Congenital diaphragmatic hernia (CDH) etiology as revealed by pathway genetics.код для вставкиСкачать
American Journal of Medical Genetics Part C (Seminars in Medical Genetics) 145C:217 – 226 (2007) A R T I C L E Congenital Diaphragmatic Hernia (CDH) Etiology as Revealed by Pathway Genetics SIBEL KANTARCI AND PATRICIA K. DONAHOE* Congenital diaphragmatic hernia (CDH) is a common birth defect with high mortality and morbidity. Two hundred seventy CDH patients were ascertained, carefully phenotyped, and classified as isolated (diaphragm defects alone) or complex (with additional anomalies) cases. We established different strategies to reveal CDHcritical chromosome loci and genes in humans. Candidate genes for sequencing analyses were selected from CDH animal models, genetic intervals of recurrent chromosomal aberration in humans, such as 15q26.1–q26.2 or 1q41–q42.12, as well as genes in the retinoic acid and related pathways and those known to be involved in embryonic lung development. For instance, FOG2, GATA4, and COUP-TFII are all needed for both normal diaphragm and lung development and are likely all in the same genetic and molecular pathway. Linkage analysis was applied first in a large inbred family and then in four multiplex families with Donnai–Barrow syndrome (DBS) associated with CDH. 10K SNP chip and microsatellite markers revealed a DBS locus on chromosome 2q23.3– q31.1. We applied array-based comparative genomic hybridization (aCGH) techniques to over 30, mostly complex, CDH patients and found a de novo microdeletion in a patient with Fryns syndrome related to CDH. Fluorescence in situ hybridization (FISH) and multiplex ligation-dependent probe amplification (MLPA) techniques allowed us to further define the deletion interval. Our aim is to identify genetic intervals and, in those, to prioritize genes that might reveal molecular pathways, mutations in any step of which, might contribute to the same phenotype. More important, the elucidation of pathways may ultimately provide clues to treatment strategies. ß 2007 Wiley-Liss, Inc. KEY WORDS: congenital diaphragmatic hernia (CDH); genetic pathways; Fryns syndrome; Donnai–Barrow syndrome; homozygosity mapping How to cite this article: Kantarci S, Donahoe PK. 2007. Congenital diaphragmatic hernia (CDH) etiology as revealed by pathway genetics. Am J Med Genet Part C Semin Med Genet 145C:217–226 INTRODUCTION The discovery of gene mutations as a cause of congenital anomalies or birth defects has been difficult, but recently progress has been made in uncovering causal or contributing mutations. His- torically, congenital anomalies were thought to be the result of accidents in utero, because experimental fetal injury models were found to produce defects at birth that resemble birth defects in humans. Some examples include in utero occlusions of the vascular supply Sibel Kantarci, Ph.D. is a Research Fellow at the Peadiatric Surgical Research Laboratories at Massachusetts General Hospital and American Board of Medical Genetics (ABMG)-approved Clinical Cytogenetics Fellow at Harvard Medical School. Dr. Kantarci has completed her ABMGapproved Clinical Molecular Genetic Training Program at Harvard Medical School. Dr. Kantarci’s research in congenital diaphragmatic hernia (CDH) in humans focuses on identifying chromosomal regions and/or genes responsible from CDH by applying molecular and cytogenetics techniques. Patricia K. Donahoe, M.D. is the Director of the Pediatric Surgical Research Laboratories at the Massachusetts General Hospital and the Martial K. Bartlett Professor of Surgery at the Harvard Medical School. She is the Principle Investigator on an NICHD funded grant to study the genetics of Congenital Diaphragmatic Hernia. Her other appointments are as an Associate Member of the Broad Institute of MIT and Harvard and on the faculty of the Harvard Stem Cell Institute. Her work on Congenital Diaphragmatic Hernia focuses on discovery of gene mutations responsible for the defect with the aim of discovering pathway defects which could lead to therapeutic strategies. *Correspondence to: Patricia K. Donahoe, M.D., Pediatric Surgical Research Laboratories, Massachusetts General Hospital, CPZN North, 6.206, Boston, MA 02114. E-mail: firstname.lastname@example.org DOI 10.1002/ajmg.c.30132 ß 2007 Wiley-Liss, Inc. of the bowel producing jejunal or ileal atresia [Louw and Barnard, 1955], or directed laser injury in chick embryos resulting in cloacal/bladder exstrophy at birth [Thomalla et al., 1985]. Clinical geneticists have for some time suspected that gene defects are responsible for nonsyndromic birth defects and have recently proven this to be the case with such disorders as Hirschsprung disease [Gabriel et al., 2002], cardiomyopathies [Ahmad et al., 2005], brain malformations [Ferland et al., 2004], and craniofacial anomalies [Jugessur and Murray, 2005]. The use of families for genetic linkage has not proven to be feasible for the study a large number of birth defects since many of them are either lethal or result in a phenotype that is disadvantageous for reproduction. Thus, it is necessary to utilize different strategies to detect candidate genes for these disorders since 218 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c large multiple affected families are unavailable for traditional linkage studies. Such is the case for congenital diaphragmatic hernia (CDH), a common human birth defect characterized by diaphragm defect, arrested lung development with pulmonary hypoplasia, and pulmonary hypertension which continues to be associated with a high mortality and morbidity. The fact that genetically altered knockout mice produced by homologous recombination or from ENU screens manifest phenotypes with CDH supports a genetic cause of the disorder, as does its appearance in small multiplex CDH families, and the fact that different chromosomal abnormalities are associated with CDH in 10% of cases (see article by Pober in this issue for further discussion). The low recurrence rate or precurrence in siblings uncovered in several studies [David and Illingworth, 1976; Czeizel and Kovacs, 1990], and recently in a 30-year active malformation surveillance program of 203 cases of CDH from a single urban academic institution [Pober et al., 2005] suggests that the mutations in these defects are likely to be de novo in countries or regions where consanguinity is low. It has been reported that vitamin A deficiency in pregnant rodents, as well as exposure to nitrofen, which suppresses a key enzyme in the vitamin A pathway, cause CDH [Andersen, 1941, 1949; Suen et al., 1994], while reintroduction of vitamin A rescues the phenotype [Wilson et al., 1953; Thebaud et al., 1999]. This scenario predicts that rescue downstream of a genetic pathway, once elucidated, may be of therapeutic value. It has been reported that vitamin A deficiency in pregnant rodents, as well as exposure to nitrofen, which suppresses a key enzyme in the vitamin A pathway, cause CDH, while reintroduction of vitamin A rescues the phenotype. This scenario predicts that rescue downstream of a genetic pathway, once elucidated, may be of therapeutic value. Such observations also indicate that common pathways may exist that explains why disruption of genes in seemingly disparate chromosomal hot spot regions can produce similar phenotypes. These heterogeneous loci, thus, may be related by affecting different steps in related pathways or networks. To study CDH patients genetically, we established an infrastructure to enroll CDH patients both at the MassGeneral Hospital for Children and Children’s Hospital in Boston. Parent support groups and the international community of pediatric genetics voluntarily engaged in an effort to detect multiple affected families worldwide. This program is funded by the National Institutes of Health under the auspices of a Birth Defects initiative sponsored by the Institute of Child Health and Development (NICHD). This effort entitled ‘‘Gene Mutation and Rescue in Human Congenital Diaphragmatic Hernia: (HD 055150) required the input of many surgeons, geneticists, and nurse and study coordinators at both institutions, and the extraordinary involvement of all families, particularly those with multiple affected sibs. We have recruited over 270 CDH patients as well as parents and healthy siblings from both institutions, with additional patients recruited through CHERUBS (The Association of Congenital Diaphragmatic Hernia Research, Advocacy, and Support) or from genetic and surgical colleagues around the world. The Institutional Review Boards at MassGeneral Hospital for Children and Children’s Hospital, Boston provided annual review and approval of consents and study protocols. Phenotypic classification of the CDH patients was performed based on information obtained from a parental questionnaire, medical record review, family pedigree, and physical examination. This information was entered into ARTICLE an Oracle based, IRB approved, HIPPA compliant database. DNA samples were obtained from various sources including blood, skin, amniotic fluid, formalinfixed pathological material, or frozen tissue. About half of CDH patients have only diaphragm malformations and are classified as isolated cases. The remaining patients have additional anomalies either as part of non-syndromic associations, chromosome abnormalities, or recognized syndromes, and are referred to as complex cases. EB virus transformed lymphoblast or primary fibroblast cell lines were established for each patient and, when necessary, for both parents and siblings. Literature and database searches were done, and 30 years of data analyzed from an active malformation single institution surveillance monitoring program conducted at the Brigham and Women’s Hospital, Boston. Recurrent chromosomal anomalies associated with CDH were catalogued from this database and from the literature, and analyzed to determine if molecular pathways would emerge indicating that CDH might have multiple molecular defects contributing to this same phenotype and explaining why the defects might vary in severity. STRATEGIES TO IDENTIFY GENETIC ABNORMALITIES RELATED TO CDH CDH is a phenotypically and genetically heterogeneous disorder. To identify chromosome loci and genes responsible from CDH requires different strategies. We used a number of different techniques to analyze patients with CDH: (A) Sequencing of multiple CDH candidate genes revealed from animal models, developmental studies, and regions of recurrent chromosomal aberrations associated with CDH. (B) If multiplex consanguineous families were available, genome wide SNP array analysis was used to determine shared homozygosity by descent in affecteds. For instance, this method was applied to a consanguineous kindred with multiple affected patients with Donnai–Barrow Syndrome (DBS) [OMIM#222448] associated with CDH. Subsequently, microsatellite markers in this and additional multiplex DBS ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c families narrowed the DBS critical locus. (C) We simultaneously performed arraybased comparative genomic hybridization (aCGH) to detect microdeletions or microduplications on DNA isolated from fresh lymphocytes or from archival pathology specimens, concentrating on syndromic, complex CDH. Intervals were narrowed using fluorescence in situ hybridization (FISH) and multiplex ligation-dependent probe amplification (MLPA) techniques. The occurance of copy number variations was compared to regions of microdeletions or microduplications. Our hypothesis was that the study of syndromic CDH or CDH associated with cytogenetic deletions detected by routine chromosomal analysis, or microdeletions detected by aCGH analysis, would provide clues about the etiology of nonsyndromic or isolated CDH. Using these techniques, we recently identified a possible critical locus, chromosome 1q41–q42.12, for Fryns syndrome [OMIM#229850] associated with CDH [Kantarci et al., 2006] and narrowed a previously detected locus of interest of 15q26.1–q26.2 Our hypothesis was that the study of syndromic CDH or CDH associated with cytogenetic deletions detected by routine chromosomal analysis, or microdeletions detected by aCGH analysis, would provide clues about the etiology of nonsyndromic or isolated CDH. Using these techniques, we recently identified a possible critical locus, chromosome 1q41–q42.12, for Fryns syndrome [OMIM#229850] associated with CDH and narrowed a previously detected locus of interest of 15q26.1–q26.2. [Klaassens et al., 2005a,b; Scott et al., 2007; Kantarci et al., in preparation]. These three approaches have yielded different loci associated with syndromic CDH. It is possible that the analysis of genes in these regions may, in turn, point us toward potential genetic pathways or networks for CDH. Identification of genes is a goal, as genetic pathways will yield therapeutic strategies that can be employed for CDH either in utero or after birth when lung development still continues in the human. Selection and Sequence Analysis of Candidate Genes Candidate genes (see Table I) were selected for mutational analysis in CDH patients, both isolated and complex, based on the following criteria: (a) Mutation of the gene in mice either by homologous recombination or in an ENU screen produced a phenotype of CDH, as observed with Slit3 [Liu et al., 2003; Yuan et al., 2003], Robo1 (the Slit receptor) [Xian et al., 2001], CouptfII [You et al., 2005], Wt1 [Kreidberg et al., 1993; Moore et al., 1998], and Fog2 [Ackerman et al., 2005]. The first causative gene in a human, FOG2, was found to be due to a de novo mutation in a patient with lung hypoplasia and a diaphragm defect in the eventration spectrum [Ackerman et al., 2005] (futher discussed in the articles by Ackerman and by Pober in this issue). (b) Genes whose expression patterns were changed at the time of branching morphogenesis during embryonic lung development, as was reported for Sonic hedgehog protein (SHH) [Bellusci et al., 1997; Litingtung et al., 1998; Pepicelli et al., 1998], its downstream transcription factors, GLI2 and GLI3 [Grindley et al., 1997], and Forkhead-related transcription factor 1 (Foxf1) [Kim et al., 2005]. (c) Genes found in chromosomal regions known to be associated with the human CDH phenotype. For example, aCGH was used to compare two patients with deletions in 219 15q26 with severe growth retardation, cardiac defects, and musculoskeletal defects, but without CDH, to seven 15q26 deletion patients with similar severe anomalies, but with CDH [Klaassens et al., 2005a]. Those with CDH had deletions limited to a region which contains at least five known genes that were chosen for subsequent sequence analysis, namely SIAT8B, CHD2, RGMA, MCTP2, and COUP-TFII (NR2F2). (d) Insulin-like growth factor I receptor (IGFR1), which is adjacent to the 15q26.2 CDH critical region [Klaassens et al., 2005b; Scott et al., 2007] was also sequenced since growth retardation is prominent in syndromic forms of CDH. Moreover, during a Drosophila tracheal development screen a candidate gene, thrombospondin, was discovered [L. Perkins, personal communication]. Thrombospondin is a coactivator with insulin and insulin like growth factor in the integrin pathway. (e) Genes in the retinoic acid (RA) pathway were selected because of their functional importance in lung [Kumar et al., 2005] and diaphragm development [Kluth et al., 1996; Greer et al., 2000; Gallot et al., 2005] and/or their location in the 15q26 locus, and included RALDH3, CRABP1, RLBP1, COUP-TFII, and RALDH2. (f) Other migration molecules sequenced (aside from RGMA and SIAT8B) included SLIT2 and SLIT3, a mutation of which caused CDH in knockout mice [Liu et al., 2003; Yuan et al., 2003], and the SLIT receptors ROBO1 and ROBO2, mutations of which also cause lung hypoplasia [Anselmo et al., 2003]. (g) Potential FOG2 pathway genes including the transcription factors GATA4 [Ackerman et al., 2006; Jay et al., 2007], GATA5, and GATA6 and COUP-TFII [Ackerman et al., 2005] which is also part of the retinoic acid pathway [Kimura et al., 2002]. GLI2 GLI3 Wingless family secreted WNT5A Receptor for SLIT Receptor for SLIT Chromodomain helicase II ROBO1 ROBO2 CHD2 ROH—RA ROH—RA Cellular binding protein for RA Retinaldehyde binding protein RA/TH receptor Complexes transcription factors GATA4, GATA5, GATA6, and COUP-TFII RALDH2 RALDH3 CRABP1 RLBP1 COUP-TFII FOG2 Retanoic acid pathway Secreted neural guidance molecule SLIT3 Gene name Secreted neural guidance molecule SLIT2 Migration molecules Sonic hedgehog, secreted SHH Gene name Insulin growth factor receptor IGF1R Growth factors/receptors Target of SHH Target of SHH GATA5 GATA6 COUP-TFII FOXF1 Gene name Zinc finger transcription factor Zinc finger transcription factor Orphan nuclear receptor Hfh8 forkhead homolog target of SHH GATA4 Transcription factors Complexes transcription factors GATA4, GATA5, GATA6, and COUP-TFII Zinc finger transcription factor FOG2 Gene name Function Modulation of retinoic acid response, retinoic metabolism An acceptor of 11-cis-retinol in the isomerization reaction of the visual cycle Retinoic acid signaling Co-repression GATA and COUP-TFII, binds RXR Enzyme converts ROH to RA Enzyme converts ROH to RA Helicase Controls midline crossing Controls midline crossing Alters cell migration Function Early branching stimulates mesenchyme, FGF10 Alveolarization, branching General homeostatsis and growth Function Transcription factor Branching morphogenesis Transcription factor Haploinsufficiency ! poor alveolarization and vascular branching Early branching Early branching Transcription factor Transcription cofactor Function Early lethal, cardiac, vascular, diaphragm defects Eventration and lung hypoplasia, cardiac defects Rhodopsin regeneration, 11-cis-retinal production, and dark adaptation after illumination are delayed Dispensable Embryonic lethal (abnormal expression in CDH nitrofen) Neonatal lethal, choanal atresia Abnormal expression CDH7-CHARGE syndrome Abnormal CNS formation Pulmonary hypoplasia, abnormal CNS formation Increased bronchial wall thickening and abnormal alveolar formation Central tendon CDH Abnormal expression Over expression lung hypoplasia phenotype Abnormal lung development Abnormal expression Abnormal lung Abnormal Lung No lung or diaphragm defect Early embryonic lethal, branching defects Cardiac, vascular, stomach, and diaphragm Thickened mesenchyme Cardiac defects, eventration of the diaphragm Eventration and lung hypoplasia, cardiac defects Abnormal expression 15q26.2 8q23.1 15q26 15q24 15q26.3 15q21.3 Gene locus 15q26.2 3p12.3 3p12 5q35-34 4p15.2 Gene locus 3p21-p24 7q36 15q26.3 Gene locus 2q14 7p13 20q13.33 18q11.1-q11.2 15q26.2 16q24 8p23.1 8q23.1 Gene locus AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c Lungs, mesenchyme, heart Mesenchyme, lung, heart, diaphragm Main bronchi and developing lamina propria of the small intestine Neural Crest (cytoplasmic, nuclear, and mitochondrial) Retina Pleuroperitoneal folds Expression Lung mesenchyme and probably epithelium Lung mesenchyme and probably epithelium Lung mesenchyme and on the basal epithelium surface. Also found in central diaphragm Lung mesenchyme and on the basal epithelium surface. Also found in central diaphragm Nuclear Expression Distal branching tips Insulin like growth factor in ligated trachea Branching tips epithelium Expression Lung mesenchyme Lung mesenchyme Liver, lung mesenchyme, diaphragm (septum transversum) Lung mesenchyme and diaphragm Lung epithelium and diaphragm Lung mesenchyme Lung epithelium and mesenchyme, later mesenchyme Lung mesenchyme and diaphragm Expression TABLE I. Candidate Genes for Congenital Diaphragmatic Hernia 220 ARTICLE ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c Since the genetics of CDH is complex, and it is probable that CDH may be caused by de novo mutations in pathway genes, identification and resequencing of these candidate genes will hopefully help us better understand the pathogenesis of CDH. Evaluation of sequence changes. Nonsynonymous SNPs (nsSNP) are analyzed by PolyPhen [http://genetics.bwh. harvard.edu/pph/] and SIFT [http:// blocks.fhcrc.org/sift/SIFT.html] programs to predict the effect of amino acid changes on physical 3D structure and protein function. SNP allele and genotype frequencies in CDH patients are then compared to an ethically matched control group recruited as part of the study. As the work progresses, with a growing sample size of both CDH patients and controls, we are focusing on nsSNPs not found in either the public databases or in our own control population. Genes are prioritized for further study based on recurring SNPs particularly those in coding regions and in highly conserved promoter or enhancer domains. SNPs are analyzed for variants associated with the CDH phenotype, loss of heterozygosity, or association studies to uncover haplotypes indicating common variants contributing to or creating susceptibility loci. Loss of heterozygous regions identified by SNPs across a gene has been evaluated with MLPA to determine whether it reflects the loss of one copy of that gene. Families With Donnai–Barrow Syndrome and Homozygosity Mapping The search to discover gene defects responsible for CDH and agenesis of the corpus callosum joined the efforts of two research teams in the study of the rare autosomal recessive multiple anomaly condition known as Donnai–Barrow syndrome (DBS). Patients with this autosomal recessive disorder may have CDH, and characteristically also have agenesis of the corpus callosum, craniofacial anomalies, hypertelorism, high myopia, sensorineural hearing loss, and developmental delay. Case reports of The search to discover gene defects responsible for CDH and agenesis of the corpus callosum joined the efforts of two research teams in the study of the rare autosomal recessive multiple anomaly condition known as Donnai–Barrow syndrome (DBS). Patients with this autosomal recessive disorder may have CDH, and characteristically also have agenesis of the corpus callosum, craniofacial anomalies, hypertelorism, high myopia, sensorineural hearing loss, and developmental delay. multiple affected siblings born to healthy parents or of affected children born to consanguineous parents suggested that DBS followed an autosomal recessive pattern of inheritance. We recruited a large inbred family including five patients with clinical features of DBS from the United Arab Emirates. The parents were healthy. The proband with characteristic craniofacial features of DBS died of complications secondary to CDH. Among the four surviving patients, each with normal chromosomal complements, one had left-sided diaphragmatic eventration and pulmonary hypoplasia. aCGH performed on two of the survivors did not reveal copy number aberrations. We also recruited four additional families previously reported with characteristic features of DBS including CDH, absence of corpus callosum, exomphalos, hypertelorism, myopia, and sensorineural deafness [Donnai and Barrow, 1993; Chassaing et al., 2003], and recently, an unreported multiplex consanguineous family from 221 Qatar. Additionally, we ascertained a previously reported family with clinically overlapping features of DBS and Facio Oculo Acoustico Renal (FOAR) [OMIM 227920] syndrome including CDH, hypertelorism, a large anterior fontanelle, high myopia, sensorineural hearing loss, macrocephaly, proteinuria, mental retardation, and developmental delay [Devriendt et al., 1998]. A genome wide screen using the Affymetrix 10K SNP array, performed on the four affected members of the large inbred UAE family for homozygosity mapping, identified homozygous regions with identical genotypes in all patients. The largest region of identity by descent was 17 cM (21Mb) on chromosome 2q23.3–q31.1. Subsequently, microsatellite marker analyses on the multiplex DBS families narrowed the DBS critical region to 18 Mb and generated a maximum single point LOD score of 3.26. Sequencing analysis of 51 candidate genes mapped to the DBS critical region is in progress. Molecular Cytogenetics and Molecular Analyses of Microdeletions in CDH Hot Spots About 10% of CDH patients have a chromosome abnormality detectable by standard cytogenetic techniques. Advanced molecular cytogenetic analyses, such as aCGH have revealed various small chromosomal abnormalities in CDH patients, such as 15q26.2– q26.3 microdeletions, in addition to those apparent in standard chromosomal analyses (see article by Pober in this issue for further discussion). It is anticipated that novel molecular analyses of CDHcritical chromosomal regions may ultimately define smaller regions and identify genes responsible for CDH. For instance, aCGH was a crucial technique in identifying the genetic basis of the CHARGE syndrome (CHARGE association-coloboma, heart anomaly, choanal atresia, retardation, genital, and ear anomalies) in a 2.3-Mb de novo overlapping microdeletion on chromosome 8q12. Subsequently, sequence analysis of genes mapped to this critical region revealed mutations in CHD7 in 222 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c other patients with CHARGE syndrome who did not have microdeletions [Vissers et al., 2004]. Deletions previously reported in unrelated CDH patients include 15q261–q26.2 [Schlembach et al., 2001; Lurie, 2003; Biggio et al., 2004; Hengstschlager et al., 2004; Tumer et al., 2004; Slavotinek et al., 2005, 2006; Klaassens et al., 2005a; Scott et al., 2007], and 1q41–q42 [Youssoufian et al., 1988; Rogers et al., 1995; Lurie, 2003; Kantarci et al., 2006; Slavotinek et al., 2006]; these regions were subjected to further analyses. Chromosome 15q26.1–q26.2 deletion. Several reports suggest that a gene critical for normal diaphragm development is likely located on chromosome 15q26.1–q26.2, based on the occurrence of CDH in patients with cytogenetically visible aberrations in this region. Recent studies using aCGH and FISH in nine patients (seven out of nine with CDH) with 15q26.1–q26.2 aberrations identified an 5Mb commonly deleted interval in the CDH cases [Klaassens Several reports suggest that a gene critical for normal diaphragm development is likely located on chromosome 15q26.1–q26.2, based on the occurrence of CDH in patients with cytogenetically visible aberrations in this region. Recent studies using aCGH and FISH in nine patients (seven out of nine with CDH) with 15q26.1–q26.2 aberrations identified an 5Mb commonly deleted interval in the CDH cases. et al., 2005a]. The 15q26.2 critical region for CDH referred to as Diaphragmatic Hernia 1 (DIH1) [OMIM #142340] contains five known genes: ST8 alpha-N-acetyl-neuraminide (SIAT8B), chromodomain helicase DNA binding protein 2 (CHD2), repulsive guidance molecule domain family member A (RGMA), multiple C2domains, transmembrane 2 (MCTP2), and chicken ovalbumin upstream promoter transcription factor 2 (COUPTFII; NR2F2). Subsequent analyses by these investigators, of only those patients with CDH, narrowed the region to 4 Mb, thereby eliminating SIAT8B from the critical region [Klaassens et al., 2005b]. Further narrowing to isolate the single critical gene associated with CDH was not possible [Castiglia et al., 2005]. A recent study showed that additional genes mapped to 15q26.1–q26.2 CDH critical region, ARRDC4, IGF1R, DMN, TTC23, HSP90B2P, and LRRC28 [Scott et al., 2007], are worthy of study. COUP-TFII, a member of the steroid/thyroid hormone nuclear receptor family, is a likely candidate gene in diaphragm development, due to its location within DIH1 as well as the fact that Couptf2 conditional null mice have diaphragmatic defects [You et al., 2005]. Of 15 cases reported to have COUP-TFII deletion, 11 have CDH [Tumer et al., 2004; Klaassens et al., 2005a; Slavotinek et al., 2006], while four do not [Tonnies et al., 2001; Castiglia et al., 2005; Glass et al., 2006]. Thus, in cases studied to date, deletion of COUP-TFII is strongly associated with CDH [Scott et al., 2007, Kantarci et al., in preparation]. Chromosome 1q41–q42.12 deletion. 1q41–1q42 aberrations were reported in five CDH patients with additional birth defects [Youssoufian et al., 1988; Smith et al., 1994; Rogers et al., 1995; Slavotinek et al., 2006], one of whom was clinically diagnosed with Fryns syndrome [Kantarci et al., 2006], a multiple malformation disorder of unknown etiology characterized by congenital abnormality of the diaphragm, coarse facial features, distal digital hypoplasia of the nails and/or terminal phalanges, pulmonary hypoplasia, genitourinary anomalies, cardio- ARTICLE vascular malformations, and orofacial clefting. We analyzed 29 CDH patients with normal karyotypes using the Spectral GenomeChipTM V1.2 arrays. (Spectral Genomics, Inc., Houston, TX), which contains 2,632 bacterial artificial chromosome (BAC) clones with 1 Mb resolution. Eleven cases were clinically classified as having isolated CDH, while 18 were classified as having complex CDH. The BAC clones previously reported in an analysis of large scale copy number variations (CNVs) [Iafrate et al., 2004] [http://projects.tcag.ca/ variation/], as well as single BAC clones, were excluded from further analysis. The patients with isolated CDH did not have additional copy number aberrations over those previously reported. However, aCGH and FISH analyses revealed a de novo deletion of three consecutive BAC clones over an interval of 5–7 Mb on chromosome 1q41– q42.12 in a new patient clinically diagnosed with Fryns syndrome who died at 1 hr of age. The postmortem examination of the patient showed a large left diaphragm hernia, mild right diaphragmatic eventration, and bilateral pulmonary hypoplasia along with other characteristics features of Fryns syndrome [Kantarci et al., 2006]. Routine chromosomal analysis was normal, but the microdeletion was confirmed independently in a CLIA-certified laboratory (Signature Genomics, Spokane, WA) using the SignatureChipTM array and FISH. Moreover, the deletion interval was further delineated by the MLPA technique [Kantarci et al., in preparation]. Although Fryns syndrome is considered to be an autosomal recessive disorder, based on case reports of parental consanguinity and of sibling recurrences, it could also be caused by (1) de novo autosomal dominant mutations as were found for the disorders Campomelic Dysplasia [OMIM #114290] and Osteogenesis Imperfecta type II [OMIM #166210], historically thought to be autososomal recessive disorders but now known to be autosomal dominant; (2) gonadal mosaicism in one of the parents; (3) deletion of a causal gene on chromosome1q41– ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c q42.12 on one allele, combined with a point mutation in the causal gene on the other allele to produce a heterogeneous compound heterozygote; (4) the appearance of phenotypically similar characteristics resulting from mutations at different genetic loci (locus heterogeneity) each resulting in haploinsufficiency which when combined could lead to increased phenotypic severity. Two reported CDH cases with 1q41–q42.12 deletions [Kantarci et al., 2006; Slavotinek et al. 2006] defined a putative CDH-critical region (6.5 Mb) containing 29 known genes. Analysis of which might provide a number of worthy CDH candidate genes. Two reported CDH cases with 1q41–q42.12 deletions defined a putative CDHcritical region (6.5 Mb) containing 29 known genes. Analysis of which might provide a number of worthy CDH candidate genes. Thus, CDH associated chromosomal hot spots (see Fig. 1) such as 15q26.1–q26.2, 1q41–q42.12, and others including: 8q23 (FOG2); 8p23.1 (GATA4); 2q23.3–q31.1 (Donnai–Barrow families); and 4p16.3 (Wolf–Hirschhorn syndrome) may contain genes which when deleted or disrupted lead to CDH. Furthermore, these hot spots may not be random, but rather reveal genes belonging to com- 223 mon pathways, which can contribute by addictive haploinsufficiency to the appearance or severity of the phenotype. Such findings may provide insight into both mechanism and potential treatment targets for CDH. Genes in these regions are being studied for such possibilities. We hypothesized that the phenotypic severity differences between and within the isolated and complex subtypes could be explained by defects in different genes in a molecular pathway, coexpression of risk factors related to sensitized backgrounds, and haploinsufficiency as a cause of variable penetrance. FUTURE DIRECTIONS In future, high resolution genome wide association studies with copy number variations could be performed on all Figure 1. The CDH candidate genes and loci located on ‘‘CDH-critical’’ chromosome regions. Arrows indicate CDH chromosomal hot spot regions. 224 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c patients with CDH and eventually, should this approach prove to be successful for CDH, it can be recommended for all patients with major congenital anomalies. Customized chips with dense SNP markers across loci and genes discovered in hot spots should be designed for CDH and other complex disorders. This will allow comparison of dense markers from, for example, the HapMap [http://www.hapmap.org/] populations may uncover common SNP signatures associated with and potentially contributing to phenotypic variations, severity, and associated anomalies in CDH. Genes in regions of interest will be analyzed for their potential contributions to a molecular pathway. Understanding such pathways will permit one to conceive downstream replacement therapies, with the ultimate aim of ameliorating or even preventing the severe CDH phenotypes. If variations of the same phenotype can be caused by multiple mutations in a molecular pathway, then the importance of a single SNP change could be missed in the larger population unless one is aware of probable heterogeneous contributions to the same phenotype, based on the principle that most mutations will be autosomal recessive. For the disease to be manifest, loss of the same gene on both alleles, or, more likely, an accumulated contribution from both parents of different allelic changes in the same or different genes in the same molecular pathway will be required. However, less common can be a single defect which causes a dominant negative effect. As discussed above, an Oracle webbased database was developed to support the data collected on patients with CDH and their families. Although this database was devised to support our ongoing CDH study, importantly, it can serve as a platform that is transportable to studies of other congenital anomalies and to monitor the progress of studies of congenital anomalies already in progress. Since the molecular genetic study of CDH and other birth defects requires multi-institutional collaboration, this type of database is a crucial element of such studies. SUMMARY Thus, the molecular and genetic etiologies of a congenital anomaly such as CDH, in the absence of large families for traditional linkage analysis, may be studied for genetic abnormalities by a combination of techniques including candidate gene sequencing, novel high resolution molecular cytogenetics, and loss of homozygosity mapping. Such strategies can reveal multiple chromosomal hot spots associated with changes which can produce the same phenotype (see Fig. 1). The challenge will be to ‘‘connect the dots’’ between each chromosomal region to determine if common molecular pathways will emerge from the analysis. This recently proved to be the case in cleft lip and palate [Lidral and Murray, 2004], Kallmann syndrome 2 [OMIM# 147950], and interstitial pulmonary fibrosis [OMIM#178500]. The later disorder, for example, can occur because of abnormalities in surfactant protein C (SPC), SPB, or the ABCA3 transporter [Bullard et al., 2006]. Murray and colleagues defined that IRF6 mutations cause van der Woude syndrome [OMIM #119300] [Kondo et al., 2002], while over-transmission of a common IRF6 variant increases the risk for isolated non-syndromic cleft lip and palate [Zucchero et al., 2004]. FOG2, GATA4, and COUP-TFII are all needed for both normal diaphragm and lung development and are likely all in the same pathway in in vivo development [Ackerman et al., 2005; You et al., 2005; Jay et al., 2007]. The importance of the retinoic acid signaling pathway in lung [Kumar et al., 2005] and diaphragm development [Kluth et al., 1996; Greer et al., 2000; Gallot et al., 2005] was highlighted by a recent report showing that mutations in STRA6, a member of ‘‘stimulated by retinoic acid’’ gene family caused the CDH phenotype in patients with distinct feature of anophthalmia and varying features of congenital heart defect, lung hypoplasia, alveolar capillary dysplasia, and mental retardation [Pasutto et al., 2007]. Accordingly, it is possible that mutations in gene(s) located in the DBS locus ARTICLE (chromosome 2q23.3–q31.1) or other loci found either by homozygosity mapping or aCGH might cause complex syndromic forms of CDH, while single nucleotide polymorphisms (SNPs) and/ or variants in the same gene(s) might lead us to an understanding of the molecular bases of isolated non-syndromic forms of CDH. The ultimate aim of this paradigm is to uncover genetic pathways which can be perturbed to provide a therapeutic intervention and to reveal the window of opportunity in which that therapeutic can be employed to alleviate the phenotype or even prevent the disorder from occurring. To pursue these technology driven approaches requires a large investment of resources, genetic and surgical talent, but most importantly, dedicated families and patients. We believe, however, that such strategies will be successful, and will bring both understanding and more effective, less invasive treatments to patients with CDH. Birth defects such as CDH occupy one third of U.S. hospital beds and place a high economic burden on the health care system. Identifying the etiology of even a single birth defect, such as CDH, will have a major impact on its own, but can also provide a paradigm for how to approach other congenital anomalies. 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