Original Article Mol Syndromol 2020;11:271–283 Received: March 5, 2020 Accepted: August 13, 2020 DOI: 10.1159/000510910 Published online: November 11, 2020 Phenotypic Characteristics and Copy Number Downloaded from http://karger.com/msy/article-pdf/11/5-6/271/3130487/000510910.pdf by Universitat Autònoma Barcelona Servei de Biblioteques user on 03 November 2023 Variants in a Cohort of Colombian Patients with VACTERL Association Olga M. Moreno a Ana I. Sánchez a, b, c Angélica Herreño a Gustavo Giraldo a Fernando Suárez a, d Juan Carlos Prieto a Ana Shaia Clavijo a Mercedes Olaya e Yaris Vargas f Javier Benítez g Jordi Surallés h Adriana Rojas a aInstituto de Genética Humana, Facultad de Medicina, Pontificia Universidad Javeriana, Bogotá, Colombia; bDepartamento Materno Infantil, Facultad de Ciencias de la Salud, Pontificia Universidad Javeriana, Cali, Colombia; cCentro Médico Imbanaco de Cali, Cali, Colombia; dUnidad de Genética Medica, Hospital Universitario de San Ignacio, Bogotá, Colombia; eServicio de Patología, Hospital Universitario de San Ignacio, Bogotá, Colombia; fServicio de Pediatría, Neonatología, Hospital Universitario de San Ignacio, Bogotá, Colombia; gCNIO: Centro Nacional de Investigaciones Oncológicas, Madrid, Spain; hDepartamento de Genética y Microbiología, Universidad Autónoma de Barcelona, Barcelona, Spain Keywords All patients (10 female and 8 male) showed a broad clinical Array-CGH · Chromosomal microalterations · Congenital spectrum: 13 (72.2%) had vertebral abnormalities, 8 (44.4%) malformations · Copy-number variants · Fanconi anemia · had anal atresia, 14 (77.8%) had heart defects, 8 (44.4%) had VACTERL association esophageal atresia, 10 (55.6%) had renal abnormalities, and 10 (55.6%) had limb defects. Chromosomal abnormalities and FA were ruled out. In 2 cases, the finding of microaltera- Abstract tions, namely del(15)(q11.2) and dup(17)(q12), explained the VACTERL association (OMIM 192350) is a heterogeneous phenotype; in 8 cases, copy number variations were classi- clinical condition characterized by congenital structural de- fied as variants of unknown significance and as not yet de- fects that include at least 3 of the following features: verte- scribed in VACTERL. These variants comprise genes related bral abnormalities, anal atresia, heart defects, tracheoesoph- to important cellular functions and embryonic develop- ageal fistula, renal malformations, and limb defects. The ment. © 2020 The Author(s) nonrandom occurrence of these malformations and some Published by S. Karger AG, Basel familial cases suggest a possible association with genetic factors such as chromosomal alterations, gene mutations, and inherited syndromes such as Fanconi anemia (FA). In this Introduction study, the clinical phenotype and its relationship with the presence of chromosomal abnormalities and FA were evalu- VATER association was first described by Quan and ated in 18 patients with VACTERL association. For this, a G- Smith in 1972 as a statistically nonrandom co-occurrence banded karyotype, array-comparative genomic hybridiza- of a group of different multisystem congenital malforma- tion, and chromosomal fragility test for FA were performed. tions, including vertebral anomalies (V), anal atresia (A), karger@karger.com © 2020 The Author(s) Olga M. Moreno www.karger.com/msy Published by S. Karger AG, Basel Instituto de Genética Humana This is an Open Access article licensed under the Creative Commons Facultad de Medicina, Pontificia Universidad Javeriana Attribution-NonCommercial-4.0 International License (CC BY-NC) Carrera 7 #40-62, Bogotá 110231 (Colombia) (http://www.karger.com/Services/OpenAccessLicense), applicable to moreno-o @ javeriana.edu.co the online version of the article only. Usage and distribution for com- mercial purposes requires written permission. tracheoesophageal (TE) fistula with or without esopha- dromes, and it is usually considered a diagnosis of exclu- geal atresia, and renal malformations (R) [Quan and sion; thus, accurate diagnosis can be quite challenging Smith, 1972, 1973]. The complete phenotype delineation [Siebel and Solomon, 2013]. Phenotypically overlapping occurred in 1996 and included cardiac defects (C) and disorders of VACTERL association include Fanconi ane- limb anomalies (L) (VACTERL) (OMIM 192350) [Rittler mia (FA), Baller-Gerold syndrome, CHARGE syndrome, et al., 1996]. VACTERL with hydrocephalus VACTERL- deletion 22q11.2 syndrome, MURCS association, oculo- H (OMIM 276950) is a more severe condition that has auriculo-vertebral syndrome, Opitz G/BBB syndrome, been associated with autosomal recessive or X-linked in- Pallister-Hall syndrome, Feingold syndrome, Holt-Oram heritance (OMIM 314390) [Porteous et al., 1992; Alter syndrome, Townes-Brocks syndrome, and VACTERL- Downloaded from http://karger.com/msy/article-pdf/11/5-6/271/3130487/000510910.pdf by Universitat Autònoma Barcelona Servei de Biblioteques user on 03 November 2023 and Rosenberg, 2013; Jung et al., 2020]. Because these H, among others [Siebel and Solomon, 2013; Chen et al., malformations have been observed to occur together 2016]. more often than would be expected by chance, the condi- In fact, patients with FA often exhibit multiple con- tion was termed an association. VACTERL association is genital malformations, suggesting a diagnosis of VAC- typically defined by the presence of at least 3 of the con- TERL association [Fiesco-Roa et al., 2019]. Furthermore, genital malformations mentioned above. VACTERL as- patients with clinically suspicious VACTERL-H have sociation is a relatively common condition, with an inci- been found to have pathogenic variants in different FANC dence estimated at 1 in 10,000–40,000 live-born infants genes, and they have accordingly been reclassified as hav- [Siebel and Solomon, 2013]. ing FA [Holden et al., 2006]. Because of the above, a com- To date, there is no known evidence for a single unify- mon pathophysiology pathway for VACTERL and FA pa- ing cause that would result in the condition. Because tients involving Shh genes has been proposed [Lubinsky, VACTERL association is likely to be causally heteroge- 2015]. As much as 5–10% of patients clinically diagnosed neous, only a few studies have sought genetic or other with VACTERL-H instead have FA; therefore, FA must causal explanations among large cohorts of affected indi- be considered one of the most important differential di- viduals [Solomon et al., 2010a, b; Solomon, 2011; Saisawat agnoses among these patients [Alter and Rosenberg, et al., 2014; Hilger et al., 2015]. It was recently identified 2013]. that recessively inherited mutations in the genes TRAP1 The aim of this study was to assess the clinical charac- and ZIC3 (the latter linked to the X chromosome), associ- teristics of a cohort of Colombian patients who met the ated with the pathophysiology of VACTERL, are a mono- diagnostic criteria for VACTERL association and to iden- genic cause of this condition [Saisawat et al., 2014; Hilger tify chromosomal rearrangements by conventional and et al., 2015]. Moreover, it was proposed that the FOXF1 molecular cytogenetics, in addition to determining the gene may also be a candidate [Stankiewicz et al., 2009; cellular phenotype of FA, to contribute to the knowledge Shaw-Smith, 2010; Hilger et al., 2015]. Based on experi- of the genetic causes of the disease. mental animal models, it has been suggested that genes related to the Sonic hedgehog (Shh) pathway may be in- volved in the phenotype [Kim et al., 2001; Tumini et al., Patients and Methods 2019], as well as genes that act on mitochondrial and cil- iary function [Thauvin-Robinet et al., 2006; Siebel and Eighteen pediatric patients with high clinical suspicion of VACTER/VACTERL association were included. Solomon, 2013; Hilger et al., 2015]. Additional evidence supporting genetic causes of Clinical Data VACTERL association is derived from case reports. These All data regarding clinical features were obtained by physical studies have also provided insight into different chromo- examination or from clinical reports; these data were analyzed to somal deletions and microduplication/microdeletion describe clinical aspects. Our analysis was based on clinical re- cords from patients who met the criteria for VACTERL. When a variants that might explain the phenotype. For example, diagnosis of VACTERL was suspected by any medical specialist, deletions at 13q and microduplications of different sizes the patient was referred to our center to be examined by medical at 1q41, 2q37.3, 8q24.3, 22q11.21, and Xq25q27.3 have geneticists. All patients received medical care and were followed been detected in patients [Schramm et al., 2011; up by different specialists. The diagnostic workup began with Dworschak et al., 2013; Hilger et al., 2013, 2015; Puvaban- the outpatient clinic visit or in the neonatal care unit where the main clinical and genealogical data were collected. Patient re- ditsin et al., 2016]. cords were collected and archived in specifically dedicated files. On the other hand, this heterogeneous condition has VACTERL association was diagnosed in patients who met at least many overlapping defects with other malformation syn- 3 of the 6 criteria (vertebral defects, anal atresia, cardiac defects, 272 Mol Syndromol 2020;11:271–283 Moreno et al. DOI: 10.1159/000510910 Table 1. Clinical characteristics of patients with VACTERL association Case ID Age Gender Vertebral Anal Heart Esophageal Tracheo- Renal Limb No. of No. of anomalies atresia defects atresia esophageal defects defects other VACTERL fistula defects birth defects 2 1m F + + – + + + + 5 6 3 5y F + + + – – + – 2 4 5 1y 8m F + – + + + – – 0 4 6 2y 5m M + – + + + – – 1 4 Downloaded from http://karger.com/msy/article-pdf/11/5-6/271/3130487/000510910.pdf by Universitat Autònoma Barcelona Servei de Biblioteques user on 03 November 2023 7 1m F + – + + + – – 0 4 8 8m F – + – + – – + 0 3 9 4y F – + + – – + + 2 4 10 18d F + – + – – – + 2 3 11 4d M – – – + + + + 1 4 12 3y 8m F + – + – – – + 4 3 13 3d F + + + – – – – 1 3 14 1m M + – + – – + – 0 3 16 3y 2m M – + + + – + + 3 5 17 1y 3m M + + – – – + – 1 3 18 15d M – – + + + – + 1 3 19 15y F + – + – – + + 3 4 20 1y 2m M + – + – – + – 2 3 21 17y M + + + – – + + 4 5 Total cases 13 8 14 8 6 10 10 14 18 y, years; m, months; d, days; F, female; M, male; +, present; –, absent. tracheoesophageal fistula/esophageal atresia, renal dysplasia, and tained by standard procedures and stained with Giemsa (Merck). limb anomalies). Common newborn findings, such as vesicoure- Chromosomal breaks and exchanges were evaluated in nonbanded teral reflux and patent ductus arteriosus, were not considered chromosomes as recommended in the literature [Auerbach, 1988]. major anomalies to warrant inclusion as a component feature At the end of the procedure, the percentage of aberrant cells (cells unless another anomaly affecting the same organ system was with chromosomal breakages, fragments, rearrangements, ex- present simultaneously. The final diagnosis of VACTERL was changes or radial figures), breakages per cell, and breakages per made after excluding other genetic syndromes with overlapping aberrant cell were analyzed, and the percentage of multiaberrant phenotypes. cells (cells with 2 or more breakages or alterations) was calculated. In addition, the total number of breakages and exchanges per cell Conventional Cytogenetics was determined. To evaluate the possible genetic causes of VACTERL associa- tion, a complete cytogenetic analysis was performed. Peripheral Molecular Cytogenetics blood lymphocytes were cultured in RPMI 1640 medium (Sigma), Genomic DNA was isolated from peripheral blood samples by 10% fetal bovine serum (Eurobio), and phytohemagglutinin (Gib- using the salting-out procedure according to the modified proto- co), synchronized with amethopterin (1 × 10−5 M; Sigma) and un- col described by Miller et al. [1988]. Lysis of red blood cells was locked with thymidine (2.4 × 10−6 g/mL; Sigma) to obtain prometa- performed with ammonium chloride and SDS. Precipitation of phase chromosomes. G-banding was performed for all patients. proteins was performed with ammonium acetate, and precipita- Chromosomes were treated with 0.025 N HCl (Merck), followed by tion of DNA was performed with isopropanol. The DNA was pre- incubation in 1× SSC buffer and Wright (Merck) staining to obtain served in TE solution (10 mM Tris-HCl, pH 1.4 and 1 mM EDTA, the G-banding. For each patient, 25 metaphases were analyzed with pH 8) and stored at −20°C until analysis. Array-comparative ge- a resolution of 550–700 bands. Chromosome breakage analyses for nomic hybridization (aCGH) was performed for each individual FA were performed by using standard procedures [Auerbach, by using Agilent®SurePrint G3 Human CGH Microarray 180K 1988]. Peripheral blood lymphocytes were cultured in RPMI me- and Cytogenomics software v. 4.0.3.12 Agilent®. Copy number dium plus 10% fetal bovine serum and phytohemagglutinin and variations were evaluated by using the following databases: http:// then incubated for 24 h at 37°C. For each individual, 2 cultures were dgvbeta.tcag.ca/dgv/app/home?ref=GRCh37/hg19, https://deci- treated with butadiene diepoxide (DEB) (100 ng/mL; Alfa Aesar) pher.sanger.ac.uk/, http://dbsearch.clinicalgenome.org/search/, and incubated for 48 h under the same conditions; 2 other cultures www.omim.org, and https://www.uniprot.org/uniprot. were not treated with DEB. Chromosomes in metaphase were ob- Phenotype and CNVs in Patients with Mol Syndromol 2020;11:271–283 273 VACTERL Association DOI: 10.1159/000510910 Hemivertebrae 8 Clinical characteristics Segmentation of vertebral bodies 3 Butterfly vertebrae 2 Scoliosis 1 Downloaded from http://karger.com/msy/article-pdf/11/5-6/271/3130487/000510910.pdf by Universitat Autònoma Barcelona Servei de Biblioteques user on 03 November 2023 Without vertebral anomaly 5 0 2 4 6 8 10 a Number of cases Atrial septal defect 8 Clinical characteristics Ventricular septal defect 4 Atrioventricular 1 Pulmonary artery (PA) dilatation 1 Interrupted aortic arch 1 Atrial septal aneurysm 1 Without cardiac defects 4 0 2 4 6 8 10 b Number of cases Renal agenesis 7 Clinical characteristics Crossed fused renal ectopia 1 Nephronophthisis 1 Hydronephrosis 1 Without renal defects 8 0 2 4 6 8 10 c Number of cases Thumb anomalies 7 Clinical characteristics Radius hypoplasia/aplasia 2 Club foot 1 Fig. 1. Type and frequency of congenital Without limb birth defects 8 malformations that occurred non-ran- domly in the 18 patients with VACTERL association. a Vertebrae congenital anom- 0 2 4 6 8 10 alies. b Heart defects. c Renal birth defects. d Number of cases d Limb birth defects. Each figure shows the number of cases presenting each character- istic. 274 Mol Syndromol 2020;11:271–283 Moreno et al. DOI: 10.1159/000510910 Genital defects 4 Single umbilical artery 2 Facial defects 2 Clinical characteristics Global developmental delay/hypotonia 2 Microphthalmia/anophthalmia/other ocular abnormalities 2 Short stature 2 Café-au-lait spots 2 Central nervous system defect 1 Downloaded from http://karger.com/msy/article-pdf/11/5-6/271/3130487/000510910.pdf by Universitat Autònoma Barcelona Servei de Biblioteques user on 03 November 2023 Rib cage defects 1 Small bowel atresia 1 Rectal/sigmoid 1 Without other type of birth defects 4 0 1 2 3 4 5 Number of cases Fig. 2. Other types of birth defects in our patients, not usually reported in the VACTERL spectrum. The figure shows the number of cases presenting each characteristic. Results the DNA of another 3 cases was not of sufficient quality for the analysis. No gene dose alterations or copy number vari- We evaluated 18 patients who met the criteria for ations (CNVs) were detected in 5 cases (cases 2, 3, 6, 8, and VACTERL association. The female to male ratio was 1.2, 20). Interestingly, 8 patients had CNVs based on aCGH re- with 10 female patients and 8 male patients. Ages at the sults classified as variants of unknown significance (VUS). time of the first clinical examination varied from 3 days This classification was obtained by using these databases: to 17 years old. Eight patients (44.4%) were under 1 year http://dgvbeta.tcag.ca/dgv/app/home?ref=GRCh37/hg19, old, 8 patients (44.4%) ranged between 1 and 5 years old, www.omim.org, https://decipher.sanger.ac.uk/, and spe- and 2 patients (11.2%) were teenagers. The clinical fea- cialized literature (online suppl. Table 1, see www.karger. tures of all patients are described in Table 1. com/doi/10.1159/000510910). These CNVs have not been Thirteen patients (72.2%) had vertebral anomalies. previously reported in individuals with phenotypes similar Anal atresia was evident in 8 patients (44.4%). Congenital to those of our patients. The regions harbor genes shown in heart defects were seen in 14 patients (77.8%). Esophageal Table 2. Two patients (cases 11 and 19) had abnormal atresia was diagnosed in 8 patients (44.4%), 6 of whom had aCGH results. Patient 11 (male) exhibited a duplication of a tracheoesophageal fistula. Ten individuals (55.6%) had 1.4 Mb at 17q12 (arr[GRCh37] 17q12(34817422_36243028) renal anomalies. Limb defects were observed in 10 patients ×3) classified as pathogenic (Fig. 3). Patient 19 (female) har- (55.6%). Each specific birth defect is provided in Figure 1. bored a deletion of 443 kb at 15q11.2 (arr[GRCh37] Fourteen of 18 patients (77.8%) showed additional system- 15q11.2(22765628_23208901)×1) classified as a VUS with ic features of the VACTERL association spectrum, with a variable expression and incomplete penetrance. Her healthy nonspecific pattern of VACTERL-type birth defects, in- mother, without any birth defect, was a carrier of the same cluding genital abnormalities, single umbilical artery, and CNV. anophthalmia. These birth defects are shown in Figure 2. All patients had normal karyotypes. The analysis of chromosomal breakages with DEB for FA was negative, Discussion with an average breakage per cell of 0.12, thus being with- in the normal range (0.02–0.37) for individuals not af- VACTERL association, which usually requires at least fected by FA. 3 component features and the absence of evidence for an Molecular cytogenetic analysis was performed for 15 pa- overlapping condition, is characterized as a heteroge- tients; in 2 cases, the parents were also analyzed, whereas neous condition with different environmental or genetic Phenotype and CNVs in Patients with Mol Syndromol 2020;11:271–283 275 VACTERL Association DOI: 10.1159/000510910 Table 2. Results of aCGH analyses in 10 patients with VACTERL association: genes involved and their function Patient Type Location Genomic coordinates, Genes Name/function of the genes Reference of CNV (size, kb) arr[GRCh37] 9 del 1p35.3 (100) chr1:28,947,613– TAF12 TATA-box binding protein associated factor 12 Gazit et al., 2009 29,047,557 Transcription and transcription regulation Detected in all tissues GMEB1 Glucocorticoid modulatory element-binding Nakagawa et al., protein 1 2008 Regulator of oxidative stress-induced apoptosis Downloaded from http://karger.com/msy/article-pdf/11/5-6/271/3130487/000510910.pdf by Universitat Autònoma Barcelona Servei de Biblioteques user on 03 November 2023 Ubiquitous expression 11 dup 17q12 (1,400) chr17:34,817,422– ZNHIT3, PIGW, 17q12 microduplication syndrome Bierhals et al., 36,243,028 ACACA, HNF1B, 2013; Mitchell and others et al., 2015 12 dup 14q24.2 (249) chr14:71,376,123– PCNX1 Pecanex-like protein 1 Yamakawa et al., 71,625,114 In Drosophila it is related to Notch signaling 2018 Expression in all tissues 13 dup 12p12.1 (41) chr12:21,580,444– PYROXD1 Pyridine nucleotide-disulfide oxidoreductase Saha et al., 2018 21,621,404 domain-containing protein 1 Oxidoreductase; cellular oxidative stress response Expression in all tissues 14 dup 18p11.32 (529) chr18:131,700– USP14 Ubiquitin carboxyl-terminal hydrolase 14 Kim et al., 2018 661,043 Proteasome-associated deubiquitinase, autophagia Expression in all tissues THOC1 THO complex subunit 1; part of TREX Wang et al., 2007; (transcription and export) complex Heath et al., 2016 Apoptosis regulator, transcription regulation Expression in all tissues COLEC12 Collectin-12 Graham et al., 2011 Promotes phagocytosis, involved in host defense Expression in endothelial cells (placenta and other tissues) Evolutionarily conserved gene in vertebrates CETN1 Centrin-1 Errabolu et al., Cell division functions: role in microtubule- 1994 organizing center structure and function, and cilia formation Cytoplasmic expression in spermatids and spermatocytes CLUL1 Clusterin-like protein 1 Talmud et al., Role in microtubule-organizing center structure 2009; Winkler and function, functional role during photoreceptor et al., 2018 differentiation Expression mainly in retina TYMSOS TYMS opposite strand protein UniProta Expression in all tissues causes not yet well understood. However, there are efforts cohort of patients. Although the literature contains some to clarify the whole phenotypic spectrum as well as the controversy regarding the inclusion of certain features for underlying genotype [Solomon et al., 2014]. the diagnosis of VACTERL [Temtamy and Miller, 1974; All patients in our study exhibited a wide clinical spec- Miller et al., 1988; Rittler et al., 1996; Solomon, 2011], ver- trum of birth defects. The results from this study high- tebrae anomalies, heart defects, and esophageal atresia light several important findings regarding VACTERL as- were observed in over half of our patients, which is in ac- sociation, which remains a poorly understood condition. cordance with the worldwide literature [Husain et al., Cardiac defects, followed by esophageal and vertebral de- 2018]. Renal defects were also seen in 55.6% of our pa- fects, were the most common types of birth defects in this tients, which is similar to the reports of other cohorts, 276 Mol Syndromol 2020;11:271–283 Moreno et al. DOI: 10.1159/000510910 Table 2 (continued) Patient Type Location Genomic coordinates, Genes Name/function of the genes Reference of CNV (size, kb) arr[GRCh37] 16b dup 14q32.33 (305) chr14:104,289,937– PPP1R13B Protein phosphatase 1 regulatory subunit 13B Samuels-Lev et al., 104,594,840 Central role in regulation of apoptosis via its 2001 interaction with p53/TP53 Evolutionarily conserved gene; ubiquitous expression ATP5MPL/ ATP synthase membrane subunit 6.8 PL, Fujikawa et al., Downloaded from http://karger.com/msy/article-pdf/11/5-6/271/3130487/000510910.pdf by Universitat Autònoma Barcelona Servei de Biblioteques user on 03 November 2023 C14orf2 mitochondrial 2014 Component of an ATP synthase complex Expression in all tissues TDRD9 ATP-dependent RNA helicase TDRD9 Arafat et al., 2017 ATP-binding RNA helicase required during spermatogenesis Highly expressed in parathyroid gland and testis RD3L Retinal degeneration 3 like UniProta Expression in heart muscle ASPG Asparaginase UniProta High expression in kidney; high expression in heart during human fetal development MIR203A, Involved in branching morphogenesis and Sonkoly et al., 2010 MIR203B differentiation of human keratinocytes. Regulate the expression of many target genes and play pivotal roles in the development 17 del 19p13.11 (146) chr19:17,733,344– UNC13A Protein unc-13 homolog A gene; gene conserved Sheu et al., 2003 17,878,860 Melanocyte differentiation, exocytosis Highly expressed in adult brain MAP1S Microtubule-associated protein 1S Xie et al., 2011 Apoptosis, autophagy, organization of the centrosomes Conserved in placental mammals FCHO1 F-BAR domain only protein 1 Henne et al., 2010 Clathrin-mediated endocytosis and involved in T-cell differentiation and function Evolutionarily conserved gene 18 dup 1p36.23 (121) chr1:7,946,655– TNFRSF9 Tumor necrosis factor receptor superfamily Jang et al., 1998 8,068,000 member 9 Immune response and apoptosis Highly expressed in blood and lymphoid tissue PARK7 Protein/nucleic acid deglycase DJ-1 Krebiehl et al., Autophagy, DNA repair, inflammatory response 2010 Ubiquitous expression Evolutionarily conserved gene 19b del 15q11.2 (443) Chr15:22,765,628– NIPA1, NIPA2, 15q11.2 microdeletion syndrome Cox and Butler, 23,208,901 CYFIP1, TUBGCP5, 2015 LOC283683, WHAMMP3 21 del 10q25.2 (210) chr10:114,316,4177– VTI1A Vesicle transport through interaction with Emperador-Melero 114,526,448 t-SNAREs homolog 1A et al., 2019 Involvement in vesicle generation and Ca2+-channel trafficking in exocytosis Evolutionarily conserved gene MIR4295 Tumor suppressor Li et al., 2016; Regulates the expression of target genes as Gao and Zheng, CDKN1A and RUNX3 2018 a https://www.uniprot.org/uniprot. b Analysis of parents: healthy mother carrying the CNV. Other databases consulted: https://www.ncbi.nlm.nih.gov/ gene/, https://www.proteinatlas.org/, http://dbsearch.clinicalgenome.org/search/, http://dgv.tcag.ca/gb2/gbrowse/dgv2_hg19/. del, deletion; dup, duplication. Phenotype and CNVs in Patients with Mol Syndromol 2020;11:271–283 277 VACTERL Association DOI: 10.1159/000510910 Downloaded from http://karger.com/msy/article-pdf/11/5-6/271/3130487/000510910.pdf by Universitat Autònoma Barcelona Servei de Biblioteques user on 03 November 2023 Fig. 3. Array-CGH for chromosome 17 in case 11. Data analysis by CytoGenomics software is shown. The left image shows the duplicated region in 17q12; the involved genes in this CNV are shown in the right image. indicating that the frequency of these types of defects causes. Interestingly, the aCGH results allowed us to de- among VACTERL patients is greater than 50% and thus tect CNVs in 10 patients: CNVs in 8 cases were classified one of the most common anomalies [Reutter et al., 2016; as VUS and are new; in the other 2 cases, CNVs were iden- Husain et al., 2018]. tified as a deletion in 15q11.2 and a duplication in 17q12. To clarify the pathophysiology, previous works have Both alterations are associated with well-known genetic suggested that patients with VACTERL association might syndromes (OMIM 615656 and 614526) not previously be subdivided into “upper” and “lower” groups: patients diagnosed in our cohort. with cardiac malformations belong to the upper group, Several CNVs have been reported as a frequent finding and patients with renal defects belong to the lower group in patients with different types of congenital malforma- [Källén et al., 2001]. However, as patients in our study tions, including those affected with VACTERL associa- with renal anomalies also had cardiac defects, it was dif- tion; however, for the vast majority of these regions, their ficult for us to separate them into these 2 groups. This co- effects on phenotypes are unknown [Nguyen et al., 2017; occurrence of both types of birth defects (cardiac and re- Zhang et al., 2017]. The CNVs found in the 8 patients of nal anomalies) has been reported in patients, and it has our cohort have not been previously related to the condi- been suggested that there may be a common genetic cause tion, and the affected genes in these regions have not been for the occurrence of both anomalies [Gabriel et al., 2018]. associated with the etiology. The CNVs detected in our The above highlights the fact that one mechanism under- patients involve genes implicated in different cellular lying the physiopathology may be common for all VAC- processes, such as apoptosis, endocytosis, energy metabo- TERL patients, including those with different clinical fea- lism, response to oxidative stress, intracellular trafficking, tures. immune response, and gene regulation, among others. Genetic factors as a cause of VACTERL association Although these CNVs have not yet been associated with have also been investigated and suggested in its etiology. the disease, these molecular processes are important in This is due to some observations: (1) the existence of fam- embryonic development [Stevenson and Hunter, 2013]. ilies with at least 2 affected members; (2) the presence of Several genes located in the regions of the CNVs have also de novo variants in some patients, found in genes that act been implicated in other diseases or have been related to during embryonic development; and (3) the presence of cancer (Table 2). On the other hand, alterations in regions chromosomal microalterations that affect gene-bearing that encode some microRNAs were also found (Table 2), regions involved in fetal development [Solomon et al., findings that are consistent with previous reports in 2010a, b; Hilger et al., 2012; Reutter et al., 2016]. In our which the regulatory role of the miRNAs is highly impli- cohort, none of the cases had a family history of similar cated in embryonic development [Allis et al., 2015; Al- phenotypes or the presence of CNVs for other clinical berti and Cochella, 2017]. 278 Mol Syndromol 2020;11:271–283 Moreno et al. DOI: 10.1159/000510910 The CNV by microdeletion at 19p13.11 in case 17 al effect on the phenotype when the gene is entirely du- (male patient with anal atresia, recto-perineal fistula, tho- plicated is unknown. racic hemivertebrae, thoracic butterfly-shaped vertebrae, The THOC1 gene was found in the duplicated 18q12 alterations of segmentation in the cervical vertebrae, su- region and encodes a component of the TREX complex. pernumerary ribs, and left renal agenesis) is a region with This complex is evolutionarily conserved across a wide 3 interesting genes involved in maintaining cellular ho- range of organisms and plays a central role in mRNA ex- meostasis. The MAP1S gene has been related to autoph- port [Wang et al., 2007]. Among other cellular functions, agy regulation in all tissues and has been associated with TREX is necessary for maintaining differentiation during renal fibrosis, heart disease, neurodegenerative diseases, embryogenesis [Heath et al., 2016]. The CLUL1 gene, lo- Downloaded from http://karger.com/msy/article-pdf/11/5-6/271/3130487/000510910.pdf by Universitat Autònoma Barcelona Servei de Biblioteques user on 03 November 2023 and some cancers, among others [Xie et al., 2011; Xu et cated in the same region, has been found to be differen- al., 2016]. Autophagy is an evolutionarily conserved pro- tially methylated (hypomethylated) in the placentas of cess in all eukaryotic organisms and plays a key role in newborns with congenital heart defects [Radhakrishna et maintaining cellular homeostasis during embryonic de- al., 2019]; however, no association with cardiac develop- velopment and differentiation [Martinet et al., 2009; Boya ment has been reported before. It reportedly overregu- et al., 2018; Levine and Kroemer, 2019]. FCHO1 is an- lates many different forms of neurodegeneration [Tal- other gene involved in clathrin-mediated endocytosis mud et al., 2009]. Notably, epigenetic alterations have and in T-cell development and function in humans [Sheu also been mentioned as a probable cause of VATER/ et al., 2003; Łyszkiewicz et al., 2020]. Silencing of fcho1 in VACTERL possibly associated with environmental fac- zebrafish embryos causes alterations in morphogenesis tors [Solomon, 2018; Lubinsky, 2019]. [Umasankar et al., 2012]. The UNC13A/MUNC13A-1 Another gene that also needs to be mentioned is the gene is evolutionarily conserved and involved in vesicle PCNX1 gene located at 14q24.2 affected by duplication maturation during exocytosis in the synaptic process in patient 12. This gene is highly conserved among spe- [Brose and Rosenmund, 2002; Magdziarek et al., 2020]. cies. Although its role in mammals is not well known, it Homozygous mutations in this gene have been associated has been studied in Drosophila, demonstrating that it is with microcephaly and abnormal cortical electrical activ- related to the NOTCH signaling pathway and may play ity [Engel et al., 2016]. It is striking that the cellular pro- a role in vesicular transport during neuroblast segrega- cesses in which these genes participate are highly active tion of the neuroectoderm [Yamakawa et al., 2018. The during embryogenesis [Mizushima and Levine, 2010]. NOTCH signaling pathway is known to be involved in We may then propose that alteration in protein levels the formation of anatomical structures affected in VAC- might affect, to some extent, the balance of the processes TERL association, such as the development of the cardio- of embryonic morphogenesis, such as those presented by vascular system and the spine [Stevenson and Hunter, the patient. 2013]. The duplication at 1p36.23 of patient 18 involves the Other genes found in the CNVs of our patients have PARK7 gene. This is an interesting gene because it is in- some important cellular functions, as described in Ta- volved in multiple cellular processes, such as in autopha- ble 2. Regarding CNVs related to known syndromes, the gy and endocytosis, as a redox-sensitive chaperone and as deletion at 15q11.2 has been reported to be a variant caus- a sensor for oxidative stress, among others [Lee et al., ing variable expression and incomplete penetrance affect- 2018]. Mutations in this gene have been highly associated ing the genes NIPA1, NIPA2, CYFIP1, and TUBGCP5 lo- with autosomal recessive Parkinson disease [Kasten et al., cated in the nonimprinted area. These genes are highly 2018]. However, in our patient, the gene was duplicated, conserved, and their alteration causes neurological, cog- and the effects of this on development are unknown. The nitive, and behavioral problems [Cox and Butler, 2015]. TAF12 gene is involved in the duplication at 1q31.1 in The phenotype of the patient displays several nonclassical case 9, and the encoded protein is reported to have DNA- clinical characteristics for this syndrome, resulting in in- binding transcription factor activity and is implicated in clusion as VACTERL. It is possible that other genetic or cancer [Tong et al., 2015]. Patient 13 carried a duplication environmental factors contributed to the etiology of the at 12p12.1, involving the PYROXD1 gene. This gene is phenotype, which must be confirmed. On the other hand, evolutionarily conserved in eukaryotes, and deficiency of microdeletions and microduplications of the 17q12 re- the protein due to mutations may cause alterations in the gion have been reported in patients with VACTERL as- proliferation, migration, and differentiation of myoblasts sociation [Zhang et al., 2017]. Our patient had esophageal [Reutter et al., 2016; Auranen et al., 2019]. The function- atresia with tracheoesophageal fistula plus renal and Phenotype and CNVs in Patients with Mol Syndromol 2020;11:271–283 279 VACTERL Association DOI: 10.1159/000510910 thumb anomalies, similar to patients reported previously. portant to rule out other genetic causes of VACTERL The HNF1B gene in this region has been implicated in phenotypes [Winberg et al., 2014]. renal anomalies and tracheoesophageal fistula in these The CNVs classified as VUS reported herein harbor patients [Smigiel et al., 2014; Zhang et al., 2017; Saha et some very interesting genes involved in cellular homeo- al., 2018]; thus, it has been suggested that this chromo- stasis; for example, MAP1S, USP14, and PARK7 are as- somal region poses a risk for expression of VATER/VAC- sociated with autophagy, FCHO1, UNC13A, and VTI1A TERL phenotypes. with endocytosis/exocytosis, COLEC12 with phagocyto- FA, which is a differential diagnosis of VACTERL-H sis, and GMEB1, PPP1R13B, THOC1, and MAP1S with association, is particularly noteworthy, as patients with apoptosis, in addition to other genes with other functions. Downloaded from http://karger.com/msy/article-pdf/11/5-6/271/3130487/000510910.pdf by Universitat Autònoma Barcelona Servei de Biblioteques user on 03 November 2023 this condition have a high risk of hematologic abnormal- Overall, our findings highlight a large number of genes ities including bone marrow failure, myelodysplastic syn- identified in CNVs with functions in processes that are drome or leukemia, and congenital anomalies that may active during embryonic development. be a part of the VACTERL-H association [Alter and It is important to note that more studies are needed to Rosenberg, 2013; Savage et al., 2016; Fiesco-Roa et al., further evaluate the chromosomal regions involved in the 2019]. The availability of chromosomal breakage tests as CNVs found in our patients because they might be re- a sensitive indicator for FA makes it especially important gions of interest due to their gene content and the com- not to miss this critical diagnosis among VACTERL-H plexity of the birth defects of some of the patients. Com- suspicious patients. Previous reports highlight the neces- plementary studies such as functional studies and/or sity of performing FA testing because compared to pa- next-generation sequencing molecular tests are also nec- tients without FA-VACTERL phenotypes, more than essary to determine the inheritance patterns of the VUS 90% of patients with FA-VACTERL phenotypes have and to elucidate the complex genetic etiology of the both renal and limb anomalies, and in more than 50%, VATER/VACTERL phenotypes in the remaining cases these findings are combined with congenital heart disease analyzed. [Alter and Rosenberg, 2013; Fiesco-Roa et al., 2019]. De- spite the fact that in our study, 6 cases (33.3%) had upper limb anomalies plus renal defects, and in 4 cases (22%), Acknowledgments these were accompanied by a cardiac defect, none of the cases showed sensitivity in in vitro DEB culture. There- We gratefully thank the patients who participated in this study at the Hospital Universitario San Ignacio and the different hospi- fore, FA was ruled out in our cohort. This finding in our tals in Bogotá city that referred the patients. Additionally, we patients should not rule out the chromosomal fragility thank the geneticists who referred some of the patients and the test for the differential diagnosis of patients with VAC- residents of the specialization in Clinical Genetics for attending to TERL. the patients and to Sistemas Genómicos for help with the sample analysis. The diversity of differential diagnoses among patients with VACTERL emphasizes the importance of clinical evaluation by a medical geneticist in all patients. Genetic Statement of Ethics testing is available for certain conditions that have fea- tures overlapping with those of VACTERL association, This study was performed in accordance with the Code of Eth- and these conditions need to be ruled out [Solomon, ics of the World Medical Association (Declaration of Helsinki) and 2011]. Such genetic testing can be helpful for assigning a approved by the Ethics Institutional Review Board of the School of diagnosis and identifying associated medical problems Medicine of Pontificia Universidad Javeriana, Bogotá and Hospital Universitario San Ignacio (approval No. 2014/107). Written in- that should be evaluated for establishing prognosis and formed consent, approved by the Ethics Committee, was obtained for genetic counseling purposes. One limitation of our from all participants. study is that not all patients underwent molecular testing for other genetic syndromes; nonetheless, all underwent clinical evaluation by a medical geneticist who did not Conflict of Interest Statement find any specific features to suggest a particular syn- drome. By molecular cytogenetic analysis, we were able The authors have no conflicts of interest to declare. to reclassify 2 patients, and VACTERL diagnosis was fi- nally ruled out. VACTERL association is still considered a diagnosis of exclusion, and aCGH genetic testing is im- 280 Mol Syndromol 2020;11:271–283 Moreno et al. DOI: 10.1159/000510910 Funding Sources Author Contributions This work was funded by Pontificia Universidad Javeriana and O.M.M., A.I.S, A.H., and A.S.C. performed cytogenetic and Hospital Universitario San Ignacio, ID 00006284, Ref: 2014-107. molecular experiments and contributed to writing the paper. G.G., A.H. also received a Young Researcher grant from COLCIENCIAS F.S., J.C.P., M.O., and Y.V. performed clinical analysis, followed (0226/2014). the patient, obtained clinical information for the paper, and con- tributed to writing the paper. O.M.M., A.R., J.B., and J.S. applied for and obtained funding for the project, directed the work, and wrote the paper. Downloaded from http://karger.com/msy/article-pdf/11/5-6/271/3130487/000510910.pdf by Universitat Autònoma Barcelona Servei de Biblioteques user on 03 November 2023 References Alberti C, Cochella L. A framework for under- Engel AG, Selcen D, Shen XM, Milone M, Harper Hilger AC, Halbritter J, Pennimpede T, van der standing the roles of miRNAs in animal devel- CM. Loss of MUNC13-1 function causes mi- Ven A, Sarma G, Braun DA, et al. Targeted opment. Development. 2017; 144(14): 2548– crocephaly, cortical hyperexcitability, and fa- resequencing of 29 candidate genes and 59. tal myasthenia. 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