Advertisement

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

Open ArchivePublished:February 06, 2020DOI:https://doi.org/10.1016/j.jmoldx.2019.12.007
      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.
      During the past decade, growing evidence on the importance of somatic mosaicism in etiology of several human genetic conditions, including cancers and neurodevelopmental diseases, has been reported.
      • Michaelson J.J.
      • Shi Y.
      • Gujral M.
      • Zheng H.
      • Malhotra D.
      • Jin X.
      • Jian M.
      • Liu G.
      • Greer D.
      • Bhandari A.
      • Wu W.
      • Corominas R.
      • Peoples A.
      • Koren A.
      • Gore A.
      • Kang S.
      • Lin G.N.
      • Estabillo J.
      • Gadomski T.
      • Singh B.
      • Zhang K.
      • Akshoomoff N.
      • Corsello C.
      • McCarroll S.
      • Iakoucheva L.M.
      • Li Y.
      • Wang J.
      • Sebat J.
      Whole-genome sequencing in autism identifies hot spots for de novo germline mutation.
      • Watson I.R.
      • Takahashi K.
      • Futreal P.A.
      • Chin L.
      Emerging patterns of somatic mutations in cancer.
      • Jamuar S.S.
      • Lam A.-T.N.
      • Kircher M.
      • D’Gama A.M.
      • Wang J.
      • Barry B.J.
      • Zhang X.
      • Hill R.S.
      • Partlow J.N.
      • Rozzo A.
      • Servattalab S.
      • Mehta B.K.
      • Topcu M.
      • Amrom D.
      • Andermann E.
      • Dan B.
      • Parrini E.
      • Guerrini R.
      • Scheffer I.E.
      • Berkovic S.F.
      • Leventer R.J.
      • Shen Y.
      • Wu B.L.
      • Barkovich A.J.
      • Sahin M.
      • Chang B.S.
      • Bamshad M.
      • Nickerson D.A.
      • Shendure J.
      • Poduri A.
      • Yu T.W.
      • Walsh C.A.
      Somatic mutations in cerebral cortical malformations.
      • Campbell I.M.
      • Shaw C.A.
      • Stankiewicz P.
      • Lupski J.R.
      Somatic mosaicism: implications for disease and transmission genetics.
      • Yang X.
      • Liu A.
      • Xu X.
      • Yang X.
      • Zeng Q.
      • Ye A.Y.
      • Yu Z.
      • Wang S.
      • Huang A.Y.
      • Wu X.
      • Wu Q.
      • Wei L.
      • Zhang Y.
      Genomic mosaicism in paternal sperm and multiple parental tissues in a Dravet syndrome cohort.
      • Demily C.
      • Hubert L.
      • Franck N.
      • Poisson A.
      • Munnich A.
      • Besmond C.
      Somatic mosaicism for SLC1A1 mutation supports threshold effect and familial aggregation in schizophrenia spectrum disorders.
      • Tarilonte M.
      • Morín M.
      • Ramos P.
      • Galdós M.
      • Blanco-Kelly F.
      • Villaverde C.
      • Rey-Zamora D.
      • Rebolleda G.
      • Muñoz-Negrete F.J.
      • Tahsin-Swafiri S.
      • Gener B.
      • Moreno-Pelayo M.-A.
      • Ayuso C.
      • Villamar M.
      • Corton M.
      Parental mosaicism in PAX6 causes intra-familial variability: implications for genetic counseling of congenital aniridia and microphthalmia.
      • de Lange I.M.
      • Koudijs M.J.
      • van ’t Slot R.
      • Sonsma A.C.M.
      • Mulder F.
      • Carbo E.C.
      • van Kempen M.J.A.
      • Nijman I.J.
      • Ernst R.F.
      • Savelberg S.M.C.
      • Knoers N.V.A.M.
      • Brilstra E.H.
      • Koeleman B.P.C.
      Assessment of parental mosaicism in SCN1A-related epilepsy by single-molecule molecular inversion probes and next-generation sequencing.
      • Legrand A.
      • Devriese M.
      • Dupuis-Girod S.
      • Simian C.
      • Venisse A.
      • Mazzella J.M.
      • Auribault K.
      • Adham S.
      • Frank M.
      • Albuisson J.
      • Jeunemaitre X.
      Frequency of de novo variants and parental mosaicism in vascular Ehlers-Danlos syndrome.
      • Wright C.F.
      • Prigmore E.
      • Rajan D.
      • Handsaker J.
      • McRae J.
      • Kaplanis J.
      • Fitzgerald T.W.
      • FitzPatrick D.R.
      • Firth H.V.
      • Hurles M.E.
      Clinically-relevant postzygotic mosaicism in parents and children with developmental disorders in trio exome sequencing data.
      • Cao Y.
      • Tokita M.J.
      • Chen E.S.
      • Ghosh R.
      • Chen T.
      • Feng Y.
      • Gorman E.
      • Gibellini F.
      • Ward P.A.
      • Braxton A.
      • Wang X.
      • Meng L.
      • Xiao R.
      • Bi W.
      • Xia F.
      • Eng C.M.
      • Yang Y.
      • Gambin T.
      • Shaw C.
      • Liu P.
      • Stankiewicz P.
      A clinical survey of mosaic single nucleotide variants in disease-causing genes detected by exome sequencing.
      However, somatic mosaic variants have been also detected in clinically unremarkable or mildly affected individuals, including parents of subjects with genetic conditions.
      • Campbell I.M.
      • Yuan B.
      • Robberecht C.
      • Pfundt R.
      • Szafranski P.
      • McEntagart M.E.
      • Nagamani S.C.S.
      • Erez A.
      • Bartnik M.
      • Wiśniowiecka-Kowalnik B.
      • Plunkett K.S.
      • Pursley A.N.
      • Kang S.-H.L.
      • Bi W.
      • Lalani S.R.
      • Bacino C.A.
      • Vast M.
      • Marks K.
      • Patton M.
      • Olofsson P.
      • Patel A.
      • Veltman J.A.
      • Cheung S.W.
      • Shaw C.A.
      • Vissers L.E.L.M.
      • Vermeesch J.R.
      • Lupski J.R.
      • Stankiewicz P.
      Parental somatic mosaicism is underrecognized and influences recurrence risk of genomic disorders.
      ,
      • Huang A.Y.
      • Xu X.
      • Ye A.Y.
      • Wu Q.
      • Yan L.
      • Zhao B.
      • Yang X.
      • He Y.
      • Wang S.
      • Zhang Z.
      • Gu B.
      • Zhao H.-Q.
      • Wang M.
      • Gao H.
      • Gao G.
      • Zhang Z.
      • Yang X.
      • Wu X.
      • Zhang Y.
      • Wei L.
      Postzygotic single-nucleotide mosaicisms in whole-genome sequences of clinically unremarkable individuals.
      The incidence and ratio of parental somatic mosaicism have important implications for the recurrence risk,
      • Ju Y.S.
      • Martincorena I.
      • Gerstung M.
      • Petljak M.
      • Alexandrov L.B.
      • Rahbari R.
      • et al.
      Somatic mutations reveal asymmetric cellular dynamics in the early human embryo.
      ,
      • Jónsson H.
      • Sulem P.
      • Arnadottir G.A.
      • Pálsson G.
      • Eggertsson H.P.
      • Kristmundsdottir S.
      • Zink F.
      • Kehr B.
      • Hjorleifsson K.E.
      • Jensson B.Ö.
      • Jonsdottir I.
      • Marelsson S.E.
      • Gudjonsson S.A.
      • Gylfason A.
      • Jonasdottir A.
      • Jonasdottir A.
      • Stacey S.N.
      • Magnusson O.T.
      • Thorsteinsdottir U.
      • Masson G.
      • Kong A.
      • Halldorsson B.V.
      • Helgason A.
      • Gudbjartsson D.F.
      • Stefansson K.
      Multiple transmissions of de novo mutations in families.
      because both affected and unaffected carriers of the pathogenic mosaic variant can transmit it to their children if it is also present in germline cells.
      • Campbell I.M.
      • Shaw C.A.
      • Stankiewicz P.
      • Lupski J.R.
      Somatic mosaicism: implications for disease and transmission genetics.
      ,
      • Wright C.F.
      • Prigmore E.
      • Rajan D.
      • Handsaker J.
      • McRae J.
      • Kaplanis J.
      • Fitzgerald T.W.
      • FitzPatrick D.R.
      • Firth H.V.
      • Hurles M.E.
      Clinically-relevant postzygotic mosaicism in parents and children with developmental disorders in trio exome sequencing data.
      ,
      • Cao Y.
      • Tokita M.J.
      • Chen E.S.
      • Ghosh R.
      • Chen T.
      • Feng Y.
      • Gorman E.
      • Gibellini F.
      • Ward P.A.
      • Braxton A.
      • Wang X.
      • Meng L.
      • Xiao R.
      • Bi W.
      • Xia F.
      • Eng C.M.
      • Yang Y.
      • Gambin T.
      • Shaw C.
      • Liu P.
      • Stankiewicz P.
      A clinical survey of mosaic single nucleotide variants in disease-causing genes detected by exome sequencing.
      However, mainly because of technical limitations, only a few systematic studies on the real incidence of somatic mosaicism in parents of affected individuals with apparent de novo events have been performed.
      • Yang X.
      • Liu A.
      • Xu X.
      • Yang X.
      • Zeng Q.
      • Ye A.Y.
      • Yu Z.
      • Wang S.
      • Huang A.Y.
      • Wu X.
      • Wu Q.
      • Wei L.
      • Zhang Y.
      Genomic mosaicism in paternal sperm and multiple parental tissues in a Dravet syndrome cohort.
      ,
      • Campbell I.M.
      • Yuan B.
      • Robberecht C.
      • Pfundt R.
      • Szafranski P.
      • McEntagart M.E.
      • Nagamani S.C.S.
      • Erez A.
      • Bartnik M.
      • Wiśniowiecka-Kowalnik B.
      • Plunkett K.S.
      • Pursley A.N.
      • Kang S.-H.L.
      • Bi W.
      • Lalani S.R.
      • Bacino C.A.
      • Vast M.
      • Marks K.
      • Patton M.
      • Olofsson P.
      • Patel A.
      • Veltman J.A.
      • Cheung S.W.
      • Shaw C.A.
      • Vissers L.E.L.M.
      • Vermeesch J.R.
      • Lupski J.R.
      • Stankiewicz P.
      Parental somatic mosaicism is underrecognized and influences recurrence risk of genomic disorders.
      ,
      • Huang A.Y.
      • Xu X.
      • Ye A.Y.
      • Wu Q.
      • Yan L.
      • Zhao B.
      • Yang X.
      • He Y.
      • Wang S.
      • Zhang Z.
      • Gu B.
      • Zhao H.-Q.
      • Wang M.
      • Gao H.
      • Gao G.
      • Zhang Z.
      • Yang X.
      • Wu X.
      • Zhang Y.
      • Wei L.
      Postzygotic single-nucleotide mosaicisms in whole-genome sequences of clinically unremarkable individuals.
      Herein, highly sensitive blocker displacement amplification (BDA), droplet digital PCR (ddPCR), quantitative PCR (qPCR), long-range PCR, and customized array comparative genomic hybridization were applied in families with alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV; Mendelian Inheritance in Man number 265380). ACDMPV is a rare neonatal lethal lung developmental disorder, characterized by unique histopathologic features.
      • Janney C.G.
      • Askin F.B.
      • Kuhn C.
      Congenital alveolar capillary dysplasia: an unusual cause of respiratory distress in the newborn.
      • Langston C.
      Misalignment of pulmonary veins and alveolar capillary dysplasia.
      • Sen P.
      • Thakur N.
      • Stockton D.W.
      • Langston C.
      • Bejjani B.A.
      Expanding the phenotype of alveolar capillary dysplasia (ACD).
      • Bishop N.B.
      • Stankiewicz P.
      • Steinhorn R.H.
      Alveolar capillary dysplasia.
      • Szafranski 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.
      To date, >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.
      • Szafranski 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.
      • Stankiewicz 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.
      • Sen 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.
      • Szafranski 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.
      • Szafranski 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.
      • Nagano 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.
      • Ma 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.
      • Everett 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.
      • Abu-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.
      • Hayasaka 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.
      • Pradhan 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.
      The vast majority of ACDMPV cases are sporadic, with de novo FOXF1 variants being detected.
      • Szafranski 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.
      ,
      • Sen 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.
      Only a few ACDMPV families with a pathogenic FOXF1 variant transmitted from a somatic mosaic or apparent heterozygous healthy parent have been reported.
      • Szafranski 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.
      ,
      • Sen 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.
      ,
      • Sen 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.
      • Reiter 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.
      • Luk 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.
      • Alsina 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.
      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.

      Materials and Methods

       Subjects

      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).
      • Szafranski 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.
      ,
      • Stankiewicz 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.
      ,
      • Sen 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.
      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).
      Figure thumbnail gr1
      Figure 1Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) families with FOXF1 variants included in this study. To precisely determine the frequency of variant allele in parental DNA, 18 families with FOXF1 variants previously indicated to be de novo, of unknown parental origin, or inherited were tested using blocker displacement amplification (BDA), droplet digital PCR (ddPCR), quantitative PCR (qPCR), long-range PCR, and array comparative genomic hybridization (array CGH). Flowchart was generated using https://www.draw.io (last accessed December 16, 2019). n = 14 (de novo variants); n = 1 (variant of unknown parental origin); n = 3 (inherited variants).

       DNA Extraction

      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.
      • Szafranski 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.
      ,
      • Sen 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.
      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.

       CNV Deletion Analysis

      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.
      • Szafranski 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.
      ,
      • Szafranski 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.
      Deletion junction fragment was amplified by long-range PCR with LA Taq DNA polymerase (TaKaRa Bio, Madison, WI), followed by Sanger sequencing.

       BDA and qPCR Experiments

      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 al
      • Wu 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.
      (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 Variants
      Family IDDNA variantProtein variantParental origin of FOXF1 pathogenic variants
      Sanger 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/A
      46c.1031_1032delp.Phe344Cysfs*66De novoDe novo0.0N/AN/A
      48c.1138T>Cp.*380Argext*73De novoDe novo0.0N/AN/A
      55c.145C>Tp.Pro49SerDe novoDe novo0.0N/AN/A
      56c.89C>Ap.Ser30*De novoDe novo0.0N/AN/A
      61c.872_879delp.Ser291*De novoDe novo0.0N/AN/A
      66c.899_903dupp.Gly302Cysfs*79De novoDe novo0.0N/AN/A
      67c.191C>Ap.Ser64*De novoDe novo0.0N/AN/A
      69c.691_698delp.Ala231Argfs*61De novoDe novo0.0N/AN/A
      76c.1139G>Cp.*380Serext*73De novoDe novo0.0N/AN/A
      83c.221T>Ap.Ile74AsnDe novoDe novo0.0N/AN/A
      85c.539C>Ap.Ser180*De novoMaternal1.5 (Blood)Maternal1.0 (Blood)
      91c.294C>Ap.His98GlnMaternalMaternal19.0 (Blood)N/AN/A
      101c.862C>Tp.Gln288*De novoDe novo0.0N/AN/A
      105c.316T>Cp.Phe106LeuUnknownPaternal0.03 (Blood)Mosaicism not confirmedN/A
      123c.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)

      <0.03 (Urine)

      0.65 (Buccal)
      N/AN/A
      182c.145C>Gp.Pro49AlaDe novoDe novo0.0N/AN/A
      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.
      • Wu 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.
      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 Sequences
      Family IDNameNucleotide sequence
      2Forward 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′-CAACAGCGGCGCCTCTTATATCA
      Reverse 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′
      BlockerNone
      182Forward 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.
      The patient, mother, and father genomic DNA samples were tested with blocker (ie, standard BDA) and without blocker (ie, forward and reverse primers only). The qPCR assays were performed using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA) with 400 nmol/L of each primer, 4 μmol/L of blocker, and 10 ng of DNA per well. For GC-rich amplicons, betaine was added to a final concentration of 1 mol/L (Sigma Aldrich, St. Louis, MO) to reduce template secondary structures (Supplemental Table S1). Reactions were performed in the final volume of 10 μL using CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA) with incubation at 95°C for 180 seconds, followed by 60 cycles of 95°C for 10 seconds and 60°C for 30 seconds. Each qPCR was repeated at least twice.
      Change in quantification cycle (ΔCq) values were calculated for each sample using Cq values obtained in both experiments (with and without blocker). First, the ΔCqsamp was calculated for each sample: ΔCqsamp = (median with blocker Cq) − (median no-blocker Cq).
      All calculated Cq and ΔCqsamp values are shown in Supplemental Table S2. A smaller ΔCqsamp indicates a higher likeliness of the sample containing a mutation.
      Next, PCR products from two replicated qPCR experiments for the parental sample with smaller ΔCqsamp were purified and Sanger sequenced. Because it is extremely unlikely that both parents carry the same pathogenic variant, the PCR product from the other parent was not sequenced. To avoid false positives caused by Taq polymerase errors, a sample was called as positive when the variant appeared in both of the duplicate Sanger results. If the presence of alternate allele was confirmed by Sanger sequencing (Figure 2), the qPCR Cq values were used to calculate the AAF. AAF was calculated as follows:
      AAF=50%2ΔCqsamp,parentΔCqsamp,patient
      (1)


      where ΔCqsamp,parent is the ΔCqsamp of the parent sample with a positive result, and ΔCqsamp,patient is the ΔCqsamp of the corresponding patient sample.
      Figure thumbnail gr2
      Figure 2Four families with detected FOXF1 somatic mosaicism. A and B: Pedigrees of families 85 (A) and 105 (B) with chromatopherograms of quantitative PCR (qPCR) Sanger sequencing [blocker displacement amplification (BDA)] and absolute quantification of the allele abundance [droplet digital PCR (ddPCR)]. Chromatopherograms display presence of alternate allele in maternal (A) and paternal (B) samples. The upper ddPCR plots show the examples of droplet counts positive for the alternate (blue bars) and wild-type (WT; yellow bars) allele and the total droplet counts (gray bars) in single reactions. The number of droplets is presented in a logarithmic scale. The lower ddPCR plots present alternate allele fraction (AAF) and example of the one-dimensional fluorescence amplitude plot of droplets for alternate allele detection. The positive droplets (blue dots), containing the alternate allele, exhibit increased fluorescence compared with negative droplets (dark gray dots). The threshold line dividing positive and negative droplets is presented (pink lines). C: Pedigree of family 91 with chromatopherograms of qPCR Sanger sequencing (BDA) showing presence of alternate allele in maternal sample. D: Pedigree of family 176 with chromatopherograms of Sanger sequencing showing presence of alternate variant in maternal blood, saliva, buccal cells, and urine. The red arrows indicate the analyzed variant of interest. MUT, mutation.
      Sanger chromatopherograms of negative (no mosaicism detected) parental samples are shown in Supplemental Figure S3.
      In family 91, calibration experiments were performed with the use of series of dilutions (1%, 0.3%, 0.1%, and 0.03%) of wild-type human genomic DNA (NA18537; Coriell Institute for Medical Research, Camden, NJ) and synthetic double-stranded DNA (gBlocks Gene Fragment; Integrated DNA Technologies, Coralville, IA) bearing the c.294C > A variant. To avoid DNA loss during dilution, the 1× Tris-EDTA buffer with 10 ng/μL carrier RNA and 0.2% Tween 20 was used for dilution of samples. BDA calibration was performed using the 30 ng of each sample. All experiments were performed in triplicate (or six replicate for 0.03% AAF). A mastermix bulk reaction mixture was made and then split into replicates to decrease the variability due to pipetting error. The assay sensitivity of BDA method was determined as 0.03% (Supplemental Figure S4).
      DNA samples from family 176 were analyzed using standard qPCR without the blocker using the same parameters as those in the BDA experiments. The forward and reverse primers were designed upstream and downstream to the approximately 7-kb deletion, respectively. The amplicon length of the variant template was 167 bp, and the amplicon length on the wild-type template was 7303 bp. qPCR was performed using non–long-range Taq polymerase with short (30 seconds) extension time. Thus, amplicons >1000 bp cannot be amplified (Supplemental Figure S3), and only variant template was detected. Duplicated Sanger sequencing was performed to confirm the correct (167-bp) length of the obtained amplicon. For positive samples, AAF was estimated as follows:
      AAF=50%2Cqmedian,parentCqmedian,patient
      (2)


      where Cqmedian,parent is the median Cq of the parent sample with a positive result, and Cqmedian,patient is the median Cq of the corresponding patient sample. Herein, the AAF in a patient is assumed to be 50% and the PCR amplification efficiency for the mutant to be two per cycle in the presence of the blocker, so that the Cq difference between parent and patient can be used to infer AAF in the parent. The assumption about PCR amplification efficiency is consistent with the ΔCqsamp in most patient samples (Supplemental Table S2), although some patient samples showed a high ΔCqsamp value, indicating the PCR yield for some mutations was lower than two with the blocker. However, even in the case of the largest ΔCqsamp, the amplification efficiency per cycle was approximately 1.75, so the above equation for AAF estimation can still be used. To further improve AAF quantitation, the PCR amplification efficiency for each different mutation can be calculated on the basis of ΔCqsamp values.

       ddPCR Assays

      To further assess the AAF in parental samples, three families were tested using the probe-based ddPCR (Table 1). The FOXF1 primers and probes specific to alternate or wild-type allele were designed using droplet digital PCR assays tool (Bio-Rad). To ensure the highest specificity between the mutant and wild-type clusters, the ddPCR assays for each variant were validated and optimized with the use of a temperature gradient and the probands' and wild-type DNA samples (positive and negative controls, respectively), as well as a nontemplate control. The droplets were classified on the basis of the fluorescence amplitude observed in the positive, negative, and nontemplate controls. In the clean reaction, there should be no mutant-positive droplets in both negative and nontemplate control wells.
      In family 105, additional calibration experiments were performed with the use of a series of dilutions of proband's DNA in the control wild-type DNA (50%, 10%, 5%, 1%, 0.1%, and 0.03%). The cutoff sensitivity of this ddPCR assay was determined as 0.1% (Supplemental Figure S5).
      The ddPCR experiments were performed using QX200 AutoDG Droplet Digital PCR System (Bio-Rad). Each 20-μL PCR contained 10 μL of 2× ddPCR Supermix for probes (Bio-Rad), 1 μL of custom-designed TaqMan probes and primers mix, and 50 to 100 ng of DNA. In addition, 1 U of HindIII (family 123) or MseI (families 85, 105, and 138) restriction enzyme (New England Biolabs, Ipswich, MA) was added to each reaction to perform restriction digestion of DNA samples directly in the ddPCR. After emulsification with Automated Droplet Generator (Bio-Rad), a plate containing ddPCR droplets was transferred to the thermocycler. Samples were denatured at 95°C for 600 seconds, followed by 40 cycles of 94°C for 30 seconds and 54°C (families 85, 123, and 138) or 56°C (family 105) for 60 seconds, and final incubation at 98°C for 600 seconds. After thermal cycling, droplets were read with the use of the QX200 Droplet Reader, followed by data analysis with Quantasoft version 1.7 Studio (Bio-Rad). Only samples with total droplets count ≥13,000 were included in calculations. Each parental sample was run in at least eight repeats.

      Results

      Mosaic FOXF1 variants in reportedly unaffected parents were identified in 4 of 18 families studied. Two of these variants were initially detected in the maternal samples (families 91 and 176) using routine molecular testing with Sanger sequencing and CNV deletion-specific PCR, as previously described.
      • Szafranski 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.
      ,
      • Sen 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.
      The AAF of studied variants, determined in parental blood samples using BDA, ddPCR, or qPCR, ranged between 0.03% and 19% (Table 1, Figure 2, Figure 3, and Supplemental Tables S2 and S3).
      Figure thumbnail gr3
      Figure 3Pedigree of family 123 suspected for maternal germline mosaicism with absolute quantification of the allele abundance by droplet digital PCR (ddPCR) assay showing no evidence of the alternate allele in the maternal blood sample. The upper ddPCR plot shows the example of droplet counts positive for the alternate (blue bars) and wild-type (yellow bars) allele and the total droplet counts (gray bars) in single reaction. The number of droplets is presented in a logarithmic scale. The lower ddPCR plot presents alternate allele fraction (AAF) and example of the one-dimensional fluorescence amplitude plot of droplets for alternate allele detection. The positive droplets (blue dots), containing the alternate allele, exhibit increased fluorescence compared with negative droplets (dark gray dots). The threshold line dividing positive and negative droplets is presented (pink line).
      A heterozygous approximately 7-kb CNV deletion involving FOXF1 was found in the patient from family 176. The same-sized junction fragment of weaker intensity was identified in the apparently healthy mother. The intertissue AAFs ranged from <0.03% to 0.65%; they were determined at 0.2% in saliva, 0.14% in redrawn saliva, 0.04% in blood, <0.03% in urine, and 0.65% in buccal cells using BDA. In the hair follicles, the deletion could not be detected; ddPCR was not performed (Figure 2D, Table 1, and Supplemental Tables S2 and S3).
      In the maternal sample of family 91, somatic mosaicism for SNV c.294C>A (p.His98Gln) was initially detected by Sanger sequencing.
      • Sen 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.
      BDA enabled precise measurement of the variant AAF at 19%; ddPCR was not performed (Figure 2C, Table 1, and Supplemental Table S2).
      The level of maternal somatic mosaicism of SNV c.539C>A (p.Ser180*) in family 85 was estimated by BDA and ddPCR at 1.5% and 1.0%, respectively (Figure 2A, Table 1, and Supplemental Table S2).
      In family 105, SNV c.316T>C (p.Phe106Leu) was found in the paternal DNA sample. The variant allele was detected using BDA method, and its ratio was estimated at 0.03% (Figure 2B, Table 1, and Supplemental Table S2). This variant was undetectable by ddPCR method.
      No evidence of parental mosaicism was found in the remaining 14 ACDMPV families, including family 123 with two children manifesting ACDMPV with the FOXF1 insertion/deletion c.849_850del (p.Ile285Glnfs*9)
      • Szafranski 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.
      (Table 1, Figure 3, and Supplemental Table S2).

      Discussion

      The incidence of somatic mosaicism varies between different diseases, genes, and type of variants. Studies in several human genetic conditions have shown that the rate of parental somatic mosaicism, explaining a familial recurrence of apparently de novo mutations, is higher than previously thought. Low-level (AAF ≤ 10%) and very-low–level (AAF ≤ 1%) parental somatic mosaicism for CNV deletions and SNVs associated with genetic disorders have been detected in 4% and 8% of families, respectively, with AAFs ranging between <9% for CNVs and 0.22% to 6.15% for SNVs.
      • Campbell I.M.
      • Yuan B.
      • Robberecht C.
      • Pfundt R.
      • Szafranski P.
      • McEntagart M.E.
      • Nagamani S.C.S.
      • Erez A.
      • Bartnik M.
      • Wiśniowiecka-Kowalnik B.
      • Plunkett K.S.
      • Pursley A.N.
      • Kang S.-H.L.
      • Bi W.
      • Lalani S.R.
      • Bacino C.A.
      • Vast M.
      • Marks K.
      • Patton M.
      • Olofsson P.
      • Patel A.
      • Veltman J.A.
      • Cheung S.W.
      • Shaw C.A.
      • Vissers L.E.L.M.
      • Vermeesch J.R.
      • Lupski J.R.
      • Stankiewicz P.
      Parental somatic mosaicism is underrecognized and influences recurrence risk of genomic disorders.
      ,
      • Acuna-Hidalgo R.
      • Bo T.
      • Kwint M.P.
      • van de Vorst M.
      • Pinelli M.
      • Veltman J.A.
      • Hoischen A.
      • Vissers L.E.L.M.
      • Gilissen C.
      Post-zygotic point mutations are an underrecognized source of de novo genomic variation.
      Rahbari et al
      • Rahbari R.
      • Wuster A.
      • Lindsay S.J.
      • Hardwick R.J.
      • Alexandrov L.B.
      • Turki S.A.
      • Dominiczak A.
      • Morris A.
      • Porteous D.
      • Smith B.
      • Stratton M.R.
      • Hurles M.E.
      UK10K Consortium
      Timing, rates and spectra of human germline mutation.
      (2016) reported 3.8% of mutations in mosaic state in at least 1% of parental blood cells. Mosaic mutations linked to SCN1A-related epilepsy have been identified in 5% of parents, whereas 2% to 3% of patients with vascular Ehlers-Danlos syndrome and presumed de novo variants in COL3A1 could have a parent with low-grade mosaicism.
      • de Lange I.M.
      • Koudijs M.J.
      • van ’t Slot R.
      • Sonsma A.C.M.
      • Mulder F.
      • Carbo E.C.
      • van Kempen M.J.A.
      • Nijman I.J.
      • Ernst R.F.
      • Savelberg S.M.C.
      • Knoers N.V.A.M.
      • Brilstra E.H.
      • Koeleman B.P.C.
      Assessment of parental mosaicism in SCN1A-related epilepsy by single-molecule molecular inversion probes and next-generation sequencing.
      ,
      • Legrand A.
      • Devriese M.
      • Dupuis-Girod S.
      • Simian C.
      • Venisse A.
      • Mazzella J.M.
      • Auribault K.
      • Adham S.
      • Frank M.
      • Albuisson J.
      • Jeunemaitre X.
      Frequency of de novo variants and parental mosaicism in vascular Ehlers-Danlos syndrome.
      Among ACDMPV families supported by the Alveolar Capillary Dysplasia Association (https://acdassociation.org), a nonprofit organization dedicated to increasing ACDMPV awareness, there are a few families with two or more affected siblings and no previous family history, suggesting the possibility of parental mosaicism. However, thus far, only five ACDMPV families with mosaic FOXF1 variants in parents have been reported.
      • Szafranski 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.
      ,
      • Reiter 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.
      ,
      • Luk 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.
      The real incidence of parental somatic or germline mosaicism, including low-level or very-low–level mosaicism, in ACDMPV families remains unknown because methods applied for standard molecular diagnostics, including Sanger sequencing, are not sensitive enough to detect low percentages of a variant allele and often fail to precisely determine the allelic ratio.
      • Gajecka M.
      Unrevealed mosaicism in the next-generation sequencing era.
      Moreover, mutational screening is usually limited to one type of tissue (ie, blood or saliva). To overcome the technical diagnostic challenges, new methods are now being implemented for more efficient detection of somatic mosaicism, including BDA.
      BDA is a relatively new PCR-based allele enrichment technology that preferably amplifies single-base variants, small insertions, and CNV deletions 1000-fold over wild-type allele, allowing for rare allele quantitation with precision similar to ddPCR, which is considered as a gold standard in rare event detection.
      • Wu 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.
      BDA does not require any chemically modified oligonucleotides or specialized instruments (only standard qPCR or PCR thermocyclers); thus, it is fast and economical for rare allele detection. It is also compatible with downstream sequence analysis methods (Sanger sequencing or next-generation sequencing) to verify the amplicon sequences. BDA's performance is consistent within an 8°C temperature window of the annealing/extension PCR step; therefore, the optimization of temperature is not required.
      Herein, 18 ACDMPV families were retrospectively analyzed, with the previously identified pathogenic or likely pathogenic variants determined by conventional molecular techniques 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). The use of BDA, ddPCR, and qPCR methods with higher sensitivity allowed us to characterize parental somatic mosaicism of FOXF1 variants detected in 4 (22%) of 18 tested ACDMPV families.
      Among four families with parental mosaicism, two were tested in parallel with the use of two different high-sensitive techniques. In family 85, the mosaic ratios of FOXF1 variant measured by BDA and ddPCR were comparable (1.5% and 1%, respectively), indicating that both methods can be used to accurately quantitate low-level mosaicism. However, in family 105, BDA turned out to be more efficient for detection of very-low–level mosaicism than ddPCR. Using BDA, the level of parental FOXF1 mosaicism was determined at 0.03%, whereas it remained undetectable by ddPCR. On the basis of calibration experiments performed for c.316T>C variant in FOXF1, the sensitivity cutoff for this particular ddPCR assay was determined as 0.1%. Because the amount of DNA available is the limiting factor for sensitivity of rare allele detection using ddPCR, the possibility that use of more DNA in calibration (proband's DNA) and actual (maternal DNA) experiment could increase the limit of detection cannot be ruled out. However, because of insufficient amount of both proband's and parental DNA samples, further experiments could not be performed.
      In family 176, the presence of somatic mosaicism in the mother was detected by junction-specific long-range PCR performed in DNA extracted from saliva and was further confirmed and quantitated in saliva and other tissues by qPCR. Although the level of somatic mosaicism was very low (<0.03% to 0.65%), the presence of CNV deletion in tissues originating from all three germinal layers suggests that it still might have occurred during early embryonic development. The observed variation in very-low–level mosaic ratios across tested cells could be a result of many different factors, including tissue-specific selection effects.
      • Campbell I.M.
      • Shaw C.A.
      • Stankiewicz P.
      • Lupski J.R.
      Somatic mosaicism: implications for disease and transmission genetics.
      ,
      • Ju Y.S.
      • Martincorena I.
      • Gerstung M.
      • Petljak M.
      • Alexandrov L.B.
      • Rahbari R.
      • et al.
      Somatic mutations reveal asymmetric cellular dynamics in the early human embryo.
      Family 123, in which two siblings had FOXF1 frameshift variant and died of ACDMPV 6 years apart,
      • Szafranski 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.
      originally screened with the use of Sanger sequencing, was now tested with ddPCR. Neither ddPCR nor Sanger screening of FOXF1 has detected the corresponding pathogenic FOXF1 variant allele in the maternal blood sample, suggesting that germline maternal mosaicism or extremely low-level maternal somatic mosaicism is the most plausible cause of the unusual recurrence of ACDMPV in this family.
      Although the use of BDA and ddPCR allowed us to detect parental mosaicism of FOXF1 variants in 22% families, the real frequency of mosaicism could be still underrecognized because of technical limitations. For example, high GC content of exon 1 of FOXF1 precluded testing FOXF1 mutations in some ACDMPV families.
      In conclusion, this research proposes that parents of children with ACDMPV who are found negative for FOXF1 variants by the routine detection techniques (eg, Sanger sequencing or array comparative genomic hybridization) may benefit from reanalyses using more sensitive and quantitative methods, including BDA or ddPCR. These techniques are shown to be efficient tools for the detection of low-level (ddPCR) or even very-low–level (BDA) parental somatic mosaicism for both SNVs and CNVs. However, given that in most cases only one type of parental tissue was available for screening, the real frequency of mosaic variants may be underestimated. Data from this study further demonstrate the need for a systematic screening of parental samples for somatic mosaicism, particularly in families in whom more than one affected carrier of the same variant was observed.
      • Campbell I.M.
      • Yuan B.
      • Robberecht C.
      • Pfundt R.
      • Szafranski P.
      • McEntagart M.E.
      • Nagamani S.C.S.
      • Erez A.
      • Bartnik M.
      • Wiśniowiecka-Kowalnik B.
      • Plunkett K.S.
      • Pursley A.N.
      • Kang S.-H.L.
      • Bi W.
      • Lalani S.R.
      • Bacino C.A.
      • Vast M.
      • Marks K.
      • Patton M.
      • Olofsson P.
      • Patel A.
      • Veltman J.A.
      • Cheung S.W.
      • Shaw C.A.
      • Vissers L.E.L.M.
      • Vermeesch J.R.
      • Lupski J.R.
      • Stankiewicz P.
      Parental somatic mosaicism is underrecognized and influences recurrence risk of genomic disorders.

      Acknowledgment

      We thank Christopher M. Grochowski for assistance in droplet digital PCR experiments.

      Supplemental Data

      • Supplemental Figure S2

        Example of quantitative PCR (qPCR) amplifications of the products of the different lengths. Amplicons >1000 bp cannot be amplified using a short extension time (30-second) qPCR protocol. Four different sets of primers designed in the FOXF1 region were used to amplify the products. In each reaction, 400 nmol/L of each primer and 20 ng of genomic DNA sample NA18537 were used. a.u., arbitrary unit.

      • Supplemental Figure S3

        An example of Sanger sequencing chromatopherograms. Some unexpected mutations observed in negative samples (eg, family 55, father, position 2 G>A) can be explained by the Taq polymerase error. Taq polymerase has an error rate of approximately 1:7000 per base per cycle; the errors in early PCR cycles can be enriched to >50% by blocker displacement amplification instead of the true mutations because these mutations only appear in one of the duplicate experiments. The red arrows indicate the analyzed variant of interest.

      • Supplemental Figure S4

        Example of a blocker displacement amplification (BDA) calibration experiment. A: Quantitative PCR (qPCR) results of the samples with the different alternate allele fraction (AAF) ratios. The SD of the triplicate (or six replicate for 0.03% AAF) Cq values for the samples with lower AAF were greater than those in the standard qPCR experiments; this is a result of sampling stochasticity. On average, the 0.03% AAF sample has 9000 copies of DNA molecules bearing wild-type (WT) alleles and three copies bearing alternate alleles in each PCR well. Assuming Poisson distribution, the SD of the copy number of alternate alleles is √3 = 1.7. Therefore, it is normal to observe an SD of approximately one PCR cycle in replicate Cq values. When testing the clinical DNA samples, only 10 ng (3000 haploid genomic copies) of DNA was loaded per PCR well, because the amount of sample is limited. Therefore, there is a physical detection limit at 0.03% AAF (ie, one DNA molecule bearing alternate allele per well) using 10 ng input. B: Calibration curve; median Cq values were plotted against AAF. The dashed line shows the Cq value of the wild-type (0% AAF) sample. C: Example of a Sanger sequencing chromatopherogram. Low level of AAF (down to 0.03%) can be detected and verified by Sanger sequencing of the BDA PCR products. The red arrow indicates the analyzed variant of interest. a.u., arbitrary unit.

      • Supplemental Figure S5

        Results of droplet digital PCR (ddPCR) calibration experiment. Experiment was performed for the c.316T>C FOXF1 variant, detected in the proband from family 105, with use of series of dilutions of proband's DNA in control wild-type DNA (50%, 10%, 5%, 1%, 0.1%, and 0.03%). Wild-type (WT) DNA and nontemplate control (NC) were used as controls. Because no droplets positive for alternate allele were observed in the 0.03% dilution of proband's DNA in control wild-type DNA, the cutoff sensitivity of this ddPCR assay was determined as 0.1%. The column chart represents droplet counts in particular dilution: positive droplets for the alternate (blue bars) and wild-type (yellow bars) allele and the total droplet counts (gray bars). The number of droplet is presented in a logarithmic scale. The linear chart shows the fractional abundance (percentage) of alternate allele (orange line).

      References

        • Michaelson J.J.
        • Shi Y.
        • Gujral M.
        • Zheng H.
        • Malhotra D.
        • Jin X.
        • Jian M.
        • Liu G.
        • Greer D.
        • Bhandari A.
        • Wu W.
        • Corominas R.
        • Peoples A.
        • Koren A.
        • Gore A.
        • Kang S.
        • Lin G.N.
        • Estabillo J.
        • Gadomski T.
        • Singh B.
        • Zhang K.
        • Akshoomoff N.
        • Corsello C.
        • McCarroll S.
        • Iakoucheva L.M.
        • Li Y.
        • Wang J.
        • Sebat J.
        Whole-genome sequencing in autism identifies hot spots for de novo germline mutation.
        Cell. 2012; 151: 1431-1442
        • Watson I.R.
        • Takahashi K.
        • Futreal P.A.
        • Chin L.
        Emerging patterns of somatic mutations in cancer.
        Nat Rev Genet. 2013; 14: 703-718
        • Jamuar S.S.
        • Lam A.-T.N.
        • Kircher M.
        • D’Gama A.M.
        • Wang J.
        • Barry B.J.
        • Zhang X.
        • Hill R.S.
        • Partlow J.N.
        • Rozzo A.
        • Servattalab S.
        • Mehta B.K.
        • Topcu M.
        • Amrom D.
        • Andermann E.
        • Dan B.
        • Parrini E.
        • Guerrini R.
        • Scheffer I.E.
        • Berkovic S.F.
        • Leventer R.J.
        • Shen Y.
        • Wu B.L.
        • Barkovich A.J.
        • Sahin M.
        • Chang B.S.
        • Bamshad M.
        • Nickerson D.A.
        • Shendure J.
        • Poduri A.
        • Yu T.W.
        • Walsh C.A.
        Somatic mutations in cerebral cortical malformations.
        N Engl J Med. 2014; 371: 733-743
        • Campbell I.M.
        • Shaw C.A.
        • Stankiewicz P.
        • Lupski J.R.
        Somatic mosaicism: implications for disease and transmission genetics.
        Trends Genet. 2015; 31: 382-392
        • Yang X.
        • Liu A.
        • Xu X.
        • Yang X.
        • Zeng Q.
        • Ye A.Y.
        • Yu Z.
        • Wang S.
        • Huang A.Y.
        • Wu X.
        • Wu Q.
        • Wei L.
        • Zhang Y.
        Genomic mosaicism in paternal sperm and multiple parental tissues in a Dravet syndrome cohort.
        Sci Rep. 2017; 7: 15677
        • Demily C.
        • Hubert L.
        • Franck N.
        • Poisson A.
        • Munnich A.
        • Besmond C.
        Somatic mosaicism for SLC1A1 mutation supports threshold effect and familial aggregation in schizophrenia spectrum disorders.
        Schizophr Res. 2018; 197: 583-584
        • Tarilonte M.
        • Morín M.
        • Ramos P.
        • Galdós M.
        • Blanco-Kelly F.
        • Villaverde C.
        • Rey-Zamora D.
        • Rebolleda G.
        • Muñoz-Negrete F.J.
        • Tahsin-Swafiri S.
        • Gener B.
        • Moreno-Pelayo M.-A.
        • Ayuso C.
        • Villamar M.
        • Corton M.
        Parental mosaicism in PAX6 causes intra-familial variability: implications for genetic counseling of congenital aniridia and microphthalmia.
        Front Genet. 2018; 9: 479
        • de Lange I.M.
        • Koudijs M.J.
        • van ’t Slot R.
        • Sonsma A.C.M.
        • Mulder F.
        • Carbo E.C.
        • van Kempen M.J.A.
        • Nijman I.J.
        • Ernst R.F.
        • Savelberg S.M.C.
        • Knoers N.V.A.M.
        • Brilstra E.H.
        • Koeleman B.P.C.
        Assessment of parental mosaicism in SCN1A-related epilepsy by single-molecule molecular inversion probes and next-generation sequencing.
        J Med Genet. 2019; 56: 75-80
        • Legrand A.
        • Devriese M.
        • Dupuis-Girod S.
        • Simian C.
        • Venisse A.
        • Mazzella J.M.
        • Auribault K.
        • Adham S.
        • Frank M.
        • Albuisson J.
        • Jeunemaitre X.
        Frequency of de novo variants and parental mosaicism in vascular Ehlers-Danlos syndrome.
        Genet Med. 2019; 21: 1568-1575
        • Wright C.F.
        • Prigmore E.
        • Rajan D.
        • Handsaker J.
        • McRae J.
        • Kaplanis J.
        • Fitzgerald T.W.
        • FitzPatrick D.R.
        • Firth H.V.
        • Hurles M.E.
        Clinically-relevant postzygotic mosaicism in parents and children with developmental disorders in trio exome sequencing data.
        Nat Commun. 2019; 10: 2985
        • Cao Y.
        • Tokita M.J.
        • Chen E.S.
        • Ghosh R.
        • Chen T.
        • Feng Y.
        • Gorman E.
        • Gibellini F.
        • Ward P.A.
        • Braxton A.
        • Wang X.
        • Meng L.
        • Xiao R.
        • Bi W.
        • Xia F.
        • Eng C.M.
        • Yang Y.
        • Gambin T.
        • Shaw C.
        • Liu P.
        • Stankiewicz P.
        A clinical survey of mosaic single nucleotide variants in disease-causing genes detected by exome sequencing.
        Genome Med. 2019; 11: 48
        • Campbell I.M.
        • Yuan B.
        • Robberecht C.
        • Pfundt R.
        • Szafranski P.
        • McEntagart M.E.
        • Nagamani S.C.S.
        • Erez A.
        • Bartnik M.
        • Wiśniowiecka-Kowalnik B.
        • Plunkett K.S.
        • Pursley A.N.
        • Kang S.-H.L.
        • Bi W.
        • Lalani S.R.
        • Bacino C.A.
        • Vast M.
        • Marks K.
        • Patton M.
        • Olofsson P.
        • Patel A.
        • Veltman J.A.
        • Cheung S.W.
        • Shaw C.A.
        • Vissers L.E.L.M.
        • Vermeesch J.R.
        • Lupski J.R.
        • Stankiewicz P.
        Parental somatic mosaicism is underrecognized and influences recurrence risk of genomic disorders.
        Am J Hum Genet. 2014; 95: 173-182
        • Huang A.Y.
        • Xu X.
        • Ye A.Y.
        • Wu Q.
        • Yan L.
        • Zhao B.
        • Yang X.
        • He Y.
        • Wang S.
        • Zhang Z.
        • Gu B.
        • Zhao H.-Q.
        • Wang M.
        • Gao H.
        • Gao G.
        • Zhang Z.
        • Yang X.
        • Wu X.
        • Zhang Y.
        • Wei L.
        Postzygotic single-nucleotide mosaicisms in whole-genome sequences of clinically unremarkable individuals.
        Cell Res. 2014; 24: 1311-1327
        • Ju Y.S.
        • Martincorena I.
        • Gerstung M.
        • Petljak M.
        • Alexandrov L.B.
        • Rahbari R.
        • et al.
        Somatic mutations reveal asymmetric cellular dynamics in the early human embryo.
        Nature. 2017; 543: 714-718
        • Jónsson H.
        • Sulem P.
        • Arnadottir G.A.
        • Pálsson G.
        • Eggertsson H.P.
        • Kristmundsdottir S.
        • Zink F.
        • Kehr B.
        • Hjorleifsson K.E.
        • Jensson B.Ö.
        • Jonsdottir I.
        • Marelsson S.E.
        • Gudjonsson S.A.
        • Gylfason A.
        • Jonasdottir A.
        • Jonasdottir A.
        • Stacey S.N.
        • Magnusson O.T.
        • Thorsteinsdottir U.
        • Masson G.
        • Kong A.
        • Halldorsson B.V.
        • Helgason A.
        • Gudbjartsson D.F.
        • Stefansson K.
        Multiple transmissions of de novo mutations in families.
        Nat Genet. 2018; 50: 1674-1680
        • Janney C.G.
        • Askin F.B.
        • Kuhn C.
        Congenital alveolar capillary dysplasia: an unusual cause of respiratory distress in the newborn.
        Am J Clin Pathol. 1981; 76: 722-727
        • Langston C.
        Misalignment of pulmonary veins and alveolar capillary dysplasia.
        Pediatr Pathol. 1991; 11: 163-170
        • Sen P.
        • Thakur N.
        • Stockton D.W.
        • Langston C.
        • Bejjani B.A.
        Expanding the phenotype of alveolar capillary dysplasia (ACD).
        J Pediatr. 2004; 145: 646-651
        • Bishop N.B.
        • Stankiewicz P.
        • Steinhorn R.H.
        Alveolar capillary dysplasia.
        Am J Respir Crit Care Med. 2011; 184: 172-179
        • Szafranski 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: 569-586
        • Stankiewicz 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: 780-791
        • Sen 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: 474-477
        • Szafranski 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: 23-33
        • Szafranski 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: 2013-2019
        • Nagano 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: 1371-1372
        • Ma 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: 672-675
        • Everett 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: 632-638
        • Abu-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: 2877-2881
        • Hayasaka 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: 477-483
        • Pradhan 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: 1045-1056
        • Sen 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: 801-811
        • Reiter 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: 921-927
        • Luk 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: 1942-1944
        • Alsina 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: 615-620
        • Wu 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: 714-723
        • Acuna-Hidalgo R.
        • Bo T.
        • Kwint M.P.
        • van de Vorst M.
        • Pinelli M.
        • Veltman J.A.
        • Hoischen A.
        • Vissers L.E.L.M.
        • Gilissen C.
        Post-zygotic point mutations are an underrecognized source of de novo genomic variation.
        Am J Hum Genet. 2015; 97: 67-74
        • Rahbari R.
        • Wuster A.
        • Lindsay S.J.
        • Hardwick R.J.
        • Alexandrov L.B.
        • Turki S.A.
        • Dominiczak A.
        • Morris A.
        • Porteous D.
        • Smith B.
        • Stratton M.R.
        • Hurles M.E.
        • UK10K Consortium
        Timing, rates and spectra of human germline mutation.
        Nat Genet. 2016; 48: 126-133
        • Gajecka M.
        Unrevealed mosaicism in the next-generation sequencing era.
        Mol Genet Genomics. 2016; 291: 513-530