Candidate genes for congenital diaphragmatic hernia from animal models: sequencing of FOG2 and PDGFRalpha reveals rare variants in diaphragmatic hernia patients

Congenital diaphragmatic hernia (CDH) is a common, life threatening birth defect. Although there is strong evidence implicating genetic factors in its pathogenesis, few causative genes have been identified, and in isolated CDH, only one de novo, nonsense mutation has been reported in FOG2 in a female with posterior diaphragmatic eventration. We report here that the homozygous null mouse for the Pdgfralpha gene has posterolateral diaphragmatic defects and thus is a model for human CDH. We hypothesized that mutations in this gene could cause human CDH. We sequenced PDGFRalpha and FOG2 in 96 patients with CDH, of which 53 had isolated CDH (55.2%), 36 had CDH and additional anomalies (37.5%), and 7 had CDH and known chromosome aberrations (7.3%). For FOG2, we identified novel sequence alterations predicting p.M703L and p.T843A in two patients with isolated CDH that were absent in 526 and 564 control chromosomes respectively. These altered amino acids were highly conserved. However, due to the lack of available parental DNA samples we were not able to determine if the sequence alterations were de novo. For PDGFRalpha, we found a single variant predicting p.L967V in a patient with CDH and multiple anomalies that was absent in 768 control chromosomes. This patient also had one cell with trisomy 15 on skin fibroblast culture, a finding of uncertain significance. Although our study identified sequence variants in FOG2 and PDGFRalpha, we have not definitively established the variants as mutations and we found no evidence that CDH commonly results from mutations in these genes.     

Array comparative genomic hybridization in patients with congenital diaphragmatic hernia: mapping of four CDH-critical regions and sequencing of candidate genes at 15q26.1-15q26.2

Congenital diaphragmatic hernia (CDH) is a common birth defect with a high mortality and morbidity. There have been few studies that have assessed copy number changes in CDH. We present array comparative genomic hybridization data for 29 CDH patients to identify and map chromosome aberrations in this disease. Three patients with 15q26.1-15q26.2 deletions had heterogeneous breakpoints that overlapped with the critical 4 Mb region previously delineated for CDH, confirming 15q26.1-15q26.2 as a critical region for CDH. The three other most compelling CDH-critical regions for genomic deletions based on these data and a literature review are located at chromosomes 8p23.1, 4p16.3-4pter, and 1q41-1q42.1. Based on these recurrent deletions at 15q26.1-15q26.2, we hypothesized that loss-of-function mutations in a gene or genes from this region could cause CDH and sequenced six candidate genes from this region in more than 100 patients with CDH. For three of these genes (CHD2, ARRDC4, and RGMA), we identified missense changes and that were not identified in normal controls; however, none of these alterations appeared unambiguously causal with CDH. These data suggest that CDH caused by chromosome deletions at 15q26.2 may arise because of a contiguous gene deletion syndrome or may have a multifactorial etiology. In addition, there is evidence for substantial genetic heterogeneity in CDH and diaphragmatic hernias can be non-penetrant in patients who have deletions involving CDH-critical regions.     

Single gene disorders associated with congenital diaphragmatic hernia

Congenital diaphragmatic hernia (CDH) is a common birth defect with a high pre- and postnatal mortality. Although the majority of diaphragmatic hernias occur as isolated malformations, additional major and minor anomalies are common and are present in more than 40% of patients. There are compelling data for the importance of genetic factors in the etiology of CDH, but the pathogenesis and the causative genes for CDH in humans remain elusive. There are more than 70 syndromes in which diaphragmatic hernias have been described, and several of these syndromes are single gene disorders for which the gene is known. One method for identifying the causative genes in isolated CDH is to study syndromes with known genes in which CDH is a recognized feature, with the rationale that those genes have a role in diaphragm development. This review discusses the syndromes that are most commonly associated with CDH, with greater attention towards syndromes in which the causative genes have been identified, including Simpson-Golabi-Behmel syndrome, Denys-Drash syndrome, spondylocostal dysostosis, craniofrontonasal syndrome, Cornelia de Lange syndrome and Marfan syndrome.     

Xq12q13.1 microduplication encompassing the EFNB1 gene in a boy with congenital diaphragmatic hernia

Congenital diaphragmatic hernia (CDH) has an incidence of around 1/3000 births. Chromosomal anomalies constitute an important etiology for non-isolated CDH, and may participate to the identification of candidate genes for diaphragm development. We report on a microduplication identified by array-CGH (comparative genomic hybridization) including five contiguous genes (OPHN1, YIPF6, STARD8, EFNB1 and PJA1) and arising de novo in a male presenting a congenital diaphragmatic hernia (CDH). Our case is the second report of EFNB1 duplication associated with CDH in a male patient, supporting its implication sensitive to gene dosage in diaphragm development.     

Prenatal detection and outcome of congenital diaphragmatic hernia (CDH) associated with deletion of chromosome 15q26: two patients and review of the literature

Congenital diaphragmatic hernia (CDH) is a severe birth defect characterized by a defect in the diaphragm with pulmonary hypoplasia and postnatal pulmonary hypertension. Approximately 50% of CDH cases are associated with other non-pulmonary congenital anomalies (so called non-isolated CDH) and in 5-10% of cases there is a chromosomal etiology. The majority of CDH cases are detected prenatally. In some cases prenatal chromosome analysis reveals a causative chromosomal anomaly, most often aneuploidy. Deletion of 15q26 is the most frequently described structural chromosomal aberration in patients with non-isolated CDH. In this paper we report on two patients with a deletion of 15q26 and phenotypes similar to other patients with CDH caused by 15q26 deletions. This phenotype consists of intra-uterine growth retardation, left-sided CDH, cardiac anomalies and characteristic facial features, similar to those seen in Fryns syndrome. We propose that when this combination of birth defects is identified, either pre- or postnatally, further investigations to confirm or exclude a deletion of 15q26 are indicated, since the diagnosis of this deletion will have major consequences for the prognosis and, therefore, can affect decision making.     

Congenital diaphragmatic hernia in a family segregating a reciprocal translocation t(5;15)(p15.3;q24)

Congenital diaphragmatic hernia (CDH) is a relatively common malformation of unknown cause with high mortality due to hypoplasia of the lungs and pulmonary hypertension. We studied a family in which two fetuses had CDH, and two pregnancies resulted in first trimester missed abortions. Both fetuses with CDH had an apparently normal karyotype. In a subsequent pregnancy, fluorescent in situ hybridization analysis of amniocytes showed a balanced translocation 46,XY, t(5;15) (p15.3;q24) also present in the mother and in a normal child, suggesting that the diaphragmatic hernia in the first two fetuses was caused by a cryptic unbalanced translocation. This hypothesis is supported by a previous observation of CDH in a distal deletion of 15q as part of a multiple congenital anomalies syndrome. It is suggested that a gene distal to 15q21 is important for the normal development of the diaphragm.     

The genetics of common disorders – Congenital diaphragmatic hernia

Congenital diaphragmatic hernia (CDH) is a common birth defect with a high mortality and morbidity. Although numerous chromosomal aberrations and gene mutations have been associated with CDH, the etiology of the diaphragmatic defect is identified in less than 50% of patients. This review discusses the some of the more frequent, recurrent karyotypic abnormalities in which CDH is a feature, including 15q26, 8p23.1 and 4p16.3 deletions and tetrasomy 12p (Pallister–Killian syndrome), together with some of the syndromes in which CDH is a relatively common feature, including Fryns syndrome, Matthew-Wood syndrome, overgrowth syndromes and Donnai–Barrow syndrome. In the era of genomic technologies, our knowledge of the genes and chromosome regions involved in pathogenesis of CDH is likely to advance significantly.     

Loss at chromosome arm 16q in retinoblastoma: confirmation of the association with diffuse vitreous seeding and refinement of the recurrently deleted region

In addition to mutations in both alleles of the retinoblastoma gene (RB1) alleles, retinoblastomas frequently show additional alterations including loss of chromosome arm 16q. In a previous study, the presence of 16q alterations was found to be associated with diffuse vitreous seeding of this tumor. This growth pattern is clinically important as it determines therapeutic decisions. The present study was designed to test this association and to narrow down the list of candidate genes in the minimal region of genomic loss on chromosome arm 16q. Our data confirm the association of 16q loss and diffuse vitreous seeding and define a minimal region of genomic loss of 6.6 Mb on 16q containing 86 known genes. As retinoblastoma is an embryonic tumor, we assumed that any gene relevant for its progression is likely to show regulated expression during retinogenesis. Microarray expression analysis of RNA from a continuous developmental series of murine retinas identified murine orthologs with regulated expression and these data helped to narrow the number of candidate genes in minimal region to 35. Analysis of gene expression in retinoblastomas with and without the loss of heterozygosity (LOH) on chromosome 16q further reduced this number to 26 candidate genes. One of these genes is cadherin 13 (CDH13) and notably, downregulation of CHD13 has previously been associated with poorer prognosis in various other cancers.     

Fryns syndrome phenotype caused by chromosome microdeletions at 15q26.2 and 8p23.1

Background: Fryns syndrome (FS) is the commonest autosomal recessive syndrome in which congenital diaphragmatic hernia (CDH) is a cardinal feature. It has been estimated that 10% of patients with CDH have FS. The autosomal recessive inheritance in FS contrasts with the sporadic inheritance for the majority of patients with CDH and renders the correct diagnosis critical for accurate genetic counselling. The cause of FS is unknown. Methods: We have used array comparative genomic hybridisation (array CGH) to screen patients who have CDH and additional phenotypic anomalies consistent with FS for cryptic chromosome aberrations. Results: We present three probands who were previously diagnosed with FS who had submicroscopic chromosome deletions detected by array CGH after normal karyotyping with G-banded chromosome analysis. Two female infants were found to have microdeletions involving chromosome band 15q26.2 and one male had a deletion of chromosome band 8p23.1. Conclusions: We conclude that phenotypes similar to FS can be caused by submicroscopic chromosome deletions and that high resolution karyotyping, including array CGH if possible, should be performed prior to the diagnosis of FS to provide an accurate recurrence risk in patients with CDH and physical anomalies consistent with FS.     

Congenital diaphragmatic hernia associated with duplication of 11q23-qter

Congenital diaphragmatic hernia (CDH) is a relatively common birth defect with a high mortality. Although little is known about its etiology, there is increasing evidence for a strong genetic contribution. Both numerical and structural chromosomal abnormalities have been described in patients with CDH. Partial trisomy 11q and partial trisomy 22 associated with the common t(11;22) has been reported in several cases of CDH. It has been assumed that the diaphragmatic defect seen in these individuals was primarily due to duplication of material from chromosome 22q11. However, in this report we describe a family with a t(11;12) in which one of two brothers with partial trisomy 11q has a left sided posterolateral CDH. This is the second case of CDH in partial trisomy 11q due to an unbalanced translocation other than t(11;22). Using array-based comparative genomic hybridization and fluorescent in situ hybridization, we mapped the breakpoints in both brothers and their mother who is a balanced translocation carrier. Our results suggest that duplication of one or more genes on a approximately 19 Mb region of 11q23.3-qter predisposes to the development of CDH. These effects may be the primary cause of CDH in individuals t(11;22) or may be additive to effects from the duplication of chromosome 22 material. We also conclude that the partial trisomy 11q syndrome has a variable phenotype and that CDH should be added to the spectrum of anomalies that can be present in this syndrome.     

Novel murine homeo box gene on chromosome 1 expressed in specific hematopoietic lineages and during embryogenesis.

We describe here a new murine homeo box gene, denoted Hlx, which is expressed within specific hematopoietic lineages. The cDNA sequence indicates that Hlx differs markedly from known vertebrate homeo box genes, and linkage analysis of an interspecific murine backcross showed that it resides at a novel homeo box locus on the distal portion of mouse chromosome 1. The Hlx homeo domain is most similar to that of the Drosophila H2.0 gene, but outside this region the two polypeptides are related only within a few short segments, the most notable being a motif (denoted Hep) also partially conserved in the engrailed and invected homeo proteins and possibly related to an octapeptide in certain paired box proteins. The presence of an intron within the Hlx homeo box at the same position as in several divergent Drosophila genes (H2.0, labial, Distal-less, proboscipedia, Abdominal-B, NK-1) suggests an ancient evolutionary relationship between these genes. RNA analysis of 67 murine hematopoietic cell lines and normal hematopoietic cells revealed Hlx expression throughout the myeloid/macrophage lineage and at early stages of B lymphocyte development but not in T lymphocytes, erythroid cells, or mast cells. Hence, Hlx is a candidate regulator of hematopoietic lineage commitment and maturation. It probably also functions outside the hematopoietic system, however, because Hlx mRNA could be detected in diverse adult tissues and in embryos from as early as day 8 of development.     

Novel mutation and three other sequence variants segregating with phenotype at keratoconus 13q32 susceptibility locus

Keratoconus (KTCN), a non-inflammatory corneal disorder characterized by stromal thinning, represents a major cause of corneal transplantations. Genetic and environmental factors have a role in the etiology of this complex disease. Previously reported linkage analysis revealed that chromosomal region 13q32 is likely to contain causative gene(s) for familial KTCN. Consequently, we have chosen eight positional candidate genes in this region: MBNL1, IPO5, FARP1, RNF113B, STK24, DOCK9, ZIC5 and ZIC2, and sequenced all of them in 51 individuals from Ecuadorian KTCN families and 105 matching controls. The mutation screening identified one mutation and three sequence variants showing 100% segregation under a dominant model with KTCN phenotype in one large Ecuadorian family. These substitutions were found in three different genes: c.2262A>C (p.Gln754His) and c.720+43A>G in DOCK9; c.2377-132A>C in IPO5 and c.1053+29G>C in STK24. PolyPhen analyses predicted that c.2262A>C (Gln754His) is possibly damaging for the protein function and structure. Our results suggest that c.2262A>C (p.Gln754His) mutation in DOCK9 may contribute to the KTCN phenotype in the large KTCN-014 family.     

Haploinsufficiency of ZNF238 is associated with corpus callosum abnormalities in 1q44 deletions

A variety of candidate genes have been proposed to cause corpus callosum abnormalities (CCAs) in patients with terminal chromosome 1q deletions. Recent data excluded AKT3 and implicated ZNF238 and/or CEP170 as genes causative of corpus callosum anomalies in patients with 1q43–1q44 deletions. We report on a girl with dysmorphic features, seizures beginning in infancy, hypotonia, marked developmental delay, and dysgenesis of the corpus callosum. Chromosomal microarray analysis detected a de novo 1.47 Mb deletion at 1q44. The deleted interval encompasses the ZNF238 gene but not the CEP170 or AKT3 genes, thus providing additional evidence for the former and against the latter as being causative of corpus callosum anomalies in patients with such deletions. © 2013 Wiley Periodicals, Inc.     

Mutational analysis of the anion exchanger 3 gene in familial paroxysmal dystonic choreoathetosis linked to chromosome 2q

Familial paroxysmal dystonic choreoathetosis (PDC) is an autosomal dominant neurological disorder characterized by episodes of involuntary movement precipitated by caffeine, alcohol, or emotional stress. The locus for PDC has recently been mapped to chromosome 2q32-36, but its causative gene has not yet been identified. PDC is most likely a kind of channelopathy, as suggested by the fact that other paroxysmal neurological disorders are caused by various ion channel mutations. Although no ion channel is located in this candidate region, anion exchanger 3 (AE3) has been mapped to 2q36 and has also been reported to be the most promising candidate gene of PDC. In this study we performed sequencing of the coding region of the AE3 gene in patients with familial PDC linked to chromosome 2q and excluded the AE3 gene as the causative gene for PDC. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 88:733-737, 1999.     

Detection of polymorphisms in the estradiol 17{beta}-hydroxysteroid dehydrogenase II gene at the EDH17B2 locus on 17q11-q21

The human estradiol 17ß-hydroxysteroid dehydrogenaseII (17ß-HSD II) gene has been assigned by somaticcell hybridization to chromosome 17q11–q21, near the regionof assignment of the gene BRCA1, which is involved in hereditarybreast-ovarian cancer. The nucleotide sequence of 17ß-HSDII was completely determined in four unrelated individuals.Direct sequencing of PCR fragments that span the complete 17ß-HSDII gene revealed a total of 11 allelic variants which were dueto single base substitutions. The presence of these variantswas then studied in twenty six additional unrelated individuals.There were nine frequent and two rare polymophisms. Seven ofthe 11 polymorphisms were in complete linkage disequilibrium.These polymorphisms in the 17ß-HSD II gene providemarkers that can be used for the genetic mapping of this locus,and may be used to establish whether 17ß-HSD II isa candidate gene for hereditary breast-ovarian cancer.     

Expanding the phenotype of craniofrontonasal syndrome: two unrelated boys with EFNB1 mutations and congenital diaphragmatic hernia

Craniofrontonasal syndrome (CFNS, MIM 304110) is an X-linked craniofacial disorder that shows paradoxically greater severity in heterozygous females than in hemizygous males. Mutations have been identified in the EFNB1 gene that encodes a member of the ephrin-B family of transmembrane ligands for Eph receptor tyrosine kinases. Here, we describe two unrelated families, in both of which a mother and her son have proven mutations in EFNB1. The mothers have classical features of CFNS; although the sons have no major craniofacial features other than telecanthus, both had a congenital diaphragmatic hernia (CDH). Our cases represent the first in which CDH has been confirmed in males with mutations in EFNB1, highlighting an important role for signalling by ephrin-B1 in the development of the diaphragm.     

Fine mapping of the locus for Shwachman-Diamond syndrome at 7q11, identification of shared disease haplotypes,and exclusion of TPST1 as a candidate gene

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterised by exocrine pancreatic dysfunction, haematological and skeletal abnormalities. We have previously defined the SDS locus as a 2.7 cM interval spanning the centromere of chromosome 7. To facilitate additional analysis of this complex and poorly characterised region, a framework of ordered genetic markers at 7p11-q11, including six newly identified, has been constructed using somatic cell, radiation hybrid and STS-content mapping. We have identified shared disease haplotypes, that recur in unrelated families of common ethnic origin, and extend across the SDS locus. Detection of ancestral and intrafamilial recombination events in patients refined the SDS locus to a 1.9 cM interval at 7q11, which contains the tyrosylprotein sulfotransferase 1 (TPST1) gene. Patients with SDS were screened for mutations in TPST1 by sequencing of exons and intron-exon junctions. Two single nucleotide polymorphisms, but no disease-causing mutations, were identified. In addition, Southern blot analysis yielded no evidence of large-scale mutations, and RT-PCR analysis failed to detect alterations in expression. These results exclude TPST1 as the causative gene for SDS. The established map of the refined SDS locus will assist in the identification and characterisation of other candidate genes for SDS.