Integrated CYP2D6 interrogation for multiethnic copy number and tandem allele detection.

AIM: To comprehensively interrogate CYP2D6 by integrating genotyping, copy number analysis and novel strategies to identify CYP2D6*36 and characterize CYP2D6 duplications. METHODS: Genotyping of 16 CYP2D6 alleles, multiplex ligation-dependent probe amplification (MLPA) and CYP2D6*36 and duplication allele-specific genotyping were performed on 427 African-American, Asian, Caucasian, Hispanic, and Ashkenazi Jewish individuals. RESULTS: A novel PCR strategy determined that almost half of all CYP2D6*10 (100C>T) alleles are actually *36 (isolated or in tandem with *10) and all identified duplication alleles were characterized. Integrated results from all testing platforms enabled the refinement of genotype frequencies across all studied populations. CONCLUSION: The polymorphic CYP2D6 gene requires comprehensive interrogation to characterize allelic variation across ethnicities, which was enabled in this study by integrating multiplexed genotyping, MLPA copy number analysis, novel PCR strategies and duplication allele-specific genotyping.

Cytogenomic identification and long-read single molecule real-time (SMRT) sequencing of a Bardet-Biedl Syndrome 9 (BBS9) deletion.

Bardet-Biedl syndrome (BBS) is a recessive disorder characterized by heterogeneous clinical manifestations, including truncal obesity, rod-cone dystrophy, renal anomalies, postaxial polydactyly, and variable developmental delays. At least 20 genes have been implicated in BBS, and all are involved in primary cilia function. We report a 1-year-old male child from Guyana with obesity, postaxial polydactyly on his right foot, hypotonia, ophthalmologic abnormalities, and developmental delay, which together indicated a clinical diagnosis of BBS. Clinical chromosomal microarray (CMA) testing and high-throughput BBS gene panel sequencing detected a homozygous 7p14.3 deletion of exons 1-4 of BBS9 that was encompassed by a 17.5 Mb region of homozygosity at chromosome 7p14.2-p21.1. The precise breakpoints of the deletion were delineated to a 72.8 kb region in the proband and carrier parents by third-generation long-read single molecule real-time (SMRT) sequencing (Pacific Biosciences), which suggested non-homologous end joining as a likely mechanism of formation. Long-read SMRT sequencing of the deletion breakpoints also determined that the aberration included the neighboring RP9 gene implicated in retinitis pigmentosa; however, the clinical significance of this was considered uncertain given the paucity of reported cases with unambiguous RP9 mutations. Taken together, our study characterized a BBS9 deletion, and the identification of this shared haplotype in the parents suggests that this pathogenic aberration may be a BBS founder mutation in the Guyanese population. Importantly, this informative case also highlights the utility of long-read SMRT sequencing to map nucleotide breakpoints of clinically relevant structural variants.

Acute Intermittent Porphyria: Predicted Pathogenicity of HMBS Variants Indicates Extremely Low Penetrance of the Autosomal Dominant Disease.

Acute intermittent porphyria results from hydroxymethylbilane synthase (HMBS) mutations that markedly decrease HMBS enzymatic activity. This dominant disease is diagnosed when heterozygotes have life-threatening acute attacks, while most heterozygotes remain asymptomatic and undiagnosed. Although >400 HMBS mutations have been reported, the prevalence of pathogenic HMBS mutations in genomic/exomic databases, and the actual disease penetrance are unknown. Thus, we interrogated genomic/exomic databases, identified non-synonymous variants (NSVs) and consensus splice-site variants (CSSVs) in various demographic/racial groups, and determined the NSV's pathogenicity by prediction algorithms and in vitro expression assays. Caucasians had the most: 58 NSVs and two CSSVs among ∼92,000 alleles, a 0.00575 combined allele frequency. In silico algorithms predicted 14 out of 58 NSVs as "likely-pathogenic." In vitro expression identified 10 out of 58 NSVs as likely-pathogenic (seven predicted in silico), which together with two CSSVs had a combined allele frequency of 0.00056. Notably, six presumably pathogenic mutations/NSVs in the Human Gene Mutation Database were benign. Compared with the recent prevalence estimate of symptomatic European heterozygotes (∼0.000005), the prevalence of likely-pathogenic HMBS mutations among Caucasians was >100 times more frequent. Thus, the estimated penetrance of acute attacks was ∼1% of heterozygotes with likely-pathogenic mutations, highlighting the importance of predisposing/protective genes and environmental modifiers that precipitate/prevent the attacks.

Long-Read Single Molecule Real-Time Full Gene Sequencing of Cytochrome P450-2D6.

The cytochrome P450-2D6 (CYP2D6) enzyme metabolizes ∼25% of common medications, yet homologous pseudogenes and copy number variants (CNVs) make interrogating the polymorphic CYP2D6 gene with short-read sequencing challenging. Therefore, we developed a novel long-read, full gene CYP2D6 single molecule real-time (SMRT) sequencing method using the Pacific Biosciences platform. Long-range PCR and CYP2D6 SMRT sequencing of 10 previously genotyped controls identified expected star (*) alleles, but also enabled suballele resolution, diplotype refinement, and discovery of novel alleles. Coupled with an optimized variant-calling pipeline, CYP2D6 SMRT sequencing was highly reproducible as triplicate intra- and inter-run nonreference genotype results were completely concordant. Importantly, targeted SMRT sequencing of upstream and downstream CYP2D6 gene copies characterized the duplicated allele in 15 control samples with CYP2D6 CNVs. The utility of CYP2D6 SMRT sequencing was further underscored by identifying the diplotypes of 14 samples with discordant or unclear CYP2D6 configurations from previous targeted genotyping, which again included suballele resolution, duplicated allele characterization, and discovery of a novel allele and tandem arrangement. Taken together, long-read CYP2D6 SMRT sequencing is an innovative, reproducible, and validated method for full-gene characterization, duplication allele-specific analysis, and novel allele discovery, which will likely improve CYP2D6 metabolizer phenotype prediction for both research and clinical testing applications.

Surface vs. solution hybridization: effects of salt, temperature, and probe type.

Hybridization thermodynamics on solid supports are compared with those in solution for two types of hybridization probe, DNA and uncharged morpholino oligonucleotides of identical sequences. Trends in hybridization affinity are discussed with respect to ionic strength, temperature, and surface behavior.

Quantitative and multiplexed DNA methylation analysis using long-read single-molecule real-time bisulfite sequencing (SMRT-BS).

BACKGROUND: DNA methylation has essential roles in transcriptional regulation, imprinting, X chromosome inactivation and other cellular processes, and aberrant CpG methylation is directly involved in the pathogenesis of human imprinting disorders and many cancers. To address the need for a quantitative and highly multiplexed bisulfite sequencing method with long read lengths for targeted CpG methylation analysis, we developed single-molecule real-time bisulfite sequencing (SMRT-BS). RESULTS: Optimized bisulfite conversion and PCR conditions enabled the amplification of DNA fragments up to ~1.5 kb, and subjecting overlapping 625-1491 bp amplicons to SMRT-BS indicated high reproducibility across all amplicon lengths (r=0.972) and low standard deviations (≤0.10) between individual CpG sites sequenced in triplicate. Higher variability in CpG methylation quantitation was correlated with reduced sequencing depth, particularly for intermediately methylated regions. SMRT-BS was validated by orthogonal bisulfite-based microarray (r=0.906; 42 CpG sites) and second generation sequencing (r=0.933; 174 CpG sites); however, longer SMRT-BS amplicons (>1.0 kb) had reduced, but very acceptable, correlation with both orthogonal methods (r=0.836-0.897 and r=0.892-0.927, respectively) compared to amplicons less than ~1.0 kb (r=0.940-0.951 and r=0.948-0.963, respectively). Multiplexing utility was assessed by simultaneously subjecting four distinct CpG island amplicons (702-866 bp; 325 CpGs) and 30 hematological malignancy cell lines to SMRT-BS (average depth of 110X), which identified a spectrum of highly quantitative methylation levels across all interrogated CpG sites and cell lines. CONCLUSIONS: SMRT-BS is a novel, accurate and cost-effective targeted CpG methylation method that is amenable to a high degree of multiplexing with minimal clonal PCR artifacts. Increased sequencing depth is necessary when interrogating longer amplicons (>1.0 kb) and the previously reported bisulfite sequencing PCR bias towards unmethylated DNA should be considered when measuring intermediately methylated regions. Coupled with an optimized bisulfite PCR protocol, SMRT-BS is capable of interrogating ~1.5 kb amplicons, which theoretically can cover ~91% of CpG islands in the human genome.

Charge-neutral morpholino microarrays for nucleic acid analysis.

A principal challenge in microarray experiments is to facilitate hybridization between probe strands on the array with complementary target strands from solution while suppressing any competing interactions that the probes and targets may experience. Synthetic DNA analogs, whose hybridization to targets can exhibit qualitatively different dependence on experimental conditions than for nucleic acid probes, open up an attractive alternative for improving selectivity of array hybridization. Morpholinos (MOs), a class of uncharged DNA analogs, are investigated as microarray probes instead of DNA. MO microarrays were fabricated by contact printing of amino-modified probes onto aldehyde slides. In addition to covalent immobilization, MOs were found to efficiently immobilize through physical adsorption; such physically adsorbed probes could be removed by post-printing washes with surfactant solutions. Hybridization of double-stranded DNA targets to MO microarrays revealed a hybridization maximum at intermediate ionic strengths. The decline in hybridization at lower ionic strengths was attributed to an electrostatic barrier accumulated from hybridized DNA targets, whereas at higher ionic strengths it was attributed to stabilization of target secondary structure in solution. These trends, which illustrate ionic strength tuning of forming on-array relative to solution secondary structure, were supported by a stability analysis of MO/DNA and DNA/DNA duplexes in solution.

Kinetic mechanisms in morpholino-DNA surface hybridization.

Morpholinos (MOs) are DNA analogues whose uncharged nature can bring fundamental advantages to surface hybridization technologies such as DNA microarrays, by using MOs as the immobilized, or "probe", species. Advancement of MO-based diagnostics, however, is challenged by limited understanding of the surface organization of MO molecules and of how this organization impacts hybridization kinetics and thermodynamics. The present study focuses on hybridization kinetics between monolayers of MO probes and DNA targets as a function of the instantaneous extent of hybridization (i.e., duplex coverage), total probe coverage, and ionic strength. Intriguingly, these experiments reveal distinct kinetic stages, none of which are consistent with Langmuir kinetics. The initial stage, in which duplex coverage remains relatively sparse, indicates confluence of two effects: blockage of target access to unhybridized probes by previously formed duplexes and deactivation of the solid support due to consumption of probe molecules. This interpretation is consistent with a surface organization in which unhybridized MO probes localize near the solid support, underneath a layer of MO-DNA duplexes. As duplex coverage builds, provided saturation is not reached first, the initial stage can transition to an unusual regime characterized by near independence of hybridization rate on duplex coverage, followed by a prolonged approach to equilibrium. The possible origins of these more complex latter behaviors are discussed. Comparison with published data for DNA and peptide nucleic acid (PNA) probes is carried out to look for universal trends in kinetics. This comparison reveals qualitative similarities when comparable surface organization of probes is expected. In addition, MO monolayers are found capable of a broad range of reactivities that span reported values for PNA and DNA probes.

Bisulfite-modified target DNA array for aberrant methylation analysis.

Aberrant DNA methylation of CpG islands is among the earliest and most frequent alterations in cancer. It is of great importance to develop simple and high-throughput methods of methylation analysis for earlier cancer diagnosis or the detection of recurrence. In this study, bisulfite-modified target DNA arrays were prepared on positively charged nylon membrane with two different procedures: fixing PCR products and fixing genomic DNA. First, a bisulfite PCR product array was prepared through fixing PCR products amplified in bisulfite sequencing primers from the bisulfite-modified genomic DNA of different clinical samples on membrane. Furthermore, bisulfite-modified genomic DNA of the different samples was directly fixed on membrane to fabricate bisulfite genomic DNA arrays. The two kinds of arrays were hybridized by probes labeled with digoxigenin, and the hybridization signals were obtained through chemiluminescent detection. The methylation statuses of the IGFBP7 gene for breast tumor and normal tissue samples and for normal human blood cell samples were detected successfully by the two procedures. It was shown that the methods are reliable and sensitive and that they have high potential in screening molecular methylation markers from a large number of clinical samples.

Microarray-based methylation analysis using dual-color fluorescence hybridization.

BACKGROUND: Aberrant DNA methylation of CpG sites is among the earliest and most frequent alterations in cancer. It is of great importance to develop simple, high-throughput and quantitative methods for methylation detection. METHODS: A high-throughput methylation analysis method has been developed based on microarray and dual-color fluorescence hybridization. The genomic DNA was treated with bisulfite, resulting in conversion of non-methylated cytosine, but not methylated cytosine, into uracil within CpG islands of interest. PCR products of the treated genomic templates were spotted and immobilized onto a poly-l-lysine coated glass slide to fabricate a microarray and then interrogated by hybridization with dual-color probes to determine the methylation status. The hybridized signals were obtained with a scanner and the results were analyzed with the software Genepix Pro 3.0. RESULTS: The methylation status of the CpG islands of IGFBP7 gene has been successfully evaluated by the microarray method for twenty-seven samples. All the investigated samples, including twenty human breast tumor tissues, six corresponding normal human breast tissues and one liver cell line, all CpG sites were found completely methylated. CONCLUSIONS: The microarray technology has been proven to have potential for high-throughput detection of the methylation status for a given gene in multi-genomic samples, which could be a novel approach for rapidly screening DNA methylation marker for early stage cancer diagnosis.