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Highly Sensitive Blocker Displacement Amplification and Droplet Digital PCR Reveal Low-Level Parental FOXF1 Somatic Mosaicism in Families with Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins

2020; Elsevier BV; Volume: 22; Issue: 4 Linguagem: Inglês

10.1016/j.jmoldx.2019.12.007

1943-7811

Justyna A. Karolak, Qian Liu, Nina G. Xie, Lucia R. Wu, Gustavo Rocha, Susana Fernandes, Luk Ho-Ming, Ivan F. M. Lo, David Mowat, Elizabeth K. Fiorino, Morris Edelman, Joyce E. Fox, Denise A. Hayes, David P. Witte, Ashley Parrott, Edwina J. Popek, Przemysław Szafrański, David Y. Zhang, Paweł Stankiewicz,

Renal and related cancers

Detection of low-level somatic mosaicism [alternate allele fraction (AAF) ≤ 10%] in parents of affected individuals with the apparent de novo pathogenic variants enables more accurate estimate of recurrence risk. To date, only a few systematic analyses of low-level parental somatic mosaicism have been performed. Herein, highly sensitive blocker displacement amplification, droplet digital PCR, quantitative PCR, long-range PCR, and array comparative genomic hybridization were applied in families with alveolar capillary dysplasia with misalignment of pulmonary veins. We screened 18 unrelated families with the FOXF1 variant previously determined to be apparent de novo (n = 14), of unknown parental origin (n = 1), or inherited from a parent suspected to be somatic and/or germline mosaic (n = 3). We identified four (22%) families with FOXF1 parental somatic mosaic single-nucleotide variants (n = 3) and copy number variant deletion (n = 1) detected in parental blood samples and an AAF ranging between 0.03% and 19%. In one family, mosaic allele ratio in tissues originating from three germ layers ranged between <0.03% and 0.65%. Because the ratio of parental somatic mosaicism have significant implications for the recurrence risk, this study further implies the importance of a systematic screening of parental samples for low-level and very-low–level (AAF ≤ 1%) somatic mosaicism using methods that are more sensitive than those routinely applied in diagnostics. Detection of low-level somatic mosaicism [alternate allele fraction (AAF) ≤ 10%] in parents of affected individuals with the apparent de novo pathogenic variants enables more accurate estimate of recurrence risk. To date, only a few systematic analyses of low-level parental somatic mosaicism have been performed. Herein, highly sensitive blocker displacement amplification, droplet digital PCR, quantitative PCR, long-range PCR, and array comparative genomic hybridization were applied in families with alveolar capillary dysplasia with misalignment of pulmonary veins. We screened 18 unrelated families with the FOXF1 variant previously determined to be apparent de novo (n = 14), of unknown parental origin (n = 1), or inherited from a parent suspected to be somatic and/or germline mosaic (n = 3). We identified four (22%) families with FOXF1 parental somatic mosaic single-nucleotide variants (n = 3) and copy number variant deletion (n = 1) detected in parental blood samples and an AAF ranging between 0.03% and 19%. In one family, mosaic allele ratio in tissues originating from three germ layers ranged between 70 distinct pathogenic or likely pathogenic single-nucleotide variants (SNVs) and 60 copy number variant (CNV) deletions, involving FOXF1 (forkhead box F1; Mendelian Inheritance in Man number 601089) or its lung-specific enhancer on 16q24.1, have been reported in 80% to 90% of ACDMPV families.20Szafranski P. Gambin T. Dharmadhikari A.V. Akdemir K.C. Jhangiani S.N. Schuette J. et al.Pathogenetics of alveolar capillary dysplasia with misalignment of pulmonary veins.Hum Genet. 2016; 135: 569-586Crossref PubMed Scopus (65) Google Scholar, 21Stankiewicz P. Sen P. Bhatt S.S. Storer M. Xia Z. Bejjani B.A. et al.Genomic and genic deletions of the FOX gene cluster on 16q24.1 and inactivating mutations of FOXF1 cause alveolar capillary dysplasia and other malformations.Am J Hum Genet. 2009; 84: 780-791Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 22Sen P. Gerychova R. Janku P. Jezova M. Valaskova I. Navarro C. Silva I. Langston C. Welty S. Belmont J. Stankiewicz P. A familial case of alveolar capillary dysplasia with misalignment of pulmonary veins supports paternal imprinting of FOXF1 in human.Eur J Hum Genet. 2013; 21: 474-477Crossref PubMed Scopus (37) Google Scholar, 23Szafranski P. Dharmadhikari A.V. Brosens E. Gurha P. Kolodziejska K.E. Zhishuo O. Dittwald P. Majewski T. Mohan K.N. Chen B. Person R.E. Tibboel D. de Klein A. Pinner J. Chopra M. Malcolm G. Peters G. Arbuckle S. Guiang S.F. Hustead V.A. Jessurun J. Hirsch R. Witte D.P. Maystadt I. Sebire N. Fisher R. Langston C. Sen P. Stankiewicz P. Small noncoding differentially methylated copy-number variants, including lncRNA genes, cause a lethal lung developmental disorder.Genome Res. 2013; 23: 23-33Crossref PubMed Scopus (109) Google Scholar, 24Szafranski P. Dharmadhikari A.V. Wambach J.A. Towe C.T. White F.V. Grady R.M. Eghtesady P. Cole F.S. Deutsch G. Sen P. Stankiewicz P. Two deletions overlapping a distant FOXF1 enhancer unravel the role of lncRNA LINC01081 in etiology of alveolar capillary dysplasia with misalignment of pulmonary veins.Am J Med Genet A. 2014; 164A: 2013-2019Crossref PubMed Scopus (43) Google Scholar, 25Nagano N. Yoshikawa K. Hosono S. Takahashi S. Nakayama T. Alveolar capillary dysplasia with misalignment of the pulmonary veins due to novel insertion mutation of FOXF1.Pediatr Int. 2016; 58: 1371-1372Crossref PubMed Scopus (6) Google Scholar, 26Ma Y. Jang M.A. Yoo H.S. Ahn S.Y. Sung S.I. Chang Y.S. Ki C.S. Park W.S. A novel de novo pathogenic variant in FOXF1 in a newborn with alveolar capillary dysplasia with misalignment of pulmonary veins.Yonsei Med J. 2017; 58: 672-675Crossref PubMed Scopus (9) Google Scholar, 27Everett K.V. Ataliotis P. Chioza B.A. Shaw-Smith C. Chung E.M.K. A novel missense mutation in the transcription factor FOXF1 cosegregating with infantile hypertrophic pyloric stenosis in the extended pedigree linked to IHPS5 on chromosome 16q24.Pediatr Res. 2017; 81: 632-638Crossref PubMed Scopus (9) Google Scholar, 28Abu-El-Haija A. Fineman J. Connolly A.J. Murali P. Judge L.M. Slavotinek A.M. Two patients with FOXF1 mutations with alveolar capillary dysplasia with misalignment of pulmonary veins and other malformations: two different presentations and outcomes.Am J Med Genet A. 2018; 176: 2877-2881Crossref PubMed Scopus (6) Google Scholar, 29Hayasaka I. Cho K. Akimoto T. Ikeda M. Uzuki Y. Yamada M. Nakata K. Furuta I. Ariga T. Minakami H. Genetic basis for childhood interstitial lung disease among Japanese infants and children.Pediatr Res. 2018; 83: 477-483Crossref PubMed Scopus (17) Google Scholar, 30Pradhan A. Dunn A. Ustiyan V. Bolte C. Wang G. Whitsett J.A. Zhang Y. Porollo A. Hu Y.-C. Xiao R. Szafranski P. Shi D. Stankiewicz P. Kalin T.V. Kalinichenko V.V. The S52F FOXF1 mutation inhibits STAT3 signaling and causes alveolar capillary dysplasia.Am J Respir Crit Care Med. 2019; 200: 1045-1056Crossref PubMed Scopus (33) Google Scholar The vast majority of ACDMPV cases are sporadic, with de novo FOXF1 variants being detected.20Szafranski P. Gambin T. Dharmadhikari A.V. Akdemir K.C. Jhangiani S.N. Schuette J. et al.Pathogenetics of alveolar capillary dysplasia with misalignment of pulmonary veins.Hum Genet. 2016; 135: 569-586Crossref PubMed Scopus (65) Google Scholar,31Sen P. Yang Y. Navarro C. Silva I. Szafranski P. Kolodziejska K.E. et al.Novel FOXF1 mutations in sporadic and familial cases of alveolar capillary dysplasia with misaligned pulmonary veins imply a role for its DNA binding domain.Hum Mutat. 2013; 34: 801-811Crossref PubMed Scopus (80) Google Scholar Only a few ACDMPV families with a pathogenic FOXF1 variant transmitted from a somatic mosaic or apparent heterozygous healthy parent have been reported.20Szafranski P. Gambin T. Dharmadhikari A.V. Akdemir K.C. Jhangiani S.N. Schuette J. et al.Pathogenetics of alveolar capillary dysplasia with misalignment of pulmonary veins.Hum Genet. 2016; 135: 569-586Crossref PubMed Scopus (65) Google Scholar,22Sen P. Gerychova R. Janku P. Jezova M. Valaskova I. Navarro C. Silva I. Langston C. Welty S. Belmont J. Stankiewicz P. A familial case of alveolar capillary dysplasia with misalignment of pulmonary veins supports paternal imprinting of FOXF1 in human.Eur J Hum Genet. 2013; 21: 474-477Crossref PubMed Scopus (37) Google Scholar,31Sen P. Yang Y. Navarro C. Silva I. Szafranski P. Kolodziejska K.E. et al.Novel FOXF1 mutations in sporadic and familial cases of alveolar capillary dysplasia with misaligned pulmonary veins imply a role for its DNA binding domain.Hum Mutat. 2013; 34: 801-811Crossref PubMed Scopus (80) Google Scholar, 32Reiter J. Szafranski P. Breuer O. Perles Z. Dagan T. Stankiewicz P. Kerem E. Variable phenotypic presentation of a novel FOXF1 missense mutation in a single family.Pediatr Pulmonol. 2016; 51: 921-927Crossref PubMed Scopus (11) Google Scholar, 33Luk H.M. Tang T. Choy K.W.R. Tong M.F.T. Wong O.K. Lo F.M.I. Maternal somatic mosaicism of FOXF1 mutation causes recurrent alveolar capillary dysplasia with misalignment of pulmonary veins in siblings.Am J Med Genet A. 2016; 170: 1942-1944Crossref PubMed Scopus (9) Google Scholar, 34Alsina Casanova M. Monteagudo-Sánchez A. Rodiguez Guerineau L. Court F. Gazquez Serrano I. Martorell L. Rovira Zurriaga C. Moore G.E. Ishida M. Castañon M. Moliner Calderon E. Monk D. Moreno Hernando J. Maternal mutations of FOXF1 cause alveolar capillary dysplasia despite not being imprinted.Hum Mutat. 2017; 38: 615-620Crossref PubMed Scopus (11) Google Scholar To examine the efficacy of the applied techniques as well as the scale and ratio of parental somatic mosaicism in families with ACDMPV, 18 families with a known FOXF1 variant were studied retrospectively. The DNA samples studied were from parents of 18 unrelated index ACDMPV patients with a known pathogenic FOXF1 SNV (n = 12), insertion/deletion (n = 5), or CNV deletion (n = 1), detected during the standard diagnostic procedure. On the basis of PCR and Sanger sequencing, these variants were originally determined to be apparent de novo (alternate allele was not detected in the parents; n = 14), of unknown parental origin (parents were not tested; n = 1), or inherited from a parent suspected to be somatic and/or germline mosaic (alternate allele was present in the parent, but the precise allelic ratio was not determined and/or alternate allele was not detected in the parents, but the family pedigree suggested the presence of germline mosaicism; n = 3) (Figure 1).20Szafranski P. Gambin T. Dharmadhikari A.V. Akdemir K.C. Jhangiani S.N. Schuette J. et al.Pathogenetics of alveolar capillary dysplasia with misalignment of pulmonary veins.Hum Genet. 2016; 135: 569-586Crossref PubMed Scopus (65) Google Scholar,21Stankiewicz P. Sen P. Bhatt S.S. Storer M. Xia Z. Bejjani B.A. et al.Genomic and genic deletions of the FOX gene cluster on 16q24.1 and inactivating mutations of FOXF1 cause alveolar capillary dysplasia and other malformations.Am J Hum Genet. 2009; 84: 780-791Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar,31Sen P. Yang Y. Navarro C. Silva I. Szafranski P. Kolodziejska K.E. et al.Novel FOXF1 mutations in sporadic and familial cases of alveolar capillary dysplasia with misaligned pulmonary veins imply a role for its DNA binding domain.Hum Mutat. 2013; 34: 801-811Crossref PubMed Scopus (80) Google Scholar Only the families in whom both parental and proband DNA samples were available, and for whom it was possible to design the BDA, ddPCR, or qPCR assays, were included in this study after obtaining informed consent. The study protocol was approved by the Institutional Review Board for Human Subject Research at Baylor College of Medicine (Houston, TX; H-8712 and H-28088). Genomic DNA was previously extracted from peripheral blood, saliva, and frozen or formalin-fixed, paraffin-embedded lung tissue using Gentra Purgene Blood Kit (Qiagen, Germantown, MD), prepIT•L2P/PT-L2P kit (DNA GenoTek, Ottawa, ON, Canada), and DNeasy Blood and Tissue Kit (Qiagen), respectively, as described.20Szafranski P. Gambin T. Dharmadhikari A.V. Akdemir K.C. Jhangiani S.N. Schuette J. et al.Pathogenetics of alveolar capillary dysplasia with misalignment of pulmonary veins.Hum Genet. 2016; 135: 569-586Crossref PubMed Scopus (65) Google Scholar,31Sen P. Yang Y. Navarro C. Silva I. Szafranski P. Kolodziejska K.E. et al.Novel FOXF1 mutations in sporadic and familial cases of alveolar capillary dysplasia with misaligned pulmonary veins imply a role for its DNA binding domain.Hum Mutat. 2013; 34: 801-811Crossref PubMed Scopus (80) Google Scholar DNA from urine, buccal cells, and hair follicles (family 176) was isolated with Gentra Purgene Blood Kit (Qiagen), prepIT•L2P/PT-L2P kit (DNA GenoTek), and QIAamp DNA Investigator Kit (Qiagen), respectively, according to the manufacturer's instructions. To study CNV deletion in family 176, array comparative genomic hybridization analysis was performed using customized 16q24.1-specific (1 Mb region flanking FOXF1) high-resolution 180K microarray (Agilent Technologies, Santa Clara, CA), as described.20Szafranski P. Gambin T. Dharmadhikari A.V. Akdemir K.C. Jhangiani S.N. Schuette J. et al.Pathogenetics of alveolar capillary dysplasia with misalignment of pulmonary veins.Hum Genet. 2016; 135: 569-586Crossref PubMed Scopus (65) Google Scholar,23Szafranski P. Dharmadhikari A.V. Brosens E. Gurha P. Kolodziejska K.E. Zhishuo O. Dittwald P. Majewski T. Mohan K.N. Chen B. Person R.E. Tibboel D. de Klein A. Pinner J. Chopra M. Malcolm G. Peters G. Arbuckle S. Guiang S.F. Hustead V.A. Jessurun J. Hirsch R. Witte D.P. Maystadt I. Sebire N. Fisher R. Langston C. Sen P. Stankiewicz P. Small noncoding differentially methylated copy-number variants, including lncRNA genes, cause a lethal lung developmental disorder.Genome Res. 2013; 23: 23-33Crossref PubMed Scopus (109) Google Scholar Deletion junction fragment was amplified by long-range PCR with LA Taq DNA polymerase (TaKaRa Bio, Madison, WI), followed by Sanger sequencing. To determine the alternate allele fraction (AAF) in parental samples, 17 families (Table 1) were tested using BDA or qPCR using the probands' DNA samples as positive controls. BDA principles were described in detail by Wu et al35Wu L.R. Chen S.X. Wu Y. Patel A.A. Zhang D.Y. Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification.Nat Biomed Eng. 2017; 1: 714-723Crossref PubMed Scopus (56) Google Scholar (2017). The workflow of BDA data analysis is shown in Supplemental Figure S1.Table 1The List of Studied Families with Distribution and Parental Origin of FOXF1 Pathogenic VariantsFamily IDDNA variantProtein variantParental origin of FOXF1 pathogenic variantsSanger sequencing (original detection method)BDA or qPCR% Of variant allele detected in parent (type of tissue)ddPCR% Of variant allele detected in parent (type of tissue)2c.225C>Ap.Tyr75*De novoDe novo0.0N/AN/A46c.1031_1032delp.Phe344Cysfs*66De novoDe novo0.0N/AN/A48c.1138T>Cp.*380Argext*73De novoDe novo0.0N/AN/A55c.145C>Tp.Pro49SerDe novoDe novo0.0N/AN/A56c.89C>Ap.Ser30*De novoDe novo0.0N/AN/A61c.872_879delp.Ser291*De novoDe novo0.0N/AN/A66c.899_903dupp.Gly302Cysfs*79De novoDe novo0.0N/AN/A67c.191C>Ap.Ser64*De novoDe novo0.0N/AN/A69c.691_698delp.Ala231Argfs*61De novoDe novo0.0N/AN/A76c.1139G>Cp.*380Serext*73De novoDe novo0.0N/AN/A83c.221T>Ap.Ile74AsnDe novoDe novo0.0N/AN/A85c.539C>Ap.Ser180*De novoMaternal1.5 (Blood)Maternal1.0 (Blood)91c.294C>Ap.His98GlnMaternalMaternal19.0 (Blood)N/AN/A101c.862C>Tp.Gln288*De novoDe novo0.0N/AN/A105c.316T>Cp.Phe106LeuUnknownPaternal0.03 (Blood)Mosaicism not confirmedN/A123c.849_850delp.Ile285Glnfs*9Probably maternalN/AN/AProbably maternal germline mosaicism0.0 (Blood)1767-kb CNV deletion involving FOXF1 chr16:86,542,131-86,549,266(hg19)MaternalMaternal0.2 (Saliva)0.14 (Redrawn saliva)0.04 (Blood) Gp.Pro49AlaDe novoDe novo0.0N/AN/AFamilies with parental somatic mosaicism determined by BDA, ddPCR, or qPCR are in bold. The array comparative genomic hybridization data were deposited in the dbVar database (https://www.ncbi.nlm.nih.gov/dbvar, accession number nstd178). The sequence variant data were submitted to the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar, submission ID SUB6388016, accessions numbers SCV001055831 to SCV001055847).BDA, blocker displacement amplification; chr, chromosome; ddPCR, droplet digital PCR; ID, identifier; N/A, not available (not tested); qPCR, quantitative PCR. Open table in a new tab Families with parental somatic mosaicism determined by BDA, ddPCR, or qPCR are in bold. The array comparative genomic hybridization data were deposited in the dbVar database (https://www.ncbi.nlm.nih.gov/dbvar, accession number nstd178). The sequence variant data were submitted to the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar, submission ID SUB6388016, accessions numbers SCV001055831 to SCV001055847). BDA, blocker displacement amplification; chr, chromosome; ddPCR, droplet digital PCR; ID, identifier; N/A, not available (not tested); qPCR, quantitative PCR. Primer and blocker sequences (Table 2) were designed according to the previously developed protocol.35Wu L.R. Chen S.X. Wu Y. Patel A.A. Zhang D.Y. Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification.Nat Biomed Eng. 2017; 1: 714-723Crossref PubMed Scopus (56) Google Scholar To prevent unspecific binding of primers to FOXF2, a highly similar genomic sequence to FOXF1, primers used in BDA experiments were not fully complementary to the FOXF2 and thus have much weaker binding energy to FOXF2. Moreover, the short extension time (30 seconds) prevented amplification of longer, potentially nonspecific amplicons (Supplemental Figure S2). Sanger sequencing of the amplified products further confirmed the specificity of all primers.Table 2BDA and qPCR Oligonucleotide SequencesFamily IDNameNucleotide sequence2Forward primer5′-GCCTGACGCTGAGCGAGATCTA-3′Reverse primer5′-AAGGCCCTTGGGTAGCTTGATG-3′Blocker5′-AGCGAGATCTACCAGTTCCTGCAGAGCCaaaa-3′46Forward primer5′-CCCAGCATGTGTGACCGAAA-3′Reverse primer5′-GCAGCCTCACATCACGCAA-3′Blocker5′-TGACCGAAAGGAGTTTGTCTTCTCTTTCAACaaat-3′48Forward primer5′-TCACCTACCAAGACATCAAGCC-3′Reverse primer5′-CGACGGTTATACCTCGAGAAGAAAG-3′Blocker5′-ATCAAGCCTTGCGTGATGTGAGGCaaaa-3′55Forward primer5′-CCGGCGCCCGGAGAA-3′Reverse primer5′-GGTAGGAGCCCCGGAAGAAG-3′Blocker5′-CGGAGAAGCCGCCCTATTCCTACAaaaa-3′56Forward primer5′-CGGCCATGGACCCCGC-3′Reverse primer5′-GCGCTTGGTGGGTGAACTCT-3′Blocker5′-CCCGCGTCGTCCGGCCCGaaat-3′61Forward primer5′-GGCGCCTCTTATATCAAGCAGCA-3′Reverse primer5′-GGCGTTGTGGCTGTTCTGGT-3′Blocker5′-AAGCAGCAGCCCCTGTCCCCCTaatt-3′66Forward primer5′-CCTGTAACCCCGCGGCCA-3′Reverse primer5′-CGGCCTCCCCACTCACCTT-3′Blocker5′-CGGCCAACCCCCTGTCCGGCAaaaa-3′67Forward primer5′-CGCGCTCATCGTCATGGC-3′Reverse primer5′-GGTAGGAGCCCCGGAAGAA-3′Blocker5′-GTCATGGCCATCCAGAGTTCACCCACatta-3′69Forward primer5′-ACATGGGCGGCTGCGG-3′Reverse primer5′-CGCCGAGCCCGAGTAGAC-3′Blocker5′-CTGCGGCGGCGCGGCGaaaa-3′76Forward primer5′-GTCACCTACCAAGACATCAAGCCT-3′Reverse primer5′-GACGGTTATACCTCGAGAAGAAAGCA-3′Blocker5′-ATCAAGCCTTGCGTGATGTGAGGCTGaaaa-3′83Forward primer5′-ACCCACCAAGCGCCTGAC-3′Reverse primer5′-AAGGCCCTTGGGTAGCTTGATG-3′Blocker5′-GCCTGACGCTGAGCGAGATCTACCAaaaa-3′85Forward primer5′-GGGCTCGGCCGGCG-3′Reverse primer5′-CGTTGGAAGGCAGGTGGGG-3′Blocker5′-CGGCGGCCTCTCGTGCCCGaaat-3′91Forward primer5′-AGGGCTGGAAGAACTCCGT-3′Reverse primer5′-CTCCTCGAACATGAACTCGCT-3′Blocker5′-AACTCCGTGCGCCACAACCTCTaaaa-3′101Forward primer5′-CAACAGCGGCGCCTCTTATATCAReverse primer5′-GGCGTTGTGGCTGTTCTGGT-3′Blocker5′-GCCTCTTATATCAAGCAGCAGCCCCTGTaaaa-3′105Forward primer5′-CCTCTCGCTCAACGAGTGC-3′Reverse primer5′-TGGCATTTCCTTCGGAAGCC-3′Blocker5′-CGAGTGCTTCATCAAGCTACCCAAGGaaaa-3′176Forward primer5′-GCCTCTCGCCCCAGCTC-3′Reverse primer5′-CGCAGTTGGGTTTCTCCTAATCA-3′BlockerNone182Forward primer5′-CCGGCGCCCGGAGAAG-3′Reverse primer5′-CTGGTAGGAGCCCCGGAAGAA-3′Blocker5′-CGGAGAAGCCGCCCTATTCCTACATCGaaaa-3′Primers and blockers are standard desalted DNA oligonucleotides with no chemical modification. Four lowercase bases at the -3′ of the blocker do not match the template, thus preventing the blocker oligonucleotide from being extended by the polymerase.BDA, blocker displacement amplification; ID, identifier; qPCR, quantitative PCR.