%0 Journal Article %J Adv Med Sci %D 2021 %T Variants in FLRT3 and SLC35E2B identified using exome sequencing in seven high myopia families from Central Europe. %A Swierkowska, Joanna %A Karolak, Justyna A %A Gambin, Tomasz %A Rydzanicz, Malgorzata %A Frajdenberg, Agata %A Mrugacz, Malgorzata %A Podfigurna-Musielak, Monika %A Stankiewicz, Pawel %A James R Lupski %A Gajecka, Marzena %K Adolescent %K Europe %K Exome %K Female %K Follow-Up Studies %K Genetic Predisposition to Disease %K Humans %K Male %K Membrane Glycoproteins %K Mutation %K Myopia %K Pedigree %K Prognosis %K Solute Carrier Proteins %X

PURPOSE: High myopia (HM) is an eye disorder with both environmental and genetic factors involved. Many genetic factors responsible for HM were recognized worldwide, but little is known about genetic variants underlying HM in Central Europe. Thus, the aim of this study was to identify rare sequence variants involved in HM in families from Central Europe to better understand the genetic basis of HM.

MATERIALS AND METHODS: We assessed 17 individuals from 7 unrelated Central European families with hereditary HM using exome sequencing (ES). Segregation of selected variants in other available family members was performed using Sanger sequencing.

RESULTS: Detected 73 rare variants were selected for verification. We observed 2 missense variants, c.938C>T in SLC35E2B - encoding solute carrier family 35 member E2B, and c.1642G>C in FLRT3 - encoding fibronectin leucine rich transmembrane protein, segregating with HM in one family.

CONCLUSIONS: FLRT3 ​and/or ​SLC35E2B ​could represent disease candidate genes and identified sequence variants might be responsible for HM in the studied family.

%B Adv Med Sci %V 66 %P 192-198 %8 2021 Mar %G eng %N 1 %1 https://www.ncbi.nlm.nih.gov/pubmed/33711669?dopt=Abstract %R 10.1016/j.advms.2021.02.005 %0 Journal Article %J Expert Rev Mol Diagn %D 2020 %T Clinical genomics and contextualizing genome variation in the diagnostic laboratory. %A James R Lupski %A Liu, Pengfei %A Stankiewicz, Pawel %A Carvalho, Claudia M B %A Posey, Jennifer E %K Clinical Laboratory Techniques %K Computational Biology %K DNA Copy Number Variations %K Gene Dosage %K Genetic Association Studies %K Genetic Predisposition to Disease %K Genetic Testing %K Genetic Variation %K Genome, Human %K Genomics %K Humans %K Molecular Sequence Annotation %K Mutation %K Phenotype %K Rare Diseases %X

INTRODUCTION: The human genome contains the instructions for the development and biological homeostasis of the human organism and the genetic transmission of traits. Genome variation in human populations is the basis of evolution; individual or personal genomes vary tremendously, making each of us truly unique.

AREAS COVERED: Assaying this individual variation using genomic technologies has many applications in clinical medicine, from elucidating the biology of disease to designing strategies to ameliorate perturbations from homeostasis. Detecting pathogenic rare variation in a genome may provide a molecular diagnosis that can be informative for patient management and family healthcare.

EXPERT OPINION: Despite the increasing clinical use of unbiased genomic testing, including chromosome microarray analysis (CMA) with array comparative genomic hybridization (aCGH) or SNP arrays, clinical exome sequencing (cES), and whole-genome sequencing (WGS), to survey genome-wide for molecular aberrations, clinical acumen paired with an understanding of the limitations of each testing type will be needed to achieve molecular diagnoses. Potential opportunities for improving case solved rates, functionally annotating the majority of genes in the human genome, and further understanding genetic contributions to disease will empower clinical genomics and the precision medicine initiative.

%B Expert Rev Mol Diagn %V 20 %P 995-1002 %8 2020 Oct %G eng %N 10 %1 https://www.ncbi.nlm.nih.gov/pubmed/32954863?dopt=Abstract %R 10.1080/14737159.2020.1826312 %0 Journal Article %J Genomics %D 2020 %T Parental somatic mosaicism for CNV deletions - A need for more sensitive and precise detection methods in clinical diagnostics settings. %A Liu, Qian %A Karolak, Justyna A %A Grochowski, Christopher M %A Wilson, Theresa A %A Rosenfeld, Jill A %A Bacino, Carlos A %A Lalani, Seema R %A Patel, Ankita %A Breman, Amy %A Smith, Janice L %A Cheung, Sau Wai %A James R Lupski %A Bi, Weimin %A Stankiewicz, Pawel %K Clinical Laboratory Techniques %K DNA Copy Number Variations %K Female %K High-Throughput Nucleotide Sequencing %K Humans %K Inheritance Patterns %K Male %K Mosaicism %K Polymerase Chain Reaction %K Sequence Analysis, DNA %X

To further assess the scale and level of parental somatic mosaicism, we queried the CMA database at Baylor Genetics. We selected 50 unrelated families where clinically relevant apparent de novo CNV-deletions were found in the affected probands. Parental blood samples screening using deletion junction-specific PCR revealed four parents with somatic mosaicism. Droplet digital PCR (ddPCR), qPCR, and amplicon-based next-generation sequencing (NGS) were applied to validate these findings. Using ddPCR levels of mosaicism ranged from undetectable to 18.5%. Amplicon-based NGS and qPCR for the father with undetectable mosaicism was able to detect mosaicism at 0.39%. In one mother, ddPCR analysis revealed 15.6%, 10.6%, 8.2%, and undetectable levels of mosaicism in her blood, buccal cells, saliva, and urine samples, respectively. Our data suggest that more sensitive and precise methods, e.g. CNV junction-specific LR-PCR, ddPCR, or qPCR may allow for a more refined assessment of the potential disease recurrence risk for an identified variant.

%B Genomics %V 112 %P 2937-2941 %8 2020 Sep %G eng %N 5 %1 https://www.ncbi.nlm.nih.gov/pubmed/32387503?dopt=Abstract %R 10.1016/j.ygeno.2020.05.003 %0 Journal Article %J Genome Med %D 2019 %T Copy number variant and runs of homozygosity detection by microarrays enabled more precise molecular diagnoses in 11,020 clinical exome cases. %A Dharmadhikari, Avinash V %A Ghosh, Rajarshi %A Bo Yuan %A Liu, Pengfei %A Dai, Hongzheng %A Al Masri, Sami %A Scull, Jennifer %A Posey, Jennifer E %A Jiang, Allen H %A He, Weimin %A Vetrini, Francesco %A Braxton, Alicia A %A Ward, Patricia %A Chiang, Theodore %A Qu, Chunjing %A Gu, Shen %A Shaw, Chad A %A Smith, Janice L %A Lalani, Seema %A Stankiewicz, Pawel %A Cheung, Sau-Wai %A Bacino, Carlos A %A Patel, Ankita %A Breman, Amy M %A Wang, Xia %A Meng, Linyan %A Xiao, Rui %A Xia, Fan %A Donna M Muzny %A Richard A Gibbs %A Beaudet, Arthur L %A Eng, Christine M %A James R Lupski %A Yang, Yaping %A Bi, Weimin %K Chromosome Aberrations %K DNA Copy Number Variations %K Exome Sequencing %K Female %K Genetic Testing %K Homozygote %K Humans %K Limit of Detection %K Male %K Microarray Analysis %X

BACKGROUND: Exome sequencing (ES) has been successfully applied in clinical detection of single nucleotide variants (SNVs) and small indels. However, identification of copy number variants (CNVs) using ES data remains challenging. The purpose of this study is to understand the contribution of CNVs and copy neutral runs of homozygosity (ROH) in molecular diagnosis of patients referred for ES.

METHODS: In a cohort of 11,020 consecutive ES patients, an Illumina SNP array analysis interrogating mostly coding SNPs was performed as a quality control (QC) measurement and for CNV/ROH detection. Among these patients, clinical chromosomal microarray analysis (CMA) was performed at Baylor Genetics (BG) on 3229 patients, either before, concurrently, or after ES. We retrospectively analyzed the findings from CMA and the QC array.

RESULTS: The QC array can detect ~ 70% of pathogenic/likely pathogenic CNVs (PCNVs) detectable by CMA. Out of the 11,020 ES cases, the QC array identified PCNVs in 327 patients and uniparental disomy (UPD) disorder-related ROH in 10 patients. The overall PCNV/UPD detection rate was 5.9% in the 3229 ES patients who also had CMA at BG; PCNV/UPD detection rate was higher in concurrent ES and CMA than in ES with prior CMA (7.2% vs 4.6%). The PCNVs/UPD contributed to the molecular diagnoses in 17.4% (189/1089) of molecularly diagnosed ES cases with CMA and were estimated to contribute in 10.6% of all molecularly diagnosed ES cases. Dual diagnoses with both PCNVs and SNVs were detected in 38 patients. PCNVs affecting single recessive disorder genes in a compound heterozygous state with SNVs were detected in 4 patients, and homozygous deletions (mostly exonic deletions) were detected in 17 patients. A higher PCNV detection rate was observed for patients with syndromic phenotypes and/or cardiovascular abnormalities.

CONCLUSIONS: Our clinical genomics study demonstrates that detection of PCNV/UPD through the QC array or CMA increases ES diagnostic rate, provides more precise molecular diagnosis for dominant as well as recessive traits, and enables more complete genetic diagnoses in patients with dual or multiple molecular diagnoses. Concurrent ES and CMA using an array with exonic coverage for disease genes enables most effective detection of both CNVs and SNVs and therefore is recommended especially in time-sensitive clinical situations.

%B Genome Med %V 11 %P 30 %8 2019 May 17 %G eng %N 1 %1 https://www.ncbi.nlm.nih.gov/pubmed/31101064?dopt=Abstract %R 10.1186/s13073-019-0639-5 %0 Journal Article %J Genome Med %D 2019 %T Interchromosomal template-switching as a novel molecular mechanism for imprinting perturbations associated with Temple syndrome. %A Carvalho, Claudia M B %A Coban-Akdemir, Zeynep %A Hijazi, Hadia %A Bo Yuan %A Pendleton, Matthew %A Harrington, Eoghan %A Beaulaurier, John %A Juul, Sissel %A Turner, Daniel J %A Kanchi, Rupa S %A Jhangiani, Shalini N %A Donna M Muzny %A Richard A Gibbs %A Stankiewicz, Pawel %A Belmont, John W %A Shaw, Chad A %A Cheung, Sau Wai %A Hanchard, Neil A %A Sutton, V Reid %A Bader, Patricia I %A James R Lupski %K Chromosome Aberrations %K Chromosome Disorders %K Chromosomes, Human, Pair 14 %K DNA Methylation %K DNA Replication %K Genomic Imprinting %K Humans %K Male %K Pedigree %K Phenotype %K Polymorphism, Single Nucleotide %K Young Adult %X

BACKGROUND: Intrachromosomal triplications (TRP) can contribute to disease etiology via gene dosage effects, gene disruption, position effects, or fusion gene formation. Recently, post-zygotic de novo triplications adjacent to copy-number neutral genomic intervals with runs of homozygosity (ROH) have been shown to result in uniparental isodisomy (UPD). The genomic structure of these complex genomic rearrangements (CGRs) shows a consistent pattern of an inverted triplication flanked by duplications (DUP-TRP/INV-DUP) formed by an iterative DNA replisome template-switching mechanism during replicative repair of a single-ended, double-stranded DNA (seDNA), the ROH results from an interhomolog or nonsister chromatid template switch. It has been postulated that these CGRs may lead to genetic abnormalities in carriers due to dosage-sensitive genes mapping within the copy-number variant regions, homozygosity for alleles at a locus causing an autosomal recessive (AR) disease trait within the ROH region, or imprinting-associated diseases.

METHODS: Here, we report a family wherein the affected subject carries a de novo 2.2-Mb TRP followed by 42.2 Mb of ROH and manifests clinical features overlapping with those observed in association with chromosome 14 maternal UPD (UPD(14)mat). UPD(14)mat can cause clinical phenotypic features enabling a diagnosis of Temple syndrome. This CGR was then molecularly characterized by high-density custom aCGH, genome-wide single-nucleotide polymorphism (SNP) and methylation arrays, exome sequencing (ES), and the Oxford Nanopore long-read sequencing technology.

RESULTS: We confirmed the postulated DUP-TRP/INV-DUP structure by multiple orthogonal genomic technologies in the proband. The methylation status of known differentially methylated regions (DMRs) on chromosome 14 revealed that the subject shows the typical methylation pattern of UPD(14)mat. Consistent with these molecular findings, the clinical features overlap with those observed in Temple syndrome, including speech delay.

CONCLUSIONS: These data provide experimental evidence that, in humans, triplication can lead to segmental UPD and imprinting disease. Importantly, genotype/phenotype analyses further reveal how a post-zygotically generated complex structural variant, resulting from a replication-based mutational mechanism, contributes to expanding the clinical phenotype of known genetic syndromes. Mechanistically, such events can distort transmission genetics resulting in homozygosity at a locus for which only one parent is a carrier as well as cause imprinting diseases.

%B Genome Med %V 11 %P 25 %8 2019 Apr 23 %G eng %N 1 %1 https://www.ncbi.nlm.nih.gov/pubmed/31014393?dopt=Abstract %R 10.1186/s13073-019-0633-y %0 Journal Article %J Genome Res %D 2018 %T Predicting human genes susceptible to genomic instability associated with /-mediated rearrangements. %A Song, Xiaofei %A Beck, Christine R %A Du, Renqian %A Campbell, Ian M %A Coban-Akdemir, Zeynep %A Gu, Shen %A Breman, Amy M %A Stankiewicz, Pawel %A Ira, Grzegorz %A Shaw, Chad A %A Lupski, James R %K Alu Elements %K DNA Copy Number Variations %K Gene Duplication %K Genome, Human %K Genomic Instability %K Humans %K Sequence Deletion %X

elements, the short interspersed element numbering more than 1 million copies per human genome, can mediate the formation of copy number variants (CNVs) between substrate pairs. These /-mediated rearrangements (AAMRs) can result in pathogenic variants that cause diseases. To investigate the impact of AAMR on gene variation and human health, we first characterized s that are involved in mediating CNVs (CNV-s) and observed that these s tend to be evolutionarily younger. We then computationally generated, with the assistance of a supercomputer, a test data set consisting of 78 million pairs and predicted ∼18% of them are potentially susceptible to AAMR. We further determined the relative risk of AAMR in 12,074 OMIM genes using the count of predicted CNV- pairs and experimentally validated the predictions with 89 samples selected by correlating predicted hotspots with a database of CNVs identified by clinical chromosomal microarrays (CMAs) on the genomes of approximately 54,000 subjects. We fine-mapped 47 duplications, 40 deletions, and two complex rearrangements and examined a total of 52 breakpoint junctions of simple CNVs. Overall, 94% of the candidate breakpoints were at least partially mediated. We successfully predicted all (100%) of pairs that mediated deletions ( = 21) and achieved an 87% positive predictive value overall when including AAMR-generated deletions and duplications. We provided a tool, AluAluCNVpredictor, for assessing AAMR hotspots and their role in human disease. These results demonstrate the utility of our predictive model and provide insights into the genomic features and molecular mechanisms underlying AAMR.

%B Genome Res %V 28 %P 1228-1242 %8 2018 Aug %G eng %N 8 %1 https://www.ncbi.nlm.nih.gov/pubmed/29907612?dopt=Abstract %R 10.1101/gr.229401.117 %0 Journal Article %J Hum Mutat %D 2017 %T Characterization of chromosomal abnormalities in pregnancy losses reveals critical genes and loci for human early development. %A Chen, Yiyun %A Bartanus, Justin %A Liang, Desheng %A Zhu, Hongmin %A Breman, Amy M %A Smith, Janice L %A Wang, Hua %A Ren, Zhilin %A Patel, Ankita %A Stankiewicz, Pawel %A Cram, David S %A Cheung, Sau Wai %A Wu, Lingqian %A Yu, Fuli %K Animals %K Chromosome Aberrations %K Chromosome Disorders %K DNA Copy Number Variations %K Embryonic Development %K Female %K Genome, Human %K Humans %K Mice %K Microarray Analysis %K Pregnancy %K Transcription Factors %K Zebrafish %X

Detailed characterization of chromosomal abnormalities, a common cause for congenital abnormalities and pregnancy loss, is critical for elucidating genes for human fetal development. Here, 2,186 product-of-conception samples were tested for copy-number variations (CNVs) at two clinical diagnostic centers using whole-genome sequencing and high-resolution chromosomal microarray analysis. We developed a new gene discovery approach to predict potential developmental genes and identified 275 candidate genes from CNVs detected from both datasets. Based on Mouse Genome Informatics (MGI) and Zebrafish model organism database (ZFIN), 75% of identified genes could lead to developmental defects when mutated. Genes involved in embryonic development, gene transcription, and regulation of biological processes were significantly enriched. Especially, transcription factors and gene families sharing specific protein domains predominated, which included known developmental genes such as HOX, NKX homeodomain genes, and helix-loop-helix containing HAND2, NEUROG2, and NEUROD1 as well as potential novel developmental genes. We observed that developmental genes were denser in certain chromosomal regions, enabling identification of 31 potential genomic loci with clustered genes associated with development.

%B Hum Mutat %V 38 %P 669-677 %8 2017 Jun %G eng %N 6 %1 https://www.ncbi.nlm.nih.gov/pubmed/28247551?dopt=Abstract %R 10.1002/humu.23207 %0 Journal Article %J Eur J Hum Genet %D 2017 %T Variants in SKP1, PROB1, and IL17B genes at keratoconus 5q31.1-q35.3 susceptibility locus identified by whole-exome sequencing. %A Karolak, Justyna A %A Gambin, Tomasz %A Pitarque, Jose A %A Molinari, Andrea %A Jhangiani, Shalini %A Stankiewicz, Pawel %A Lupski, James R %A Gajecka, Marzena %K Chromosomes, Human, Pair 5 %K Exome %K Female %K Gene Frequency %K Genetic Linkage %K Genetic Predisposition to Disease %K Genome, Human %K Hexokinase %K High-Throughput Nucleotide Sequencing %K Humans %K Interleukin-17 %K Keratoconus %K Male %K Pedigree %K Phenotype %K Proteins %K S-Phase Kinase-Associated Proteins %X

Keratoconus (KTCN) is a protrusion and thinning of the cornea, resulting in impairment of visual function. The extreme genetic heterogeneity makes it difficult to discover factors unambiguously influencing the KTCN phenotype. In this study, we used whole-exome sequencing (WES) and Sanger sequencing to reduce the number of candidate genes at the 5q31.1-q35.3 locus and to prioritize other potentially relevant variants in an Ecuadorian family with KTCN. We applied WES in two affected KTCN individuals from the Ecuadorian family that showed a suggestive linkage between the KTCN phenotype and the 5q31.1-q35.3 locus. Putative variants identified by WES were further evaluated in this family using Sanger sequencing. Exome capture discovered a total of 173 rare (minor allele frequency <0.001 in control population) nonsynonymous variants in both affected individuals. Among them, 16 SNVs were selected for further evaluation. Segregation analysis revealed that variants c.475T>G in SKP1, c.671G>A in PROB1, and c.527G>A in IL17B in the 5q31.1-q35.3 linkage region, and c.850G>A in HKDC1 in the 10q22 locus completely segregated with the phenotype in the studied KTCN family. We demonstrate that a combination of various techniques significantly narrowed the studied genomic region and reduced the list of the putative exonic variants. Moreover, since this locus overlapped two other chromosomal regions previously recognized in distinct KTCN studies, our findings suggest that this 5q31.1-q35.3 locus might be linked with KTCN.

%B Eur J Hum Genet %V 25 %P 73-78 %8 2017 Jan %G eng %N 1 %1 https://www.ncbi.nlm.nih.gov/pubmed/27703147?dopt=Abstract %R 10.1038/ejhg.2016.130 %0 Journal Article %J Neurol Neurochir Pol %D 2016 %T CAV3 mutation in a patient with transient hyperCKemia and myalgia. %A Macias, Anna %A Gambin, Tomasz %A Szafranski, Przemyslaw %A Jhangiani, Shalini N %A Kolasa, Anna %A Obersztyn, Ewa %A Lupski, James R %A Stankiewicz, Pawel %A Kaminska, Anna %K Caveolin 3 %K Colitis, Ulcerative %K Creatine Kinase %K Frameshift Mutation %K Humans %K Klinefelter Syndrome %K Magnetic Resonance Imaging %K Male %K Membrane Proteins %K Middle Aged %K Muscle, Skeletal %K Muscular Diseases %K Mutation, Missense %K Myalgia %K Phenotype %K Sjogren's Syndrome %K Thigh %X

Mutations in caveolin-3 (CAV3) can lead to different clinical phenotypes affecting skeletal or cardiac muscles. Here, we describe a patient with Klinefelter syndrome, ulcerative colitis and Sjögren syndrome, who developed transient hyperCKemia, myalgia and mild muscular weakness. Using whole exome sequencing (WES), a missense mutation G169A was found in the CAV3 gene. In addition, we identified a homozygous frameshift deletion in MS4A12 that may contribute to inflammatory bowel disease, further demonstrating usefulness of WES in dual molecular diagnoses.

%B Neurol Neurochir Pol %V 50 %P 468-473 %8 2016 Nov-Dec %G eng %N 6 %1 https://www.ncbi.nlm.nih.gov/pubmed/27772553?dopt=Abstract %R 10.1016/j.pjnns.2016.06.008 %0 Journal Article %J Trends Genet %D 2016 %T Erratum to: Somatic Mosaicism: Implications for Disease and Transmission Genetics. %A Campbell, Ian M %A Shaw, Chad A %A Stankiewicz, Pawel %A Lupski, James R %B Trends Genet %V 32 %P 138 %8 2016 Feb %G eng %N 2 %1 https://www.ncbi.nlm.nih.gov/pubmed/29482722?dopt=Abstract %R 10.1016/j.tig.2015.07.004 %0 Journal Article %J Am J Hum Genet %D 2015 %T Absence of heterozygosity due to template switching during replicative rearrangements. %A Carvalho, Claudia M B %A Pfundt, Rolph %A King, Daniel A %A Lindsay, Sarah J %A Zuccherato, Luciana W %A Macville, Merryn V E %A Liu, Pengfei %A Johnson, Diana %A Stankiewicz, Pawel %A Brown, Chester W %A Shaw, Chad A %A Hurles, Matthew E %A Ira, Grzegorz %A Hastings, P J %A Brunner, Han G %A Lupski, James R %K Base Sequence %K DNA Copy Number Variations %K DNA Repair %K DNA Replication %K Gene Rearrangement %K Humans %K In Situ Hybridization, Fluorescence %K Loss of Heterozygosity %K Models, Genetic %K Molecular Sequence Data %K Netherlands %K Polymerase Chain Reaction %K Polymorphism, Single Nucleotide %K Sequence Analysis, DNA %K Uniparental Disomy %X

We investigated complex genomic rearrangements (CGRs) consisting of triplication copy-number variants (CNVs) that were accompanied by extended regions of copy-number-neutral absence of heterozygosity (AOH) in subjects with multiple congenital abnormalities. Molecular analyses provided observational evidence that in humans, post-zygotically generated CGRs can lead to regional uniparental disomy (UPD) due to template switches between homologs versus sister chromatids by using microhomology to prime DNA replication-a prediction of the replicative repair model, MMBIR. Our findings suggest that replication-based mechanisms might underlie the formation of diverse types of genomic alterations (CGRs and AOH) implicated in constitutional disorders.

%B Am J Hum Genet %V 96 %P 555-64 %8 2015 Apr 02 %G eng %N 4 %1 https://www.ncbi.nlm.nih.gov/pubmed/25799105?dopt=Abstract %R 10.1016/j.ajhg.2015.01.021 %0 Journal Article %J BMC Genomics %D 2015 %T Assessing structural variation in a personal genome-towards a human reference diploid genome. %A English, Adam C %A Salerno, William J %A Hampton, Oliver A %A Gonzaga-Jauregui, Claudia %A Ambreth, Shruthi %A Ritter, Deborah I %A Beck, Christine R %A Davis, Caleb F %A Dahdouli, Mahmoud %A Ma, Singer %A Carroll, Andrew %A Veeraraghavan, Narayanan %A Bruestle, Jeremy %A Drees, Becky %A Hastie, Alex %A Lam, Ernest T %A White, Simon %A Mishra, Pamela %A Wang, Min %A Han, Yi %A Zhang, Feng %A Stankiewicz, Pawel %A Wheeler, David A %A Reid, Jeffrey G %A Muzny, Donna M %A Rogers, Jeffrey %A Sabo, Aniko %A Worley, Kim C %A Lupski, James R %A Boerwinkle, Eric %A Gibbs, Richard A %K Computational Biology %K Databases, Genetic %K Diploidy %K Genome, Human %K Genomic Structural Variation %K Humans %K Sequence Analysis, DNA %K Software %X

BACKGROUND: Characterizing large genomic variants is essential to expanding the research and clinical applications of genome sequencing. While multiple data types and methods are available to detect these structural variants (SVs), they remain less characterized than smaller variants because of SV diversity, complexity, and size. These challenges are exacerbated by the experimental and computational demands of SV analysis. Here, we characterize the SV content of a personal genome with Parliament, a publicly available consensus SV-calling infrastructure that merges multiple data types and SV detection methods.

RESULTS: We demonstrate Parliament's efficacy via integrated analyses of data from whole-genome array comparative genomic hybridization, short-read next-generation sequencing, long-read (Pacific BioSciences RSII), long-insert (Illumina Nextera), and whole-genome architecture (BioNano Irys) data from the personal genome of a single subject (HS1011). From this genome, Parliament identified 31,007 genomic loci between 100 bp and 1 Mbp that are inconsistent with the hg19 reference assembly. Of these loci, 9,777 are supported as putative SVs by hybrid local assembly, long-read PacBio data, or multi-source heuristics. These SVs span 59 Mbp of the reference genome (1.8%) and include 3,801 events identified only with long-read data. The HS1011 data and complete Parliament infrastructure, including a BAM-to-SV workflow, are available on the cloud-based service DNAnexus.

CONCLUSIONS: HS1011 SV analysis reveals the limits and advantages of multiple sequencing technologies, specifically the impact of long-read SV discovery. With the full Parliament infrastructure, the HS1011 data constitute a public resource for novel SV discovery, software calibration, and personal genome structural variation analysis.

%B BMC Genomics %V 16 %P 286 %8 2015 Apr 11 %G eng %N 1 %1 https://www.ncbi.nlm.nih.gov/pubmed/25886820?dopt=Abstract %R 10.1186/s12864-015-1479-3 %0 Journal Article %J Trends Genet %D 2015 %T Somatic mosaicism: implications for disease and transmission genetics. %A Campbell, Ian M %A Shaw, Chad A %A Stankiewicz, Pawel %A Lupski, James R %K Animals %K Genetic Diseases, Inborn %K Genome, Human %K Humans %K Mosaicism %K Mutagenesis, Insertional %K Mutation %K Polymorphism, Single Nucleotide %K Risk %K Trinucleotide Repeat Expansion %X

Nearly all of the genetic material among cells within an organism is identical. However, single-nucleotide variants (SNVs), small insertions/deletions (indels), copy-number variants (CNVs), and other structural variants (SVs) continually accumulate as cells divide during development. This process results in an organism composed of countless cells, each with its own unique personal genome. Thus, every human is undoubtedly mosaic. Mosaic mutations can go unnoticed, underlie genetic disease or normal human variation, and may be transmitted to the next generation as constitutional variants. We review the influence of the developmental timing of mutations, the mechanisms by which they arise, methods for detecting mosaic variants, and the risk of passing these mutations on to the next generation.

%B Trends Genet %V 31 %P 382-92 %8 2015 Jul %G eng %N 7 %1 https://www.ncbi.nlm.nih.gov/pubmed/25910407?dopt=Abstract %R 10.1016/j.tig.2015.03.013 %0 Journal Article %J Am J Hum Genet %D 2014 %T The Alu-rich genomic architecture of SPAST predisposes to diverse and functionally distinct disease-associated CNV alleles. %A Boone, Philip M %A Bo Yuan %A Campbell, Ian M %A Scull, Jennifer C %A Withers, Marjorie A %A Baggett, Brett C %A Beck, Christine R %A Shaw, Christine J %A Stankiewicz, Pawel %A Moretti, Paolo %A Goodwin, Wendy E %A Hein, Nichole %A Fink, John K %A Seong, Moon-Woo %A Seo, Soo Hyun %A Park, Sung Sup %A Karbassi, Izabela D %A Batish, Sat Dev %A Ordóñez-Ugalde, Andrés %A Quintáns, Beatriz %A Sobrido, María-Jesús %A Stemmler, Susanne %A James R Lupski %K Adenosine Triphosphatases %K Alu Elements %K Base Sequence %K Cation Transport Proteins %K Cell Line, Transformed %K DNA Copy Number Variations %K Genotype %K Humans %K Protein Isoforms %K Recombinant Fusion Proteins %K Sequence Analysis, DNA %K Sequence Deletion %K Spastic Paraplegia, Hereditary %K Spastin %X

Intragenic copy-number variants (CNVs) contribute to the allelic spectrum of both Mendelian and complex disorders. Although pathogenic deletions and duplications in SPAST (mutations in which cause autosomal-dominant spastic paraplegia 4 [SPG4]) have been described, their origins and molecular consequences remain obscure. We mapped breakpoint junctions of 54 SPAST CNVs at nucleotide resolution. Diverse combinations of exons are deleted or duplicated, highlighting the importance of particular exons for spastin function. Of the 54 CNVs, 38 (70%) appear to be mediated by an Alu-based mechanism, suggesting that the Alu-rich genomic architecture of SPAST renders this locus susceptible to various genome rearrangements. Analysis of breakpoint Alus further informs a model of Alu-mediated CNV formation characterized by small CNV size and potential involvement of mechanisms other than homologous recombination. Twelve deletions (22%) overlap part of SPAST and a portion of a nearby, directly oriented gene, predicting novel chimeric genes in these subjects' genomes. cDNA from a subject with a SPAST final exon deletion contained multiple SPAST:SLC30A6 fusion transcripts, indicating that SPAST CNVs can have transcriptional effects beyond the gene itself. SLC30A6 has been implicated in Alzheimer disease, so these fusion gene data could explain a report of spastic paraplegia and dementia cosegregating in a family with deletion of the final exon of SPAST. Our findings provide evidence that the Alu genomic architecture of SPAST predisposes to diverse CNV alleles with distinct transcriptional--and possibly phenotypic--consequences. Moreover, we provide further mechanistic insights into Alu-mediated copy-number change that are extendable to other loci.

%B Am J Hum Genet %V 95 %P 143-61 %8 2014 Aug 07 %G eng %N 2 %1 https://www.ncbi.nlm.nih.gov/pubmed/25065914?dopt=Abstract %R 10.1016/j.ajhg.2014.06.014 %0 Journal Article %J Genome Res %D 2013 %T Deletions of recessive disease genes: CNV contribution to carrier states and disease-causing alleles. %A Boone, Philip M %A Campbell, Ian M %A Baggett, Brett C %A Soens, Zachry T %A Rao, Mitchell M %A Hixson, Patricia M %A Patel, Ankita %A Bi, Weimin %A Cheung, Sau Wai %A Lalani, Seema R %A Beaudet, Arthur L %A Stankiewicz, Pawel %A Shaw, Chad A %A Lupski, James R %K Alleles %K Comparative Genomic Hybridization %K Databases, Genetic %K DNA Copy Number Variations %K Gene Deletion %K Gene Frequency %K Genes, Dominant %K Genes, Recessive %K Genetic Diseases, Inborn %K Homozygote %K Humans %X

Over 1200 recessive disease genes have been described in humans. The prevalence, allelic architecture, and per-genome load of pathogenic alleles in these genes remain to be fully elucidated, as does the contribution of DNA copy-number variants (CNVs) to carrier status and recessive disease. We mined CNV data from 21,470 individuals obtained by array-comparative genomic hybridization in a clinical diagnostic setting to identify deletions encompassing or disrupting recessive disease genes. We identified 3212 heterozygous potential carrier deletions affecting 419 unique recessive disease genes. Deletion frequency of these genes ranged from one occurrence to 1.5%. When compared with recessive disease genes never deleted in our cohort, the 419 recessive disease genes affected by at least one carrier deletion were longer and located farther from known dominant disease genes, suggesting that the formation and/or prevalence of carrier CNVs may be affected by both local and adjacent genomic features and by selection. Some subjects had multiple carrier CNVs (307 subjects) and/or carrier deletions encompassing more than one recessive disease gene (206 deletions). Heterozygous deletions spanning multiple recessive disease genes may confer carrier status for multiple single-gene disorders, for complex syndromes resulting from the combination of two or more recessive conditions, or may potentially cause clinical phenotypes due to a multiply heterozygous state. In addition to carrier mutations, we identified homozygous and hemizygous deletions potentially causative for recessive disease. We provide further evidence that CNVs contribute to the allelic architecture of both carrier and recessive disease-causing mutations. Thus, a complete recessive carrier screening method or diagnostic test should detect CNV alleles.

%B Genome Res %V 23 %P 1383-94 %8 2013 Sep %G eng %N 9 %1 https://www.ncbi.nlm.nih.gov/pubmed/23685542?dopt=Abstract %R 10.1101/gr.156075.113 %0 Journal Article %J Genet Med %D 2013 %T Incidental copy-number variants identified by routine genome testing in a clinical population. %A Boone, Philip M %A Soens, Zachry T %A Campbell, Ian M %A Stankiewicz, Pawel %A Cheung, Sau Wai %A Patel, Ankita %A Beaudet, Arthur L %A Plon, Sharon E %A Shaw, Chad A %A McGuire, Amy L %A Lupski, James R %K Age of Onset %K Base Sequence %K Chromosome Mapping %K Comparative Genomic Hybridization %K DNA Copy Number Variations %K Female %K Gene Order %K Genetic Predisposition to Disease %K Humans %K Inheritance Patterns %K Male %K Reproducibility of Results %X

PURPOSE: Mutational load of susceptibility variants has not been studied on a genomic scale in a clinical population, nor has the potential to identify these mutations as incidental findings during clinical testing been systematically ascertained.

METHODS: Array comparative genomic hybridization, a method for genome-wide detection of DNA copy-number variants, was performed clinically on DNA from 9,005 individuals. Copy-number variants encompassing or disrupting single genes were identified and analyzed for their potential to confer predisposition to dominant, adult-onset disease. Multigene copy-number variants affecting dominant, adult-onset cancer syndrome genes were also assessed.

RESULTS: In our cohort, 83 single-gene copy-number variants affected 40 unique genes associated with dominant, adult-onset disorders and unrelated to the patients' referring diagnoses (i.e., incidental) were found. Fourteen of these copy-number variants are likely disease-predisposing, 25 are likely benign, and 44 are of unknown clinical consequence. When incidental copy-number variants spanning up to 20 genes were considered, 27 copy-number variants affected 17 unique genes associated with dominant, adult-onset cancer predisposition.

CONCLUSION: Copy-number variants potentially conferring susceptibility to adult-onset disease can be identified as incidental findings during routine genome-wide testing. Some of these mutations may be medically actionable, enabling disease surveillance or prevention; however, most incidentally observed single-gene copy-number variants are currently of unclear significance to the patient.

%B Genet Med %V 15 %P 45-54 %8 2013 Jan %G eng %N 1 %1 https://www.ncbi.nlm.nih.gov/pubmed/22878507?dopt=Abstract %R 10.1038/gim.2012.95 %0 Journal Article %J Am J Hum Genet %D 2013 %T TM4SF20 ancestral deletion and susceptibility to a pediatric disorder of early language delay and cerebral white matter hyperintensities. %A Wiszniewski, Wojciech %A Hunter, Jill V %A Hanchard, Neil A %A Willer, Jason R %A Shaw, Chad %A Tian, Qi %A Illner, Anna %A Wang, Xueqing %A Cheung, Sau W %A Patel, Ankita %A Campbell, Ian M %A Gelowani, Violet %A Hixson, Patricia %A Ester, Audrey R %A Azamian, Mahshid S %A Potocki, Lorraine %A Zapata, Gladys %A Hernandez, Patricia P %A Ramocki, Melissa B %A Santos-Cortez, Regie L P %A Wang, Gao %A York, Michele K %A Justice, Monica J %A Chu, Zili D %A Bader, Patricia I %A Omo-Griffith, Lisa %A Madduri, Nirupama S %A Scharer, Gunter %A Crawford, Heather P %A Yanatatsaneejit, Pattamawadee %A Eifert, Anna %A Kerr, Jeffery %A Bacino, Carlos A %A Franklin, Adiaha I A %A Goin-Kochel, Robin P %A Simpson, Gayle %A Immken, Ladonna %A Haque, Muhammad E %A Stosic, Marija %A Williams, Misti D %A Morgan, Thomas M %A Pruthi, Sumit %A Omary, Reed %A Boyadjiev, Simeon A %A Win, Kay K %A Thida, Aye %A Hurles, Matthew %A Hibberd, Martin Lloyd %A Khor, Chiea Chuen %A Van Vinh Chau, Nguyen %A Gallagher, Thomas E %A Mutirangura, Apiwat %A Stankiewicz, Pawel %A Beaudet, Arthur L %A Maletic-Savatic, Mirjana %A Rosenfeld, Jill A %A Shaffer, Lisa G %A Davis, Erica E %A Belmont, John W %A Dunstan, Sarah %A Simmons, Cameron P %A Bonnen, Penelope E %A Leal, Suzanne M %A Katsanis, Nicholas %A Lupski, James R %A Lalani, Seema R %K Age of Onset %K Aging, Premature %K Asian People %K Base Sequence %K Brain %K Child %K Child, Preschool %K Chromosomes, Human, Pair 2 %K Exons %K Female %K Genetic Predisposition to Disease %K Humans %K Language Development Disorders %K Leukoencephalopathies %K Magnetic Resonance Imaging %K Male %K Molecular Sequence Data %K Pedigree %K Sequence Analysis, DNA %K Sequence Deletion %K Tetraspanins %X

White matter hyperintensities (WMHs) of the brain are important markers of aging and small-vessel disease. WMHs are rare in healthy children and, when observed, often occur with comorbid neuroinflammatory or vasculitic processes. Here, we describe a complex 4 kb deletion in 2q36.3 that segregates with early childhood communication disorders and WMH in 15 unrelated families predominantly from Southeast Asia. The premature brain aging phenotype with punctate and multifocal WMHs was observed in ~70% of young carrier parents who underwent brain MRI. The complex deletion removes the penultimate exon 3 of TM4SF20, a gene encoding a transmembrane protein of unknown function. Minigene analysis showed that the resultant net loss of an exon introduces a premature stop codon, which, in turn, leads to the generation of a stable protein that fails to target to the plasma membrane and accumulates in the cytoplasm. Finally, we report this deletion to be enriched in individuals of Vietnamese Kinh descent, with an allele frequency of about 1%, embedded in an ancestral haplotype. Our data point to a constellation of early language delay and WMH phenotypes, driven by a likely toxic mechanism of TM4SF20 truncation, and highlight the importance of understanding and managing population-specific low-frequency pathogenic alleles.

%B Am J Hum Genet %V 93 %P 197-210 %8 2013 Aug 08 %G eng %N 2 %1 https://www.ncbi.nlm.nih.gov/pubmed/23810381?dopt=Abstract %R 10.1016/j.ajhg.2013.05.027 %0 Journal Article %J N Engl J Med %D 2010 %T Whole-genome sequencing in a patient with Charcot-Marie-Tooth neuropathy. %A Lupski, James R %A Reid, Jeffrey G %A Gonzaga-Jauregui, Claudia %A Rio Deiros, David %A Chen, David C Y %A Nazareth, Lynne %A Bainbridge, Matthew %A Dinh, Huyen %A Jing, Chyn %A Wheeler, David A %A McGuire, Amy L %A Zhang, Feng %A Stankiewicz, Pawel %A Halperin, John J %A Yang, Chengyong %A Gehman, Curtis %A Guo, Danwei %A Irikat, Rola K %A Tom, Warren %A Fantin, Nick J %A Muzny, Donna M %A Gibbs, Richard A %K Adult %K Aged %K Aged, 80 and over %K Charcot-Marie-Tooth Disease %K Codon, Nonsense %K Female %K Genes, Recessive %K Genetic Association Studies %K Genome, Human %K Genotype %K Humans %K Male %K Middle Aged %K Mutation, Missense %K Pedigree %K Phenotype %K Polymorphism, Single Nucleotide %K Sequence Analysis, DNA %X

BACKGROUND: Whole-genome sequencing may revolutionize medical diagnostics through rapid identification of alleles that cause disease. However, even in cases with simple patterns of inheritance and unambiguous diagnoses, the relationship between disease phenotypes and their corresponding genetic changes can be complicated. Comprehensive diagnostic assays must therefore identify all possible DNA changes in each haplotype and determine which are responsible for the underlying disorder. The high number of rare, heterogeneous mutations present in all humans and the paucity of known functional variants in more than 90% of annotated genes make this challenge particularly difficult. Thus, the identification of the molecular basis of a genetic disease by means of whole-genome sequencing has remained elusive. We therefore aimed to assess the usefulness of human whole-genome sequencing for genetic diagnosis in a patient with Charcot-Marie-Tooth disease.

METHODS: We identified a family with a recessive form of Charcot-Marie-Tooth disease for which the genetic basis had not been identified. We sequenced the whole genome of the proband, identified all potential functional variants in genes likely to be related to the disease, and genotyped these variants in the affected family members.

RESULTS: We identified and validated compound, heterozygous, causative alleles in SH3TC2 (the SH3 domain and tetratricopeptide repeats 2 gene), involving two mutations, in the proband and in family members affected by Charcot-Marie-Tooth disease. Separate subclinical phenotypes segregated independently with each of the two mutations; heterozygous mutations confer susceptibility to neuropathy, including the carpal tunnel syndrome.

CONCLUSIONS: As shown in this study of a family with Charcot-Marie-Tooth disease, whole-genome sequencing can identify clinically relevant variants and provide diagnostic information to inform the care of patients.

%B N Engl J Med %V 362 %P 1181-91 %8 2010 Apr 01 %G eng %N 13 %1 https://www.ncbi.nlm.nih.gov/pubmed/20220177?dopt=Abstract %R 10.1056/NEJMoa0908094