Supplementary Material
Pathogenetics of Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins
Przemyslaw Szafranski, Tomasz Gambin, Avinash V. Dharmadhikari,Kadir Caner Akdemir, Shalini N. Jhangiani, Jennifer Schuette, NihalGodiwala, Svetlana A.Yatsenko, Jessica Sebastian,Suneeta Madan-Khetarpal, Urvashi Surti,Rosanna G.Abellar, David A. Bateman, Ashley L. Wilson,Melinda H. Markham, Jill Slamon, Fernando Santos-Simarro, MaríaPalomares, Julián Nevado, Pablo Lapunzina,Brian Chung Hon-Yin, Wong Wai-Lap, Yoyo Wing Yiu Chu, Gary Tsz Kin Mok,EitanKerem, Joel Reiter, NamasivayamAmbalavanan, Scott A. Anderson, David R. Kelly, Joseph Shieh, Taryn C. Rosenthal, Kristin Scheible, Laurie Steiner, M. Anwar Iqbal, Margaret L. McKinnon, Sara Jane Hamilton, KamillaSchlade-Bartusiak, Dawn English, Glenda Hendson, Elizabeth R. Roeder, Thomas S.DeNapoli, Rebecca Okashah Littlejohn, Daynna J. Wolff, Carol L. Wagner, Alison Yeung, David Francis, Elizabeth K.Fiorino, Morris Edelman, Joyce Fox, Denise A. Hayes, Sandra Janssens, Elfride De Baere, BjörnMenten, Anne Loccufier, LieveVanwalleghem, Philippe Moerman, Yves Sznajer, Amy S. Lay, Jennifer L.Kussmann,Jasneek Chawla,Diane J. Payton, Gael E. Phillips, Erwin Brosens,Dick Tibboel, Annelies de Klein, Isabelle Maystadt, Richard Fisher, Neil Sebire, Alison Male, Maya Chopra, Jason Pinner, Girvan Malcolm, Gregory Peters, Susan Arbuckle,Melissa Lees, Zoe Mead,Oliver Quarrell, Richard Sayers, Martina Owens, Charles Shaw-Smith,Janet Lioy, Eileen McKay, Nicole de Leeuw, Ilse Feenstra, LiesbethSpruijt,Frances Elmslie, Timothy Thiruchelvam, Carlos A.Bacino, Claire Langston, James R.Lupski, Partha Sen, Edwina Popek, Paweł Stankiewicz
Author affiliations
P. Szafranski, T. Gambin, A. V. Dharmadhikari, S. N. Jhangiani, C. A. Bacino, J. R. Lupski, P. Stankiewicz
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
A. V. Dharmadhikari, P. Stankiewicz
Interdepartmental Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, TX, USA
P. Stankiewicz
Institute of Mother and Child, Warsaw, Poland
S. N. Jhangiani, J. R. Lupski
Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX, USA
J. R. Lupski
Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA
K. C. Akdemir
Genomic Medicine Department, MD Anderson Cancer Center, Houston, TX, USA
J. Schuette
Division of PediatricAnesthesia and Critical Care Medicine, Johns Hopkins Medical Institutions, Baltimore, MD, USA
N. Godiwala
Division of Critical Care Medicine, Children’s National Health System, Washington, DC, USA
S. A. Yatsenko, U. Surti
Department of Obstetrics, Gynecology, and Reproductive Sciences, Center for Medical Genetics and Genomics, Magee-Womens Hospital of UPMC, Pittsburgh, PA, USA
Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
U. Surti
Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, PA, USA
J. Sebastian, S. Madan-Khetarpal
Division of Medical Genetics, Children’s Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA
R. G. Abellar
Department of Pathology, Columbia University Medical Center, New York, NY, USA
D. A. Bateman
Department of Pediatrics, Columbia University Medical Center, New York, NY, USA
A. L. Wilson
Children's Hospital of New York-Presbyterian, New York, NY, USA
M. H. Markham
Division of Neonatology, Division of Pediatrics, Vanderbilt University Medical Center, Nashville, TN, USA
J. Slamon
Department of Obstetrics and Gynecology, Division of Maternal Fetal Medicine, Vanderbilt University Medical Center, Nashville, TN, USA
F. Santos-Simarro M. Palomares, J. Nevado, P. Lapunzina
INGEMM,Instituto deGenéticaMédica y Molecular, IdiPAZ, Madrid, Spain
CIBERER, ISCIII, Madrid, Spain
B. H.-Y. Chung, W.-L.Wong, Y. W. Y. Chu, G. T. K. Mok
Department of Paediatrics and Adolescent Medicine, The University of Hong Kong, Hong Kong, China
B. H.-Y. Chung
Department of Obstetrics and Gynaecology, and Centre for Genomic Sciences, The University of Hong Kong, Hong Kong, China
E. Karem, J. Reiter
Pediatric Pulmonary Unit, Department of Pediatrics, Hadassah-Hebrew University Medical Center, Jerusalem, Israel
N. Ambalavanan
Departments of Pediatrics, University of Alabama at Birmingham, Alabama, USA
Cell Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, USA
S. A. Anderson
Department of Surgery, Division of Pediatric Surgery, University of Alabama at Birmingham and Children's of Alabama, Birmingham, AL, USA
D. R. Kelly
Department of Pathology, University of Alabama at Birmingham and Pathology and Laboratory Medicine Service, Children's of Alabama, Birmingham, AL, USA
J. Shieh
Division of Medical Genetics, Department of Pediatrics, and Institute for Human Genetics, University of California San Francisco, San Francisco, CA, USA
T. C. Rosenthal
Genetics Department, Kaiser Permanente San Jose Medical Center, San Jose, CA, USA
K. Scheible
Department of Pediatrics, University of Rochester, Rochester, NY, USA
L. Steiner
Division of Neonatology, University of Rochester, Rochester, NY, USA
M. A. Iqbal
Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA
M. L. McKinnon, S. J. Hamilton, K. Schlade-Bartusiak, D. English
Department of Medical Genetics, University of British Columbia, Vancouver, Canada
G. Hendson
Department of Pathology, University of British Columbia, Vancouver, Canada
E. R. Roeder, R. O. Littlejohn
Department of Pediatrics, Baylor College of Medicine, San Antonio, TX, USA
E. R. Roeder
Department of Molecular and Human Genetics, Baylor College of Medicine, San Antonio, TX, USA
T. S. DeNapoli
Department of Pathology, Children’s Hospital of San Antonio, San Antonio, TX, USA
D. J. Wolff
Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC, USA
C. L. Wagner
Department of Pediatrics, Medical University of South Carolina, Charleston, SC, USA
A. Yeung, D. Francis
Victorian Clinical Genetics Services, Murdoch Childrens Research Institute, Parkville, VIC, Australia
E. K. Fiorino
Division of Pediatric Pulmonary Medicine, The Children's Heart Center Steven and Alexandra Cohen Children's Medical Center of New York, New York, NY, USA
M. Edelman
Division of Pediatric Pathology, The Children's Heart Center Steven and Alexandra Cohen Children's Medical Center of New York, New York, NY, USA
J. Fox
Division of Medical Genetics, Steven and Alexandra Cohen Children's Medical Center of New York, New Hyde Park, New York, NY, USA
D. A. Hayes
Pediatric Cardiology, The Children's Heart Center Steven and Alexandra Cohen Children's Medical Center of New York, New York, NY, USA
S. Janssens, E. De Baere, B. Menten
Center for Medical Genetics, Ghent University and Ghent University Hospital, Ghent, Belgium
A. Loccufier
Department of Obstetrics, Gynaecology, and Fertility, AZ St Jan Brugge, Brugge, Begium
L. Vanwalleghem
Department of Anatomopathology, AZ St Jan Brugge, Brugge, Begium
P. Moerman
Department of Pathology UZ Leuven, Leuven, Belgium
Y. Sznajer
Center for Human Genetics, CliniquesUniversitaires St-Luc, UniversiteCatholique de Louvain, Brussels, Belgium
A. S. Lay
Division of Pediatric Cardiology, Children’s Mercy Hospital, Kansas City, MS, USA
J. L. Kussmann
Division of Clinical Genetics, Children’s Mercy Hospital, Kansas City, MS, USA
J. Chawla
Division of Paediatric Respiratory & Sleep Medicine, Lady Cilento Children’s Hospital, Children’s Health Queensland Hospital and HealthService, Brisbane, QLD, Australia
The University of Queensland, Brisbane, QLD, Australia
D. J. Payton, G. E. Phillips
Division of Anatomical Pathology, Lady Cilento Children’s Hospital, Children’s Health Queensland Hospital and HealthService, Brisbane, QLD, Australia
Pathology Queensland, Brisbane, QLD, Australia
E. Brosens, A. de Klein
Clinical Genetics Department, Erasmus MC-Sophia, Rotterdam, Netherlands
E. Brosens,D. Tibboel
Paediatric Surgery, Erasmus MC-Sophia, Rotterdam, Netherlands
I. Maystadt
Centre de GénétiqueHumaine, Institut de Pathologie et de Génétique, Gosselies, Belgium
R. Fisher
James Cook University Hospital, Middlesborough, UK
N. Sebire
Department of Paediatric Histopathology, Great Ormond Street Hospital for Children and UCL Institute of Child Health, London, UK
A. Male, M. Lees
Clinical Genetics Unit, Great Ormond Street Hospital for Children and UCL Institute of Child Health, London, UK
M. Chopra, J. Pinner,
Department of Medical Genomics, Royal Prince Alfred Hospital, Sydney, NSW, Australia
G. Malcolm
Department of Newborn Care, Royal Prince Alfred Hospital, Sydney, NSW, Australia
G. Peters
Cytogenetics Department, The Children’s Hospital at Westmead, Westmead, NSW, Australia
S. Arbuckle
Histopathology Department, The Children’s Hospital at Westmead, Westmead, NSW, Australia
Z. Mead
Department of Histopathology, Addenbrooke’s NHS Trust Pathology Department, Addenbrooke’s Hospital, Cambridge, UK
O. Quarrell, R. Sayers
Department of Clinical Genetics, Sheffield Children's Hospital, Sheffield, UK
M. Owens, C. Shaw-Smith
Molecular Genetics Department, Royal Devon and Exeter NHS Foundation Trust, Exeter, UK
J. Lioy
Division of Neonatology, The Children's Hospital of Philadelphia, The University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, USA
E. McKay
Department of Pathology, The Children's Hospital of Philadelphia,Philadelphia, PA, USA
N. de Leeuw, I. Feenstra, L. Spruijt
Department of Human Genetics, Radboud University Medical Center, Nijmegen, the Netherlands
F. Elmslie
South West Thames Regional Genetics Service, St George's University Hospital, London, UK
T. Thiruchelvam
Critical Care and Cardiorespiratory Unit, Great Ormond Street Hospital NHS Trust, London, UK
C. A. Bacino, J. R. Lupski
Texas Children’s Hospital, Houston, TX, USA
C. Langston, E. Popek
Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX, USA
P. Sen
Department of Pediatrics, Northwestern University, Chicago, IL, USA
METHODS
DNA isolation and sequencing
DNA was extracted from FFPE lung tissue or peripheral blood using MasterPure Complete DNA & RNA Purification Kit (Epicentre, Madison, WI, USA) or GentraPurgene Kit (Qiagen, Germantown, MD, USA), respectively. PCR products were treated with ExoSAP-IT (USB, Cleveland, OH, USA), and directly sequenced by the Sanger method (Lone Star Labs, Houston, TX, USA). Sequences were assembled using Sequencher v4.8 (GeneCodes, Ann Arbor, MI, USA).
RNA isolation and RT-qPCR
Gene expression was measured by estimating relative levels of mRNA. Total RNA was isolated from normal or transfected human foetal lung fibroblasts IMR-90 (ATCC, Manassas, VA, USA) using miRNeasy Mini Kit (Qiagen, Hilden, Germany). RNA preps were treated with DNase using DNA-free Kit (Ambion, Austin, TX, USA), and converted to cDNA using SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). RT-qPCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) or Power SYBR Green kit (Applied Biosystems, Warrington, UK). qPCR conditions included 40 cycles of 95°C for 15 sec and 60°C for 1 min.For relative quantification of a transcript, the comparative CT method was used.
Cloning of the 16q24.1 region deleted in patient 122 and of the FOXF1 promoter
To determine whether the deleted region can function as a promoter of LINC01082, we cloned the deleted 4.1 kb DNA fragment and measured its transcriptional activity in the IMR-90 cell line. For comparison, we also cloned the 3 kb FOXF1 promoter region, which exhibits residual promoter activity in the absence of enhancers. The 4.1 kb noncoding fragment (chr16:86,216,561-86,220,676) deleted in patient 122.3, was amplified from the normal human DNA using the following LR-PCR primers: 5`-AAGGTACCGGCATTTCTGTCACTCATTCAACAATCTGA-3` and 5`-AAGCTAGCGAGGTATGTTAGAGGAATAGAAGGACTGCCTTG-3`, which included the restriction sites for KpnI and NheI, respectively. PCR was performed using LA Taq DNA polymerase, applying 25 cycles of incubation at 94°C for 30 sec and at 68°C for 4 min. The amplified region was cut with KpnI and NheI, and cloned into KpnI and NheI sites of the multiple cloning site (MCS) of the promoter-less vector pGL4.10 (Promega) to generate the pGL4.10.122 construct.
An ~ 5.5 kb 16q24.1 region containing the FOXF1 promoter and the FOXF1 ATG initiation codon (chr16:86,538,679–86,544,175) was amplified from the normal human DNA using the LR-PCR primers: 5`-CTAGCTAGCACATTTCCTCATATTCTGTGTAGAGAGCACCT-3` and 5`-TTGCGCCGATTCGAACGGGTGGCTGCTG-3` that included the restriction sites for NheI and BstBI, respectively. PCR was run using LA Taq DNA polymerase in the presence of 20% betaine, applying 25 cycles of incubation at 94°C for 30 sec and at 68°C for 5 min. The amplified FOXF1 promoter region was subsequently cut with BstBI, blunt ends generated with DNA polymerase Klenow fragment, cut with NheI, and cloned into the EcoRV and NheI sites of the MCS of the pGL4.10 to generate the pGL4.10FOXpr construct.
Transcriptional activity assay
Transfections of the IMR-90 cells, were performed on the sub-confluent cells grown in 12-well plates. The IMR-90 cultures were maintained in the Eagle’s minimum essential medium (EMEM) supplemented with 2 mM L-glutamine and 10% FBS (ATCC). The cells were transfected with 1 μg of either pGL4.10.122, pGL4.10 (negative control), or pGL4.10FOXpr (positive control) using Lipofectamine 3000 with p3000 (Invitrogen). Total RNA was prepared 48 h after transfection following lysis of the cells in Triazol, and converted to cDNA.
The transcriptional activity of the cloned fragments was assayed by qPCR by measuring relative quantity of Renillaluciferase (Luc) cDNA. At least three biological replicates for each construct were analysed. The Luc cDNA levels were normalized to cDNA levels of the pGL4.10 Amp gene. qPCR primers (lucF 5`-GCACATATCGAGGTGGACATTA-3`, lucR 5`-CCACGATCCGATGGTTTGTAT-3`; ampF 5`-GCTGTCGTGATGCTAGAGTAAG-3`, ampR 5`-AGAGTTGAACGAAGCCATACC-3`) were designed using PrimerQuest (IDT, Coralville, IA, USA). cDNA synthesized using RNA isolated from IMR-90 cells transfected with the pGL4.10 plasmid was designated as a calibrator.
Bioinformatic analysis of the distant upstream enhancer region
Reference DNA sequences, chromatin modification, location of CpG islands, and ChIP-seq data for the selected transcription regulators were accessed using the UCSC Genome Browser ( GRCh37/hg19).
High-throughput chromosome conformation capture (Hi-C)(Lieberman-Aiden et al.2009) interaction datasets for Lymphoblastoid cell line from B-lymphocytes (GM12878), Human Umbilical Vein Endothelial Cells (HUVEC), Normal Human Epidermal Keratinocytes (NHEK), and Human Mammary Epithelial Cells (HMEC), and human fetal lung fibroblasts (IMR-90) cell lines were downloaded from the GEO database (GSE63525). Normalized 25 kb resolution Hi-C interaction matrices of chromosome 16 for the aforementioned five cell lines were generated by multiplying Knight and Ruiz normalization scores for two contacting loci and dividing raw observed values (MAPQGE30 filtered reads) at the interacting positions with this calculated normalization-score (Rao et al. 2014). IMR-90 cell line Pol2 ChIP-Seq was downloaded from UCSC Genome Browser ENCODE portal. Chromatin states calls (ChromHMM) were downloaded from 2015). HiCPlotter ( was used to plot Hi-C data with chromatin states(Akdemir et al. 2015).
Microarray analyses
In addition to custom aCGH, a number of probands were studied using various microarrays: Illumina CytoSNP-850k BeadChip (Illumina, San Diego, CA, USA) (pt 120.3), AffymetrixCytoscan 750K (pt 122.3), AffymetrixCytoScan HD array platform (Affymetrix Inc., Santa Clara,CA, USA) (pts 125.3, 126.3, 135.3, and 136.3), 12x135K array (Roche-NimbleGen, Madison, WI, USA) (pt 127.3), ISCA v.1 180K (Agilent Technologies, Santa Clara, CA, USA) (pts 128.3 and 133.3), and 4x180 K CGH-SNP array (Agilent Technologies) (pt. 139.3).
Whole exome sequencing
WES was performed at Baylor College of Medicine Human Genome Sequencing Center through the Baylor Hopkins Center for Mendelian Genomics as previously described (pts 114.3, 121.3)(Lupskiet al. 2013) and at the University of California Los Angeles (pt 128.3) (Lee et al. 2014). WES in family 138 was performed as described [Reiter et al. manuscript submitted].DNA sample, prepared into Illumina paired-end libraries, underwent whole exome capture using VCRome 2.1 design (Roche NimbleGen, Madison, WI, USA), followed by sequencing on the Illumina HiSeq 2000 platform (Illumina, San Diego, USA) with 78 paired-end reads coverage. Raw sequence data were post-processed using the Mercury pipeline(Reid et al. 2014), which performs conversion of raw sequencing data (bcl files) to the fastq format using Casava, mapping of the short reads against a human genome reference sequence (GRCh37) by the Burrows-Wheeler Alignment (BWA), recalibration using GATK(McKenna et al. 2010), and variant calling using the Atlas2 suite(Challis et al. 2012). Variants were annotated using the in-house-developed “Cassandra” annotation pipeline(Bainbridge et al. 2011). Gene expression data were analysed through a gene annotation portal BioGPS (
To search for regions of absence of heterozygosity (AOH) in the WES data, we calculated B-allele frequency as a ratio of variants reads to total reads. These data were then processed using the Circular Binary Segmentation algorithm (CBS)(Olshen et al. 2004). To detect CNVs in families sequenced at BH-CMG (121, 124), we processed WES data using CoNIFER software(Krumm et al. 2012). Control set required by CoNIFER was created based on WES data from 200 samples sequenced at BH-CMG using the same platform and processed with pipeline as ACDMPV families.
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