Development of DNA Confirmatory and High-Risk Diagnostic Testing for Newborns Using Targeted Next-Generation DNA Sequencing

PurposeGenetic testing is routinely used for second-tier confirmation of newborn sequencing results to rule out false positives and to confirm diagnoses in newborns undergoing inpatient and outpatient care. We developed a targeted next-generation sequencing panel coupled with a variant processing pipeline and demonstrated utility and performance benchmarks across multiple newborn disease presentations in a retrospective clinical study.MethodsThe test utilizes an in silico gene filter that focuses directly on 126 genes related to newborn screening diseases and is applied to the exome or a next-generation sequencing panel called NBDx. NBDx targets the 126 genes and additional newborn-specific disorders. It integrates DNA isolation from minimally invasive biological specimens, targeted next-generation screening, and rapid characterization of genetic variation.ResultsWe report a rapid parallel processing of 8 to 20 cases within 105 hours with high coverage on our NBDx panel. Analytical sensitivity of 99.8% was observed across known mutation hotspots. Concordance calls with or without clinical summaries were 94% and 75%, respectively.ConclusionRapid, automated targeted next-generation sequencing and analysis are practical in newborns for second-tier confirmation and neonatal intensive care unit diagnoses, laying a foundation for future primary DNA-based molecular screening of additional disorders and improving existing molecular testing options for newborns.Genet Med 17 5, 337–347.     

Next-generation sequencing for the diagnosis of hereditary breast and ovarian cancer using genomic capture targeting multiple candidate genes

To optimize the molecular diagnosis of hereditary breast and ovarian cancer (HBOC), we developed a next-generation sequencing (NGS)-based screening based on the capture of a panel of genes involved, or suspected to be involved in HBOC, on pooling of indexed DNA and on paired-end sequencing in an Illumina GAIIx platform, followed by confirmation by Sanger sequencing or MLPA/QMPSF. The bioinformatic pipeline included CASAVA, NextGENe, CNVseq and Alamut-HT. We validated this procedure by the analysis of 59 patients'' DNAs harbouring SNVs, indels or large genomic rearrangements of BRCA1 or BRCA2. We also conducted a blind study in 168 patients comparing NGS versus Sanger sequencing or MLPA analyses of BRCA1 and BRCA2. All mutations detected by conventional procedures were detected by NGS. We then screened, using three different versions of the capture set, a large series of 708 consecutive patients. We detected in these patients 69 germline deleterious alterations within BRCA1 and BRCA2, and 4 TP53 mutations in 468 patients also tested for this gene. We also found 36 variations inducing either a premature codon stop or a splicing defect among other genes: 5/708 in CHEK2, 3/708 in RAD51C, 1/708 in RAD50, 7/708 in PALB2, 3/708 in MRE11A, 5/708 in ATM, 3/708 in NBS1, 1/708 in CDH1, 3/468 in MSH2, 2/468 in PMS2, 1/708 in BARD1, 1/468 in PMS1 and 1/468 in MLH3. These results demonstrate the efficiency of NGS in performing molecular diagnosis of HBOC. Detection of mutations within other genes than BRCA1 and BRCA2 highlights the genetic heterogeneity of HBOC.     

CACNA1C haploinsufficiency accounts for the common features of interstitial 12p13.33 deletion carriers

We identified a de novo 44.7 Kb interstitial 12p13.33 micro-deletion that involves solely the first exon of the CACNA1C (MIM 114205), using microarray-based comparative genomic hybridization (aCGH). The associated main phenotype is characterized by expressive language impairment, tremors, fine motor-skills delay, muscular hypotonia, and joint laxity. A careful comparison between the clinical and genomic characteristics between our proband and 20 previously reported patients, led us to propose CACNA1C haploinsufficiency as the main cause of both expressive language delay and motor-skills impairment. Pathogenic variants of CACNA1C have been associated to a plethora of clinical phenotypes, such as Timothy syndrome (TS, OMIM 601005), Brugada syndrome (BRGDA3, OMIM 611875) and a variety of neuropsychiatric disorders (bipolar disorder, major depression, schizophrenia, autism spectrum disorder, psychotic manifestations). In this report we describe a 12p13.33 micro-deletion involving one coding gene only, in contrast with previous studies that mostly concluded that a multi-genes deletion in the 12p13.33 sub-telomeric region is responsible of the minimum clinical phenotype of patients with 12p13.33 monosomy. Certainly, larger deletions spanning multiple Mb in 12p13.33 are responsible for more severe phenotypes, associated to a variable degree of dysmorphic features.     

Treacher Collins syndrome: a clinical and molecular study based on a large series of patients

PurposeTreacher Collins/Franceschetti syndrome (TCS; OMIM 154500) is a disorder of craniofacial development belonging to the heterogeneous group of mandibulofacial dysostoses. TCS is classically characterized by bilateral mandibular and malar hypoplasia, downward-slanting palpebral fissures, and microtia. To date, three genes have been identified in TCS:,TCOF1, POLR1D, and POLR1C.MethodsWe report a clinical and extensive molecular study, including TCOF1, POLR1D, POLR1C, and EFTUD2 genes, in a series of 146 patients with TCS. Phenotype–genotype correlations were investigated for 19 clinical features, between TCOF1 and POLR1D, and the type of mutation or its localization in the TCOF1 gene.ResultsWe identified 92/146 patients (63%) with a molecular anomaly within TCOF1, 9/146 (6%) within POLR1D, and none within POLR1C. Among the atypical negative patients (with intellectual disability and/or microcephaly), we identified four patients carrying a mutation in EFTUD2 and two patients with 5q32 deletion encompassing TCOF1 and CAMK2A in particular. Congenital cardiac defects occurred more frequently among patients with TCOF1 mutation (7/92, 8%) than reported in the literature.ConclusionEven though TCOF1 and POLR1D were associated with extreme clinical variability, we found no phenotype–genotype correlation. In cases with a typical phenotype of TCS, 6/146 (4%) remained with an unidentified molecular defect.     

Applicability of partial characterization of cytochrome oxidase I in identification of forensically important flies (Diptera) from China and Egypt

Precise species identification of every insect sample collected from criminal scenes play an essential role in the accurate estimation of postmortem interval. The morphological similarity poses a great challenge for forensic entomologists. DNA-based method can be used as a supplemental means of morphological method. In the present study, we demonstrate the applicability of the 304-bp cytochrome oxidase I gene fragment in molecular identification of forensically important Diptera. We analyzed 75 specimens belonging to 19 species of 3 families originating from China (Calliphoridae: Chrysomya megacephala, Chrysomya albiceps, Chrysomya rufifacies, Chrysomya nigripes, Aldrichina grahami; Lucilia bazini, Lucilia caesar, Lucilia cuprina, Lucilia sericata, Lucilia porphyrina; Muscidae: Musca autumnalis, Musca domestica, Fannia canicularis, Stomoxys calcitrans; Sarcophagidae: Sarcophaga albiceps, Sarcophaga dux, Helicophagella melanura) and Egypt (Calliphoridae: C. megacephala, C. albiceps, L. sericata; Muscidae: M. domestica, F. canicularis, S. calcitrans, Synthesiomyia nudiseta; Sarcophagidae: Sarcophaga argyrostoma). This region was amplified using polymerase chain reaction followed by direct sequencing of the amplification products. Nucleotide sequence divergences were calculated using the Kimura two-parameter distance model and a neighbor-joining phylogenetic tree generated. Intraspecific variation ranged from 0–0.8 % and interspecific variation occurred between 1–19 %. Although all examined specimens were assigned to the correct species and formed distinct monophyletic clades, the data of the phylogenetic analysis were not completely in accordance with the traditional morphological classification. As both C. nigripes and A. grahami unexpectedly joined with Muscidae and Sarcophagidae groups respectively. Moreover, both Calliphorinae and Luciliinae clades failed to represent Calliphoridae as a separate group. Therefore, although molecular methods are beneficial especially if the available evidence are immature stage or degraded fragments of insect, we suggest using this fragment only in addition to other identification methods.     

Under the mask of Kabuki syndrome: Elucidation of genetic-and phenotypic heterogeneity in patients with Kabuki-like phenotype

Kabuki syndrome is mainly caused by dominant de-novo pathogenic variants in the KMT2D and KDM6A genes. The clinical features of this syndrome are highly variable, making the diagnosis of Kabuki-like phenotypes difficult, even for experienced clinical geneticists. Herein we present molecular genetic findings of causal genetic variation using array comparative genome hybridization and a Mendeliome analysis, utilizing targeted exome analysis focusing on regions harboring rare disease-causing variants in Kabuki-like patients which remained KMT2D/KDM6A-negative. The aCGH analysis revealed a pathogenic CNV in the 14q11.2 region, while targeted exome sequencing revealed pathogenic variants in genes associated with intellectual disability (HUWE1, GRIN1), including a gene coding for mandibulofacial dysostosis with microcephaly (EFTUD2). Lower values of the MLL2-Kabuki phenotypic score are indicative of Kabuki-like phenotype (rather than true Kabuki syndrome), where aCGH and Mendeliome analyses have high diagnostic yield. Based on our findings we conclude that for new patients with Kabuki-like phenotypes it is possible to choose a specific molecular testing approach that has the highest detection rate for a given MLL2-Kabuki score, thus fostering more precise patient diagnosis and improved management in these genetically- and phenotypically heterogeneous clinical entities.     

Prevalence of mutations in a panel of breast cancer susceptibility genes in BRCA1/2-negative patients with early-onset breast cancer

PurposeClinical testing for germ-line variation in multiple cancer susceptibility genes is available using massively parallel sequencing. Limited information is available for pretest genetic counseling regarding the spectrum of mutations and variants of uncertain significance in defined patient populations.MethodsWe performed massively parallel sequencing using targeted capture of 22 cancer susceptibility genes in 278 BRCA1/2-negative patients with early-onset breast cancer (diagnosed at younger than 40 years of age).ResultsThirty-one patients (11%) were found to have at least one deleterious or likely deleterious variant. Seven patients (2.5% overall) were found to have deleterious or likely deleterious variants in genes for which clinical guidelines exist for management, namely TP53 (4), CDKN2A (1), MSH2 (1), and MUTYH (double heterozygote). Twenty-four patients (8.6%) had deleterious or likely deleterious variants in a cancer susceptibility gene for which clinical guidelines are lacking, such as CHEK2 and ATM. Fifty-four patients (19%) had at least one variant of uncertain significance, and six patients were heterozygous for a variant in MUTYH.ConclusionThese data demonstrate that massively parallel sequencing identifies reportable variants in known cancer susceptibility genes in more than 30% of patients with early-onset breast cancer. However, only few patients (2.5%) have definitively actionable mutations given current clinical management guidelines.Genet Med17 8, 630–638.     

Caution in interpretation of disease causality for heterozygous loss-of-function variants in the MYH8 gene associated with autosomal dominant disorder

To date, the NM_002472.2(MYH8):c.2021G>A (p.Arg674Gln) missense variant in the MYH8 gene is the only known genetic change in individuals with autosomal dominant trismus-pseudocamptodactyly syndrome with unknown molecular mechanism. Next-generation sequencing (NGS), including targeted gene panels and whole-exome sequencing, is routinely performed in many clinical diagnostic laboratories as standard-of-care testing aimed at identifying disease-causing genomic variants. Whole-exome sequencing has revealed loss-of-function variants in the MYH8 gene. To properly classify the MYH8 loss-of-function variants, we either retrieved them from public databases or retrospectively collected them from individuals genetically tested by custom NGS panels or by whole-exome sequencing and confirmed using Sanger sequencing. We further evaluated the respective clinical presentations of these individuals with the MYH8 loss-of-function variants. Heterozygous loss-of-function variants in the MYH8 gene were detected in 16 individuals without trismus-pseudocamptodactyly syndrome. Four of these 16 individuals had a pathogenic or likely pathogenic variant detected in another gene that could explain their clinical presentation. Moreover, there are ∼100 MYH8 heterozygous protein-truncating and splice site variants in the ExAC database in different populations. Our results, combined with the population data, indicate that loss-of-function variants in the MYH8 gene do not cause autosomal dominant trismus-pseudocamptodactyly syndrome, and the clinical significance of these variants remains unknown at present. This result highlights the importance of considering the molecular mechanism of disease, variants published in the medical literature, and population genomic data for the correct interpretation of loss-of-function variants in genes associated with autosomal dominant diseases.     

Identity-by-descent–guided mutation analysis and exome sequencing in consanguineous families reveals unusual clinical and molecular findings in retinal dystrophy

PurposeAutosomal recessive retinal dystrophies are clinically and genetically heterogeneous, which hampers molecular diagnosis. We evaluated identity-by-descent–guided Sanger sequencing or whole-exome sequencing in 26 families with nonsyndromic (19) or syndromic (7) autosomal recessive retinal dystrophies to identify disease-causing mutations.MethodsPatients underwent genome-wide identity-by-descent mapping followed by Sanger sequencing (16) or whole-exome sequencing (10). Whole-exome sequencing data were filtered against identity-by-descent regions and known retinal dystrophy genes. The medical history was reviewed in mutation-positive families.ResultsWe identified mutations in 14 known retinal dystrophy genes in 20/26 (77%) families: ABCA4, CERKL, CLN3, CNNM4, C2orf71, IQCB1, LRAT, MERTK, NMNAT1, PCDH15, PDE6B, RDH12, RPGRIP1, and USH2A. Whole-exome sequencing in single individuals revealed mutations in either the largest or smaller identity-by-descent regions, and a compound heterozygous genotype in NMNAT1. Moreover, a novel deletion was found in PCDH15. In addition, we identified mutations in CLN3, CNNM4, and IQCB1 in patients initially diagnosed with nonsyndromic retinal dystrophies.ConclusionOur study emphasized that identity-by-descent–guided mutation analysis and/or whole-exome sequencing are powerful tools for the molecular diagnosis of retinal dystrophy. Our approach uncovered unusual molecular findings and unmasked syndromic retinal dystrophies, guiding future medical management. Finally, elucidating ABCA4, LRAT, and MERTK mutations offers potential gene-specific therapeutic perspectives.     

Isolation and Enrichment of Cryptosporidium DNA and Verification of DNA Purity for Whole-Genome Sequencing

Whole-genome sequencing of Cryptosporidium spp. is hampered by difficulties in obtaining sufficient, highly pure genomic DNA from clinical specimens. In this study, we developed procedures for the isolation and enrichment of Cryptosporidium genomic DNA from fecal specimens and verification of DNA purity for whole-genome sequencing. The isolation and enrichment of genomic DNA were achieved by a combination of three oocyst purification steps and whole-genome amplification (WGA) of DNA from purified oocysts. Quantitative PCR (qPCR) analysis of WGA products was used as an initial quality assessment of amplified genomic DNA. The purity of WGA products was assessed by Sanger sequencing of cloned products. Next-generation sequencing tools were used in final evaluations of genome coverage and of the extent of contamination. Altogether, 24 fecal specimens of Cryptosporidium parvum, C. hominis, C. andersoni, C. ubiquitum, C. tyzzeri, and Cryptosporidium chipmunk genotype I were processed with the procedures. As expected, WGA products with low (<16.0) threshold cycle (C_T) values yielded mostly Cryptosporidium sequences in Sanger sequencing. The cloning-sequencing analysis, however, showed significant contamination in 5 WGA products (proportion of positive colonies derived from Cryptosporidium genomic DNA, ≤25%). Following this strategy, 20 WGA products from six Cryptosporidium species or genotypes with low (mostly <14.0) C_T values were submitted to whole-genome sequencing, generating sequence data covering 94.5% to 99.7% of Cryptosporidium genomes, with mostly minor contamination from bacterial, fungal, and host DNA. These results suggest that the described strategy can be used effectively for the isolation and enrichment of Cryptosporidium DNA from fecal specimens for whole-genome sequencing.     

Exploring concordance and discordance for return of incidental findings from clinical sequencing

PurposeThe aim of this study was to explore specific conditions and types of genetic variants that specialists in genetics recommend should be returned as incidental findings in clinical sequencing.MethodsSixteen specialists in clinical genetics and/or molecular medicine selected variants in 99 common conditions to return to the ordering physician if discovered incidentally through whole-genome sequencing. For most conditions, the specialists independently considered three molecular scenarios for both adults and minor children: a known pathogenic mutation, a truncating variant presumed pathogenic (where other truncating variants are known to be pathogenic), and a missense variant predicted in silico to be pathogenic.ResultsOn average, for adults and children, respectively, each specialist selected 83.5 and 79.0 conditions or genes of 99 in the known pathogenic mutation categories, 57.0 and 53.5 of 72 in the truncating variant categories, and 33.4 and 29.7 of 72 in the missense variant categories. Concordance in favor of disclosure within the adult/known pathogenic mutation category was 100% for 21 conditions or genes and 80% or higher for 64 conditions or genes.ConclusionSpecialists were highly concordant for the return of findings for 64 conditions or genes if discovered incidentally during whole-exome sequencing or whole-genome sequencing.Genet Med 2012:14(4):405–410     

Targeted next-generation sequencing makes new molecular diagnoses and expands genotype–phenotype relationship in Ehlers–Danlos syndrome

PurposeEhlers–Danlos syndrome (EDS) comprises a group of overlapping hereditary disorders of connective tissue with significant morbidity and mortality, including major vascular complications. We sought to identify the diagnostic utility of a next-generation sequencing (NGS) panel in a mixed EDS cohort.MethodsWe developed and applied PCR-based NGS assays for targeted, unbiased sequencing of 12 collagen and aortopathy genes to a cohort of 177 unrelated EDS patients. Variants were scored blind to previous genetic testing and then compared with results of previous Sanger sequencing.ResultsTwenty-eight pathogenic variants in COL5A1/2, COL3A1, FBN1, and COL1A1 and four likely pathogenic variants in COL1A1, TGFBR1/2, and SMAD3 were identified by the NGS assays. These included all previously detected single-nucleotide and other short pathogenic variants in these genes, and seven newly detected pathogenic or likely pathogenic variants leading to clinically significant diagnostic revisions. Twenty-two variants of uncertain significance were identified, seven of which were in aortopathy genes and required clinical follow-up.ConclusionUnbiased NGS-based sequencing made new molecular diagnoses outside the expected EDS genotype–phenotype relationship and identified previously undetected clinically actionable variants in aortopathy susceptibility genes. These data may be of value in guiding future clinical pathways for genetic diagnosis in EDS.Genet Med 18 11, 1119–1127.     

Read clouds uncover variation in complex regions of the human genome

Although an increasing amount of human genetic variation is being identified and recorded, determining variants within repeated sequences of the human genome remains a challenge. Most population and genome-wide association studies have therefore been unable to consider variation in these regions. Core to the problem is the lack of a sequencing technology that produces reads with sufficient length and accuracy to enable unique mapping. Here, we present a novel methodology of using read clouds, obtained by accurate short-read sequencing of DNA derived from long fragment libraries, to confidently align short reads within repeat regions and enable accurate variant discovery. Our novel algorithm, Random Field Aligner (RFA), captures the relationships among the short reads governed by the long read process via a Markov Random Field. We utilized a modified version of the Illumina TruSeq synthetic long-read protocol, which yielded shallow-sequenced read clouds. We test RFA through extensive simulations and apply it to discover variants on the NA12878 human sample, for which shallow TruSeq read cloud sequencing data are available, and on an invasive breast carcinoma genome that we sequenced using the same method. We demonstrate that RFA facilitates accurate recovery of variation in 155 Mb of the human genome, including 94% of 67 Mb of segmental duplication sequence and 96% of 11 Mb of transcribed sequence, that are currently hidden from short-read technologies.Although next-generation sequencing (NGS) technologies have enabled whole-genome sequencing (WGS) of many individuals to identify variation, current large-scale and cost-effective resequencing platforms produce reads of limited length (Shendure and Ji 2008; Metzker 2010); and as a result, variant identification within repeated sequences remains challenging. The 1000 Genomes Project Consortium has reported that nearly 6% of the GRCh37 human genome reference is inaccessible by short-read technologies (The 1000 Genomes Project Consortium 2012). Further studies have shown that as much as 10% of GRCh37 cannot be aligned to for the purpose of accurate variant discovery (Lee and Schatz 2012).The portion of the human genome that is currently dark to short-read technologies is significant in both its size and phenotypic effect. Recent segmental duplications (also referred to as low copy repeats), consisting of regions >5 kbp in size and >94% sequence identity, have been identified as making up 130.5 Mb, or ∼4.35% of the human genome (Bailey et al. 2002). These regions tend to be hotspots of structural and copy number variants (CNVs) (Coe et al. 2014; Chaisson et al. 2015) that in aggregate affect a larger fraction of the genome than that affected by single nucleotide polymorphisms (SNPs) (Conrad et al. 2010). CNVs have been associated with diseases such as autism (Sebat et al. 2007; Pinto et al. 2010), Crohn''s disease (Wellcome Trust Case Control Consortium et al. 2010), schizophrenia (Stefansson et al. 2008; McCarthy et al. 2009), and neurocognitive disorders (Coe et al. 2014). However, current short-read technologies are unable to identify precise nucleotide variation in these regions.In principle, longer sequencing reads provide an opportunity to disambiguate repeated sequences. Technologies such as Pacific Biosciences (PacBio) (McCarthy 2010) and Oxford Nanopore (Ashton et al. 2014) produce long reads, but at much higher per-base error rate. PacBio has been leveraged for improved bacterial reference genome assemblies (Koren et al. 2013) and for targeted de novo assembly of the complex 1.3 Mb of 17q21.31 (Huddleston et al. 2014). However, these technologies are currently substantially lower in throughput and higher in cost than short-read technologies and so cannot currently be used to cost-effectively uncover variation in repeated regions of the genome.An alternative approach used in LFR (Peters et al. 2012), CPT-seq (Amini et al. 2014), and Illumina TruSeq Synthetic Long-Reads (previously known as Moleculo) (Kuleshov et al. 2014) utilizes accurate short-read sequencing of long DNA fragments in order to obtain long-range information at high nucleotide accuracy. The Illumina TruSeq protocol is able to produce 10-kbp long reads, retaining the benefits of the highly accurate and cost-effective Illumina technology (Kuleshov et al. 2014) and enabling human genome phasing (Kuleshov et al. 2014) and de novo assembly of complex genomes (Voskoboynik et al. 2013; McCoy et al. 2014).Under Illumina''s synthetic long-read protocol, DNA sequencing libraries are prepared as follows: First, the genomic DNA is sheared into long (≥10 kbp) fragments and ligated with amplification adapters at both ends; second, these molecules are diluted into wells so that each well receives only a small fraction (1%–2%) of the genome; third, molecules are amplified, sheared into short fragments, and uniquely barcoded within each well (Kuleshov et al. 2014). The individual wells are then pooled and sequenced together. Demultiplexing the resulting reads by well barcode and aligning them to the reference genome yields clusters of short reads, which we call read clouds, each of which originated from a single long DNA molecule (Fig. 1A). Additionally, short reads that originate from the endpoints of a read cloud will overlap the original adapters ligated to the long molecules and serve as end-markers of the original long molecule.Open in a separate windowFigure 1.Read clouds (RC) and synthetic long reads (SLR) obtained by Illumina TruSeq Synthetic Long-Read sequencing. Each well initially contains long molecules that represent a small fraction of the target genome; reads from each long molecule are separated in genomic coordinates within the target genome, and therefore, clusters of such reads (read clouds) are formed with each cluster originating from one source fragment. Blue reads denote end-markers of the source fragments and may not always be present as sequenced short reads. (A) In the RC approach, long fragments from several wells w_n are sequenced to a shallow depth and aligned to the reference to obtain read clouds. Pooling of reads across several read clouds allows inference of the variation in the underlying long fragments. (B) In the SLR approach, long fragments are sequenced to a much higher depth to enable de novo assembly of synthetic long reads. For the same total sequencing budget C, the RC approach covers proportionally more target genome space than the SLR approach.A read cloud approach has two key parameters for genome coverage (Fig. 1): coverage of the genome with long DNA fragments, C_F, and coverage of each long fragment with short reads, C_R. The total sequencing depth is then C = C_F × C_R. The choice of C_F and C_R for a given short-read sequencing budget C heavily influences the ability of the read cloud approach to accurately discover variation within a target genome. Both C_F and C have to be sufficiently high; in particular, C_F has to be high enough so that both haplotypes of a diploid genome are covered with a sufficient number of long fragments (Lander and Waterman 1988). The original protocol (McCoy et al. 2014) required for each well to be sequenced at a high depth (C_R = 50×) in order to first de novo assemble synthetic long reads (SLR) of the original source long fragments (Fig. 1B; Voskoboynik et al. 2013). However, performing WGS with this approach requires an exorbitant amount of total sequencing in order to obtain a sufficiently high C_F. For example, if C_R = 50× (Voskoboynik et al. 2013) and C_F = 20×, C = 50 × 20 = 1000×, or the equivalent of 33 whole human genomes sequenced at the currently standard 30× coverage.The alternative strategy to true SLR approaches is to bypass the requirement for actual assembly of the original long fragments and to minimize short-read coverage (C_R ≤ 2×). This strategy allows a sufficiently high C_F in order to cover a genome at a reasonable coverage budget C. Choosing C_R = 1.5 and C_F = 20×, C = 1.5 × 20 = 30×, would yield valuable long-range information for the same total sequencing cost as the currently standard short-read WGS approach.In this work, we present RFA (Random Field Aligner), a novel methodology that utilizes the high C_F, low C_R read cloud approach to confidently map short reads within repetitive regions. In RFA, we directly model the short-read generative process from source long molecules in order to capture the dependencies of short reads through the hidden source long molecules. Using this probabilistic approach, we reduce the problem of finding optimal short-read alignments to optimizing a Markov Random Field (MRF). The resulting alignments tend to cluster the mapped reads into read clouds that fit the properties of the synthetic long-read sequencing protocol. The model naturally favors alignment of a read cloud to the specific copy of a repeated sequence that minimizes the sequence variation of the read cloud to the copy.To our knowledge, RFA is the first attempt to take advantage of the long-range information present in shallow read cloud sequencing to improve the resulting short-read alignments and also to use read clouds to directly genotype an individual. Prior implementations of read clouds to provide molecular-phased genotypes for a single individual require known genotypes as input (Kitzman et al. 2011; Amini et al. 2014; Kuleshov et al. 2014) and typically align the resulting read clouds with standard short-read aligners in order to observe the allele at a known SNV within each read cloud. As genotypes are typically determined with a standard whole-genome 30× shotgun sequencing, in which a short-read workflow would be used, variants in complex regions would remain unresolved.We demonstrate the utility of our approach using shallow-sequenced read clouds (C_R = 1.5×) obtained from the Illumina TruSeq synthetic long-read protocol (henceforth referred to as TruSeq read clouds to avoid confusion with the Illumina product that uses deep sequencing to assemble synthetic long reads). We tested our approach on simulated read cloud wells, on TruSeq read cloud libraries for the cell line GM12878 for which assembled synthetic long reads are also available for direct validation (Genomes Moleculo NA12878, 2014, ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/phase3/integrated_sv_map/supporting/NA12878/moleculo/), and on a high coverage cancer sample that we sequenced. Evaluation of the results confirmed that our method accurately recovers precise nucleotide variation within a significant fraction of the human genome that was previously dark to current short-read technologies. We are able to leverage the read cloud strategy to recover this variation at a fraction of the cost of the original protocol and eliminate the need for first assembling synthetic long reads.     

High frequency of mosaic pathogenic variants in genes causing epilepsy-related neurodevelopmental disorders

PurposeMosaicism probably represents an underreported cause of genetic disorders due to detection challenges during routine molecular diagnostics. The purpose of this study was to evaluate the frequency of mosaicism detected by next-generation sequencing in genes associated with epilepsy-related neurodevelopmental disorders.MethodsWe conducted a retrospective analysis of 893 probands with epilepsy who had a multigene epilepsy panel or whole-exome sequencing performed in a clinical diagnostic laboratory and were positive for a pathogenic or likely pathogenic variant in one of nine genes (CDKL5, GABRA1, GABRG2, GRIN2B, KCNQ2, MECP2, PCDH19, SCN1A, or SCN2A). Parental results were available for 395 of these probands.ResultsMosaicism was most common in the CDKL5, PCDH19, SCN2A, and SCN1A genes. Mosaicism was observed in GABRA1, GABRG2, and GRIN2B, which previously have not been reported to have mosaicism, and also in KCNQ2 and MECP2. Parental mosaicism was observed for pathogenic variants in multiple genes including KCNQ2, MECP2, SCN1A, and SCN2A.ConclusionMosaic pathogenic variants were identified frequently in nine genes associated with various neurological conditions. Given the potential clinical ramifications, our findings suggest that next-generation sequencing diagnostic methods may be utilized when testing these genes in a diagnostic laboratory.     

Voriconazole resistance genes in Aspergillus flavus clinical isolates

ObjectiveThe present study was designed to discover novel biomarkers involved in voriconazole resistance in clinical isolates of Aspergillus flavus.Materials and methodsTwo voriconazole non-wild-type and two voriconazole-wild-type A. flavus clinical isolates were selected to evaluate possible molecular mechanism involved in A. flavus resistance to voriconazole using the mutation assessment, Quantitative real- time PCR of cyp51A and cyp51C genes and complementary DNA- amplified fragment length polymorphism technique.ResultsNo mutations were seen in the cyp51A and cyp51C genes in voriconazole non-wild-type isolates compared to wild- type and reference strains. Regarding to mRNA expression results, no changes were observed in expression fold of cyp51A and cyp51C mRNA expression level in first non- wild- type isolate compared to wild-type isolate. For second isolate cyp51C mRNA expression level was down regulated (5.6 fold). The set of genes including ABC fatty acid transporter XM- 002375835 and aldehydereductase XM- 002376518 and three unknown functional genes were identified. Based on results, the over-expression of AKR1 and ABC fatty acid transporter in the voriconazole non- wild- type isolates suggests these genes could represent a novel molecular marker linked to the voriconazole resistance in A. flavus.ConclusionThe results obtained in this study showed a novel finding as the authors identified AKR1 and ABC fatty acid transporter genes as possible voriconazole target genes in Iranian clinical isolates of A. flavus.     

Solving the molecular diagnostic testing conundrum for Mendelian disorders in the era of next-generation sequencing: single-gene,gene panel,or exome/genome sequencing

Next-generation sequencing is changing the paradigm of clinical genetic testing. Today there are numerous molecular tests available, including single-gene tests, gene panels, and exome sequencing or genome sequencing. As a result, ordering physicians face the conundrum of selecting the best diagnostic tool for their patients with genetic conditions. Single-gene testing is often most appropriate for conditions with distinctive clinical features and minimal locus heterogeneity. Next-generation sequencing–based gene panel testing, which can be complemented with array comparative genomic hybridization and other ancillary methods, provides a comprehensive and feasible approach for heterogeneous disorders. Exome sequencing and genome sequencing have the advantage of being unbiased regarding what set of genes is analyzed, enabling parallel interrogation of most of the genes in the human genome. However, current limitations of next-generation sequencing technology and our variant interpretation capabilities caution us against offering exome sequencing or genome sequencing as either stand-alone or first-choice diagnostic approaches. A growing interest in personalized medicine calls for the application of genome sequencing in clinical diagnostics, but major challenges must be addressed before its full potential can be realized. Here, we propose a testing algorithm to help clinicians opt for the most appropriate molecular diagnostic tool for each scenario.Genet Med17 6, 444–451.     

Exome-wide copy number variation analysis identifies a COL9A1 in frame deletion that is associated with hearing loss

Pathogenic variants in COL9A1 are primarily associated with autosomal recessive Stickler syndrome. Patients with COL9A1-associated Stickler syndrome (STL) present hearing loss (HL), ophthalmic manifestations and skeletal abnormalities. However, the clinical spectrum of patients with COL9A1 variants can also include multiple epiphyseal dysplasia, as well as non-syndromic HL that was observed in one previously reported proband. Exome sequencing was performed on the genomic DNA of an Iranian patient and his affected brother who both report non-syndromic HL. A 44.6 kb homozygous in-frame deletion spanning exons 6 to 33 of COL9A1 was detected via exome-based copy number variation analysis. The deleted exons were confirmed by PCR in the patient and his affected brother, who both have non-syndromic HL. Segregation analysis via qPCR confirmed the parents as heterozygous deletion carriers. Breakpoint analysis mapped the homozygous deletion spanning introns 5 to 33 (g.70,948,188_70,997,277del, NM_001851.4(COL9A1):c.697–3754_2112+769del, p.(Phe233_Ser704del), with an additional 67 bp of inserted intronic sequence that may have originated due to a fork stalling and template switching/microhomology-mediated break-induced replication (FoSTeS/MMBIR) mechanism. This mechanism has not been previously implicated in HL or STL. This is also the first reported copy number variation in COL9A1 that was identified through an exome data set in an Iranian family with apparent non-syndromic HL. The present study emphasizes the importance of exome-wide copy number variation analysis in molecular diagnosis and provides supporting evidence to associate COL9A1 with autosomal recessive non-syndromic HL.     

The role of combined SNV and CNV burden in patients with distal symmetric polyneuropathy

PurposeCharcot-Marie-Tooth (CMT) disease is a heterogeneous group of genetic disorders of the peripheral nervous system. Copy-number variants (CNVs) contribute significantly to CMT, as duplication of PMP22 underlies the majority of CMT1 cases. We hypothesized that CNVs and/or single-nucleotide variants (SNVs) might exist in patients with CMT with an unknown molecular genetic etiology.MethodsTwo hundred patients with CMT, negative for both SNV mutations in several CMT genes and for CNVs involving PMP22, were screened for CNVs by high-resolution oligonucleotide array comparative genomic hybridization. Whole-exome sequencing was conducted on individuals with rare, potentially pathogenic CNVs.ResultsPutatively causative CNVs were identified in five subjects (~2.5%); four of the five map to known neuropathy genes. Breakpoint sequencing revealed Alu-Alu-mediated junctions as a predominant contributor. Exome sequencing identified MFN2 SNVs in two of the individuals.ConclusionNeuropathy-associated CNV outside of the PMP22 locus is rare in CMT. Nevertheless, there is potential clinical utility in testing for CNVs and exome sequencing in CMT cases negative for the CMT1A duplication. These findings suggest that complex phenotypes including neuropathy can potentially be caused by a combination of SNVs and CNVs affecting more than one disease-associated locus and contributing to a mutational burden.     

The phenotypic spectrum of WWOX-related disorders: 20 additional cases of WOREE syndrome and review of the literature

PurposeGermline WWOX pathogenic variants have been associated with disorder of sex differentiation (DSD), spinocerebellar ataxia (SCA), and WWOX-related epileptic encephalopathy (WOREE syndrome). We review clinical and molecular data on WWOX-related disorders, further describing WOREE syndrome and phenotype/genotype correlations.MethodsWe report clinical and molecular findings in 20 additional patients from 18 unrelated families with WOREE syndrome and biallelic pathogenic variants in the WWOX gene. Different molecular screening approaches were used (quantitative polymerase chain reaction/multiplex ligation-dependent probe amplification [qPCR/MLPA], array comparative genomic hybridization [array-CGH], Sanger sequencing, epilepsy gene panel, exome sequencing), genome sequencing.ResultsTwo copy-number variations (CNVs) or two single-nucleotide variations (SNVs) were found respectively in four and nine families, with compound heterozygosity for one SNV and one CNV in five families. Eight novel missense pathogenic variants have been described. By aggregating our patients with all cases reported in the literature, 37 patients from 27 families with WOREE syndrome are known. This review suggests WOREE syndrome is a very severe epileptic encephalopathy characterized by absence of language development and acquisition of walking, early-onset drug-resistant seizures, ophthalmological involvement, and a high likelihood of premature death. The most severe clinical presentation seems to be associated with null genotypes.ConclusionGermline pathogenic variants in WWOX are clearly associated with a severe early-onset epileptic encephalopathy. We report here the largest cohort of individuals with WOREE syndrome.