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 14/58 NSVs as “likely-pathogenic”. In vitro expression identified 10/58 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 to 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.

The 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 non-reference 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 (CYP2D6*36+*41). 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.

BackgroundDNA 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).ResultsOptimized 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.ConclusionsSMRT-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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1572-7) contains supplementary material, which is available to authorized users.

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.