of relative types for any two given DNA samples (e.g., full siblings, parental, etc) indicated that for outbred populations the panel performs sufficiently to classify relationship in extended families and therefore also for smaller structures such as trios and for twin zygosity testing. Additionally, familial relationships do not significantly diminish the (mean match) probability of sharing SNP genotypes in pedigrees, further indicating the uniqueness of the “barcode.” Simulation using these SNPs for an African American case-control disease association study demonstrated that population stratification, even in complex admixed samples, can be adequately corrected under a range of disease models using the SNP panel.

Conclusion

The panel has been validated for use in a variety of human disease genetics research tasks including sample barcoding, relationship verification, population substructure detection and statistical correction. Given the ease of genotyping our specific assay contained herein, this panel represents a useful and economical panel for human geneticists.     

Molecular characterization of adeno-associated viruses infecting children

Although adeno-associated virus (AAV) infection is common in humans, the biology of natural infection is poorly understood. Since it is likely that many primary AAV infections occur during childhood, we set out to characterize the frequency and complexity of circulating AAV isolates in fresh and archived frozen human pediatric tissues. Total cellular DNA was isolated from 175 tissue samples including freshly collected tonsils (n = 101) and archived frozen samples representing spleen (n = 21), lung (n = 16), muscle (n = 15), liver (n = 19), and heart (n = 3). Samples were screened for the presence of AAV and adenovirus sequences by PCR using degenerate primers. AAV DNA was detected in 7 of 101 (7%) tonsil samples and two of 74 other tissues (one spleen and one lung). Adenovirus sequences were identified in 19 of 101 tonsils (19%), but not in any other tissues. Complete capsid gene sequences were recovered from all nine AAV-positive tissues. Sequence analyses showed that eight of the capsid sequences were AAV2-like (approximately 98% amino acid identity), while the single spleen isolate was intermediate between serotypes 2 and 3. Comparison to the available AAV2 crystal structure revealed that the majority of the amino acid substitutions mapped to surface-exposed hypervariable domains. To further characterize the AAV capsid structure in these samples, we used a novel linear rolling-circle amplification method to amplify episomal AAV DNA and isolate infectious molecular clones from several human tissues. Serotype 2-like viruses were generated from these DNA clones and interestingly, failed to bind to a heparin sulfate column. Inspection of the capsid sequence from these two clones (and the other six AAV2-like isolates) revealed that they lacked arginine residues at positions 585 and 588 of the capsid protein, which are thought to be essential for interaction with the heparin sulfate proteoglycan coreceptor. These data provide a framework with which to explore wild-type AAV persistence in vivo and provide additional tools to further define the biodistribution and form of AAV in human tissues.     

The dileucine motif at the COOH terminus of human multidrug resistance P-glycoprotein is important for folding but not activity

P-glycoprotein (P-gp, ABCB1) actively transports a broad range of cytotoxic compounds out of the cell. The COOH terminus of P-gp contains a dileucine motif (Leu(1260)-Leu(1261)) and a conserved phenylalanine (Phe(1268)). Similar residues in SUR1 (ABCC8) were reported to be important plasma membrane-targeting signals (Sharma, N., Crane, A., Clement, J. P. t., Gonzalez, G., Babenko, A. P., Bryan, J., and Aguilar-Bryan, L. (1999) J. Biol. Chem. 274, 20628-20632). Here, we used alanine-scanning mutagenesis to test whether these residues were essential for trafficking of P-gp to the cell surface. Mutant L1260A expressed a 150-kDa immature protein that did not reach the cell surface and was sensitive to digestion by Endo H(f). By contrast, mutants L1261A, F1268A, and wild-type P-gps expressed the 170-kDa mature proteins at the cell surface. Mutation of Leu(1260) to Gly, Ile, Trp, Lys, or Glu also resulted in the expression of the 150-kDa immature protein. All of the mutants, however, expressed the 170-kDa protein in the presence of the drug substrate/specific chemical chaperone cyclosporin A. Mutant L1260A P-gp exhibited drug-stimulated ATPase activities similar to that of wild-type enzyme after rescue with cyclosporin A. Deletion of the last 22 amino acids (Q(1259)-Q(1280)) also caused misprocessing. The mutant, however, was rescued by expression in the presence of cyclosporin A and conferred resistance to colchicine in transfected cells. These results show that the dileucine motif is not a plasma membrane targeting signal. The COOH terminus is required for proper folding of P-gp but not for activity.     

Multivalent interactions of calcium/calmodulin-dependent protein kinase II with the postsynaptic density proteins NR2B, densin-180, and alpha-actinin-2

Dendritic calcium/calmodulin-dependent protein kinase II (CaMKII) is dynamically targeted to the synapse. We show that CaMKIIalpha is associated with the CaMKII-binding proteins densin-180, the N-methyl-D-aspartate receptor NR2B subunit, and alpha-actinin in postsynaptic density-enriched rat brain fractions. Residues 819-894 within the C-terminal domain of alpha-actinin-2 constitute the minimal CaMKII-binding domain. Similar amounts of Thr286-autophosphorylated CaMKIIalpha holoenzyme [P-T286]CaMKII bind to alpha-actinin-2 as bind to NR2B (residues 1260-1339) or to densin-180 (residues 1247-1495) in glutathione-agarose cosedimentation assays, even though the CaMKII-binding domains share no amino acid sequence similarity. Like NR2B, alpha-actinin-2 binds to representative splice variants of each CaMKII gene (alpha, beta, gamma, and delta), whereas densin-180 binds selectively to CaMKIIalpha. In addition, C-terminal truncated CaMKIIalpha monomers can interact with NR2B and alpha-actinin-2, but not with densin-180. Soluble alpha-actinin-2 does not compete for [P-T286]CaMKII binding to immobilized densin-180 or NR2B. However, soluble densin-180, but not soluble NR2B, increases CaMKII binding to immobilized alpha-actinin-2 by approximately 10-fold in a PDZ domain-dependent manner. A His6-tagged NR2B fragment associates with GST-densin or GST-actinin but only in the presence of [P-T286]CaMKII. Similarly, His6-tagged densin-180 or alpha-actinin fragments associate with GST-NR2B in a [P-T286]CaMKII-dependent manner. In addition, GST-NR2B and His6-tagged alpha-actinin can bind simultaneously to monomeric CaMKII subunits. In combination, these data support a model in which [P-T286]CaMKIIalpha can simultaneously interact with multiple dendritic spine proteins, possibly stabilizing the synaptic localization of CaMKII and/or nucleating a multiprotein synaptic signaling complex.     

Disulfide cross-linking analysis shows that transmembrane segments 5 and 8 of human P-glycoprotein are close together on the cytoplasmic side of the membrane

Human P-glycoprotein (P-gp) transports a wide variety of structurally diverse compounds out of the cell. Knowledge about the packing of the transmembrane (TM) segments is essential for understanding the mechanism of drug recognition and transport. We used cysteine-scanning mutagenesis and disulfide cross-linking analysis to determine which TM segment in the COOH half of P-gp was close to TMs 5 and 6 since these segments in the NH(2) half are important for drug binding. An active Cys-less P-gp mutant cDNA was used to generate 240 double cysteine mutants that contained 1 cysteine in TMs 5 or 6 and another in TMs 7 or 8. The mutants were subjected to oxidative cross-linking analysis. No disulfide cross-linking was observed in the 140 TM6/TM7 or TM6/TM8 mutants. By contrast, cross-linking was detected in several P-gp TM5/TM8 mutants. At 4 degrees C, when thermal motion is low, P-gp mutants N296C(TM5)/G774C(TM8), I299C(TM5)/F770C(TM8), I299C(TM5)/G774C(TM8), and G300C(TM5)/F770C(TM8) showed extensive cross-linking with oxidant. These mutants retained drug-stimulated ATPase activity, but their activities were inhibited after treatment with oxidant. Similarly, disulfide cross-linking was inhibited by vanadate trapping of nucleotide. These results indicate that significant conformational changes must occur between TMs 5 and 8 during ATP hydrolysis. We revised the rotational symmetry model for TM packing based on our results and by comparison to the crystal structure of MsbA (Chang, G. (2003) J. Mol. Biol. 330, 419-430) such that TM5 is adjacent to TM8, TM2 is adjacent to TM11, and TMs 1 and 7 are next to TMs 6 and 12, respectively.     

Examination of potential overlap in autism and language loci on chromosomes 2, 7, and 13 in two independent samples ascertained for specific language impairment

Specific language impairment is a neurodevelopmental disorder characterized by impairments essentially restricted to the domain of language and language learning skills. This contrasts with autism, which is a pervasive developmental disorder defined by multiple impairments in language, social reciprocity, narrow interests and/or repetitive behaviors. Genetic linkage studies and family data suggest that the two disorders may have genetic components in common. Two samples, from Canada and the US, selected for specific language impairment were genotyped at loci where such common genes are likely to reside. Significant evidence for linkage was previously observed at chromosome 13q21 in our Canadian sample (HLOD 3.56) and was confirmed in our US sample (HLOD 2.61). Using the posterior probability of linkage (PPL) to combine evidence for linkage across the two samples yielded a PPL over 92%. Two additional loci on chromosome 2 and 7 showed weak evidence for linkage. However, a marker in the cystic fibrosis transmembrane conductance regulator (7q31) showed evidence for association to SLI, confirming results from another group (O'Brien et al. 2003). Our results indicate that using samples selected for components of the autism phenotype may be a useful adjunct to autism genetics.     

Rapid determination of the synthetic pyrethroid insecticide, deltamethrin, in rat plasma and tissues by HPLC

Deltamethrin (DLM), [(S)-alpha-cyano-d-phenoxybenzyl-(1R,3R)-e-(2,2 dibromovinyl)-2,2-dimethylcyclo-propane-1-carboxylate], is a pyrethroid insecticide widely used in agriculture and households. There are several methods for analysis of DLM in biological fluids and tissues, but these methods are time consuming. They generally involve the extraction of DLM with lipid-soluble solvents such as n-pentane, n-hexane, diethylether or acetone, and subsequent evaporation of the solvent. A more rapid and sensitive high-performance liquid chromatography (HPLC) method to analyze DLM in plasma and tissues (liver, kidney, and brain) was developed and validated according to U.S. Food and Drug Administration (U.S. FDA) and International Conference on Harmonization (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines. The limit of detection (S/N of 3/1) for DLM was 0.01 microg/ml for plasma, liver, kidney and brain. The method performances were shown to be selective for DLM and linear over the concentration range 0.01-20.0 microg/ml. For five replications of samples at 0.05, 0.1, 0.2, 1.5 and 4.0 microg/ml, intraday precision and accuracy values were in the range of 0.7-13.1% relative standard deviation (%R.S.D.) and 1.8-14.1%Error, respectively. Interday (n = 15) precision and accuracy values at 0.05, 0.1, 0.2, 1.5, and 4.0 microg/ml were in the range of 3.2-15.2% (%R.S.D.) and 3.7-14.8%Error, respectively. The absolute recoveries of DLM ranged from 93 to 103% for plasma, 95 to 114% for liver, 97 to 108% for kidney, and 95 to 108% for brain. This method can be quite useful for DLM pharmacokinetic and tissue distribution studies, for which multiple plasma and tissue samples have to be analyzed quickly with high reproducibility.     

Pulsatile flow and mass transport over an array of cylinders: gas transfer in a cardiac-driven artificial lung

The pulsatile flow and gas transport of a Newtonian passive fluid across an array of cylindrical microfibers are numerically investigated. It is related to an implantable, artificial lung where the blood flow is driven by the right heart. The fibers are modeled as either squared or staggered arrays. The pulsatile flow inputs considered in this study are a steady flow with a sinusoidal perturbation and a cardiac flow. The aims of this study are twofold: identifying favorable array geometry/spacing and system conditions that enhance gas transport; and providing pressure drop data that indicate the degree of flow resistance or the demand on the right heart in driving the flow through the fiber bundle. The results show that pulsatile flow improves the gas transfer to the fluid compared to steady flow. The degree of enhancement is found to be significant when the oscillation frequency is large, when the void fraction of the fiber bundle is decreased, and when the Reynolds number is increased; the use of a cardiac flow input can also improve gas transfer. In terms of array geometry, the staggered array gives both a better gas transfer per fiber (for relatively large void fraction) and a smaller pressure drop (for all cases). For most cases shown, an increase in gas transfer is accompanied by a higher pressure drop required to power the flow through the device.