Yaping Qian

Combined Immune Deficiency in a Patient with a Novel NFKB2 Mutation

NFKB2 encodes the p100/p52 protein, a critical mediator of the canonical and noncanonical NFkB signaling pathways. Here we report the comprehensive immune evaluation of a child with a novel NFKB2 mutation and provide evidence that aberrant NFKB2 signaling not only causes humoral immune deficiency, but also interferes with the TCR-mediated proliferation of T cells. These observations expand the known phenotype associated with NFKB2 mutations.

The struggle to find reliable results in exome sequencing data: filtering out Mendelian errors

Next Generation Sequencing studies generate a large quantity of genetic data in a relatively cost and time efficient manner and provide an unprecedented opportunity to identify candidate causative variants that lead to disease phenotypes. A challenge to these studies is the generation of sequencing artifacts by current technologies. To identify and characterize the properties that distinguish false positive variants from true variants, we sequenced a child and both parents (one trio) using DNA isolated from three sources (blood, buccal cells, and saliva). The trio strategy allowed us to identify variants in the proband that could not have been inherited from the parents (Mendelian errors) and would most likely indicate sequencing artifacts. Quality control measurements were examined and three measurements were found to identify the greatest number of Mendelian errors. These included read depth, genotype quality score, and alternate allele ratio. Filtering the variants on these measurements removed ~95% of the Mendelian errors while retaining 80% of the called variants. These filters were applied independently. After filtering, the concordance between identical samples isolated from different sources was 99.99% as compared to 87% before filtering. This high concordance suggests that different sources of DNA can be used in trio studies without affecting the ability to identify causative polymorphisms. To facilitate analysis of next generation sequencing data, we developed the Cincinnati Analytical Suite for Sequencing Informatics (CASSI) to store sequencing files, metadata (eg. relatedness information), file versioning, data filtering, variant annotation, and identify candidate causative polymorphisms that follow either de novo, rare recessive homozygous or compound heterozygous inheritance models. We conclude the data cleaning process improves the signal to noise ratio in terms of variants and facilitates the identification of candidate disease causative polymorphisms.

Deep Sequencing Reveals Novel Genetic Variants in Children with Acute Liver Failure and Tissue Evidence of Impaired Energy Metabolism

Background & Aims The etiology of acute liver failure (ALF) remains elusive in almost half of affected children. We hypothesized that inherited mitochondrial and fatty acid oxidation disorders were occult etiological factors in patients with idiopathic ALF and impaired energy metabolism. Methods Twelve patients with elevated blood molar lactate/pyruvate ratio and indeterminate etiology were selected from a retrospective cohort of 74 subjects with ALF because their fixed and frozen liver samples were available for histological, ultrastructural, molecular and biochemical analysis. Results A customized next-generation sequencing panel for 26 genes associated with mitochondrial and fatty acid oxidation defects revealed mutations and sequence variants in five subjects. Variants involved the genes ACAD9, POLG, POLG2, DGUOK, and RRM2B; the latter not previously reported in subjects with ALF. The explanted livers of the patients with heterozygous, truncating insertion mutations in RRM2B showed patchy micro- and macrovesicular steatosis, decreased mitochondrial DNA (mtDNA) content <30% of controls, and reduced respiratory chain complex activity; both patients had good post-transplant outcome. One infant with severe lactic acidosis was found to carry two heterozygous variants in ACAD9, which was associated with isolated complex I deficiency and diffuse hypergranular hepatocytes. The two subjects with heterozygous variants of unknown clinical significance in POLG and DGUOK developed ALF following drug exposure. Their hepatocytes displayed abnormal mitochondria by electron microscopy. Conclusion Targeted next generation sequencing and correlation with histological, ultrastructural and functional studies on liver tissue in children with elevated lactate/pyruvate ratio expand the spectrum of genes associated with pediatric ALF.

A Review of DNA Enrichment Technologies

Next-generation sequencing (NGS) technologies, by sequencing hundreds of thousands to millions of DNA templates in parallel, resulted in higher throughput (Gb scale) and lowered sequencing cost (Mardis 2008; Shendure and Ji 2008). This has permitted the definition of entire genome as well as the differences that exist between them. The ultimate goal is to routinely perform whole-genome sequencing to allow us to gain a deeper understanding of genetic variation and to define its role in phenotypic variation and the pathogenesis of complex traits (Mamanova et al. 2010). Due to the cost and time limitations, it is not yet feasible to sequence large numbers of complex genomes. Therefore, a significant effort has focused on the development of “target enrichment” methods, in which genomic regions are selectively captured from a DNA sample before sequencing (Fig. 3.1). This approach is more time- and cost-effective, and the resulting data are considerably less cumbersome to analyze, except in the case of exome capture (Chap. 8). Several approaches to target enrichment have been developed, and the performance parameters vary from one to another: (1) sensitivity, or the percentage of the target bases that are represented by one or more sequence reads; (2) specificity, or the percentage of sequences that map to the intended targets; (3) uniformity, or the variability in sequence coverage across target regions; (4) reproducibility, or how closely results obtained from replicate experiments correlate; (5) cost; (6) ease of use; and (7) amount of DNA required per experiment, or per megabase of target (Mamanova et al. 2010).

Sanger Sequencing Principles, History, and Landmarks

The first DNA sequencing (1968) was performed 15 years after the discovery of the double helix (1953) (Hutchison 2007). However, the chemical method of Maxam and Gilbert and the dideoxy method of Sanger began in the mid-1970s (Fig. 1.1). The profound insights into genetic organization were shown by Nicklen and Coulson with the first complete DNA sequence of phage ϕX174. As sequencing output improved larger molecules greater than 200 kb (human cytomegalovirus) were sequenced and computational analysis and bioinformatics was born. Sequencing efforts reached new heights with the initiation of the US Human Genome Project culminating in the first “sequencing factory” by 1992 (Hutchison 2007). With this effort came the sequencing of the first bacterial genome, by 1995, and other small eubacterial, archaebacterial, and eukaryotic genomes soon thereafter. Published in 2001, the working draft of the human genome sequence was the result of the competition between the public Human Genome Project and Celera Genomics (Fig. 1.1). The new “massively parallel” sequencing methods (Chap. 2) are greatly increasing sequencing capacity, but further innovations are needed to achieve the “thousand dollar genome” that many feel is the prerequisite to personalized genomic medicine (Fig. 1.1). These advances will also allow new approaches to a variety of problems in biology, evolution, and the environment.

Exome Sequencing as a Discovery and Diagnostic Tool

The estimated size of the human genome is 2,872 Mbps consisting of genes and noncoding sequences of DNA. Approximately 1.5 % of the human genome is known to code for proteins and this portion is the exome. This coding sequence has been shown to be more evolutionary-conserved, thus more sensitive to change (Birney et al. 2007). The decreasing cost of sequencing, due to emerging next-generation sequencing (NGS) technologies, provides an opportunity to screen the exome at an affordable cost for gene discovery and diagnostic purposes. The great amount of information generated from the human genome sequencing, 1000 genomes project, HapMap, and whole exome sequencing (WES) projects has allowed us to interpret sequence changes with a higher level of confidence (Abecasis et al. 2012, 2010; Tennessen et al. 2012). To deal with the large sequencing datasets, a variety of bioinformatics tools have been developed to automate the process of annotation and prediction of sequence changes (Wang et al. 2010b). Due to the massive parallel nature of NGS, research and clinical applications of NGS include the sequencing of many genes, as targeted panels, exomes, and even genomes. An increase in published findings has allowed cataloging of polymorphisms and disease-associated mutations at various databases that include the database of single nucleotide polymorphisms (dbSNP), the human gene mutation database (HGMD), ENSEMBL, the 1000 genomes project database (http://www.1000genomes.org/), and the exome sequencing project database (http://evs.gs.washington.edu/EVS/) to mention a few. The large data is evident in dbSNP that has close to 53 million records and the number of new submissions has been exponentially increasing (Wheeler et al. 2007).

Next-Generation–Sequencing-Based Noninvasive Prenatal Diagnosis

Prenatal diagnosis is important part of obstetric practice (Tounta et al. 2011). Traditionally, fetal DNA is obtained by invasive techniques, namely, amniocentesis and chorionic villus sampling. Such invasive procedure leads to a miscarriage rate of about 1 % and is reserved only for high risk pregnancies for specific genetic conditions which include fetal chromosomal aneuploidies and monogenic disorders with relatively high prevalence in the relevant populations. The ultimate goal for early prenatal diagnosis, while decreasing the miscarriage rate, is to employ noninvasive testing using maternal peripheral blood as a source of fetal genetic material (Tounta et al. 2011). Multiple studies indicate that both intact fetal cells and cell-free fetal nucleic acids (cffNA) cross the placenta and can be found in the maternal circulation. Intact fetal cells present an attractive target for noninvasive prenatal diagnosis (NIPD) of fetal chromosomal abnormalities (Lo et al. 1996). Isolation and analysis of fetal cells from maternal circulation have been extensively investigated and several methods for fetal cell enrichment have been developed (Bianchi 1999; Jackson 2003; Sekizawa et al. 2007). However, due to the lack of cells in the maternal circulation and low efficiency of enrichment methods results have not been promising. In addition, it has been challenging to perform Fluorescent In Situ Hybridization (FISH) because of the presence of apoptotic nuclei of fetal cells (Bianchi et al. 1997).

Application of Next-Generation–Sequencing to the Diagnosis of Genetic Disorders: A Brief Overview

Next-generation sequencing (NGS) holds a number of advantages over traditional Sanger sequencing, the most obvious being able to do panel testing in a shorter span of time at a lower cost (Hu et al. 2009). A number of phenotypically similar diseases can have a number of different genetic causes (Hoischen et al. 2010). This genetic heterogeneity, seen in congenital muscular dystrophies (Chap. 6), congenital disorders of glycosylation (CDG), and hearing loss (Chap. 7), can be addressed by NGS by simply sequencing all genes related to specific phenotypes (Rehman et al. 2010; Lim et al. 2011; Valencia et al. 2012). In the case of CDG, NGS is a time- and cost-effective tool for comprehensive mutations screening of metabolic diseases caused by mutations in different genes of a common pathway. In addition, its use can be applied to disorders that have a variable presentation but can raise flags for a certain set of diseases such as mitochondrial defects. One distinct advantage can be seen in the field of cancer genetics where panel testing can save a significant amount of time by reaching a diagnosis (Chan et al. 2012). An interesting application of NGS is in the application of noninvasive prenatal diagnosis of aneuploidies and trisomies 21, 18, and 13 (Chap. 5; Chiu et al. 2008).

A Survey of Next-Generation–Sequencing Technologies

Innovative application of new technologies in research is one of the major factors driving advances in knowledge acquisition. In 1977, Maxam and Gilbert reported an approach in which terminally labeled DNA fragments were subjected to base-specific chemical cleavage and the reaction products were separated by gel electrophoresis (Maxam and Gilbert 1977). In an alternative approach, Sanger described the use of chain-terminating dideoxynucleotide analogs that caused base-specific termination of primed DNA synthesis (Sanger et al. 1977; Chap. 1). Improvements of the Sanger method led to utilization in the research community and eventually in the clinical diagnosis of many genetic disorders (http://www.ncbi.nlm.nih.gov/sites/GeneTests/). In a factory-based format, Sanger sequencing was the method of choice for the first human genome at an estimated cost of

Diagnosis of Inherited Neuromuscular Disorders by Next-Generation–Sequencing

2.7 billion (Fig. 1.1; Chap. 1). In 2008, by comparison, the genome of Dr. James Watson was sequenced over a 2-month period for less than