Exome survey of individuals affected by VATER/VACTERL with renal phenotypes identifies phenocopies and novel candidate genes

Abstract

Introduction:

The acronym VATER/VACTERL refers to the rare non-random association of the following component features (CFs): vertebral defects (V), anorectal malformations (ARM) (A), cardiac anomalies (C), tracheoesophageal fistula with or without esophageal atresia (TE), renal malformations (R), and limb anomalies (L). For the clinical diagnosis the presence of at least three CFs is required, individuals presenting with only two CFs have been categorized as VATER/VACTERL-like. The majority of VATER/VACTERL individuals displays a renal phenotype. Hitherto, variants in FGF8, FOXF1, HOXD13, LPP, TRAP1, PTEN and ZIC3 have been associated with the VATER/VACTERL association; however, large-scale re-sequencing could only confirm TRAP1 and ZIC3 as VATER/VACTERL disease genes, both associated with a renal phenotype.

Methods:

In this study, we performed exome sequencing in 21 individuals and their families with a renal VATER/VACTERL or VATER/VACTERL-like phenotype to identify potentially novel genetic causes.

Results:

Exome analysis identified biallelic and X-chromosomal hemizygous potentially pathogenic variants in six individuals (29%) in B9D1, FREM1, ZNF157, SP8, ACOT9, and TTLL11, respectively. The online tool GeneMatcher revealed another individual with a variant in ZNF157.

Conclusion:

Our study suggests six biallelic and X-chromosomal hemizygous VATER/VACTERL disease gene implicating all six genes in the expression of human renal malformations.

INTRODUCTION

The acronym VATER/VACTERL (Online Mendelian Inheritance in Man (OMIM) #192350) refers to the rare non-random association of at least three of the following component features (CFs): vertebral defects (V), anorectal malformations (ARM) (A), cardiac anomalies (C), tracheoesophageal fistula with or without esophageal atresia (TE), renal malformations (R), and limb anomalies (L) (Solomon et al., 2014). Individuals presenting with only two CFs are diagnosed as VATER/VACTERL-like (Solomon et al., 2014). The diagnosis of VATER/VACTERL is mainly made on the basis of the clinical phenotype. The VATER/VACTERL association occurs in 1/10,000 – 40,000 live births (Solomon et al., 2011).

Even though most cases of VATER/VACTERL occur sporadically, the involvement of genetic factors in the pathogenesis of the VATER/VACTERL association is supported by reports of familial segregation (Solomon et al., 2011). However, only seven likely monogenic causes have been identified to date, comprising FGF8 (Zeidler et al., 2014), FOXF1 (Hilger et al., 2015), HOXD13 (Garcia-Barceló et al., 2008), LPP (Arrington et al., 2010), TRAP1 (Saisawat et al., 2014), PTEN (Chen et al., 2013), and ZIC3 (Hilger et al., 2015; Reutter et al., 2016). All of these potential VATER/VACTERL disease genes have been associated with a renal phenotype, which is the most frequent CF found in individuals with VATER/VACTERL and comprises a large spectrum of congenital anomalies of the kidneys and urinary tract (CAKUT). (Reutter et al., 2016; Quan et al., 1973).

These findings suggest that the identification of novel genetic causes of the VATER/VACTERL association allows insights into the genetic causes of CAKUT per se (Reutter et al., 2016). However, large-scale re-sequencing could only confirm TRAP1 and ZIC3 as VATER/VACTERL disease genes.

Here, we aimed to identify potentially novel genetic causes of the VATER/VACTERL association with a renal phenotype performing survey of the exome in 21 individuals and their families.

METHODS

Human Subjects

Affected individuals and their families were recruited through the network for congenital uro-rectal malformations (CURE-Net) and the GREAT-consortium (Genetic risk of esophageal atresia). The participating families provided written informed consents. The Ethics Committees of the Medical Faculty of the University of Bonn and Boston Children’s Hospital approved this study. DNA of the individuals and their families was extracted from saliva samples and blood which were gained peripherally by the families’ physician or by the families themselves with the Oragene® DNA self-collection kit (following the Oragene TM DNA Purification Protocol for saliva samples).

A clinical VATER/VACTERL phenotype with at least three component features (CF) was present in 16, a VATER/VACTERL-like phenotype with only two CFs in 5 affected individuals. All of the 21 individuals presented with a renal phenotype, ranging from duplex, horseshoe and pelvic kidneys over ureteric-pelvic junction obstruction and dysplastic and polycystic kidneys to renal agenesis. Due to the recruitment procedure, 20 of our 21 individuals had ARM (Schramm et al., 2011). Clinical characteristics of all families are outlined in Table 1 and in Table S1.

Table 1.

Information on clinical phenotype and identified potentially pathogenic variants in novel VACTERL candidate genes.

Family	Phenotypes	Gene	Pathway/ Complex	Mode of transmission	Nucleotide change	State	Amino acid change	Evolutionary conservationA	PP2 SIFT MT CADD	SNP ID	gnomad B	ACMG C	Segregation
381_501	A, C, R	B9D1	Sonic hedgehog signaling, cilia organization	recessive	c.324G>A	het	p.(Val89Met)	D. rerio	0.982 Del. D.C. 27.9	rs777500443	0/2/251 074	VUS (PM2, PP3)	Variant inherited from healthy father
		B9D1	Sonic hedgehog signaling, cilia organization	recessive	c.71_72del	het	p.(Pro24fs)	/	/	rs748661746	0/2/150 750	Likely pathogenic (PVS1 ,PM2)	NA*
794_ 501	V, A, R	FREM1	Fraser complex	recessive	c.1408C>T	het	p.(Leu470Phe)	M. musculus	0.359 Del. D.C. 23.1	rs1418448401	0/0/245 792	VUS (PM2, PP3)	Variant inherited from healthy father
		FREM1	Fraser complex	recessive	c.56C>T	het	p.(Ala19Val)	/	0.001 Tol. Poly. 7.947	rs368794455	0/6/232 056	VUS (PM2)	Variant inherited from healthy mother
358_ 501	V, R	ZNF157	Kruppel family linking motifs, DNA transcription regulation	NA	c.764T>C	hemi	p.(Phe255Ser)	X. tropicalis	0.999 Del. Poly. 10.51	/	/	VUS (PM2, PP3)	Variant inherited from healthy mother
GE_ 052	V, R	ZNF157	Kruppel family linking motifs, DNA transcription regulation	NA	c.667T>G	hemi	p.(Cys223Gly)	D. rerio	0.999 Del. D.C. 15.48	/	/	VUS (PM2, PP3)	Variant inherited from healthy mother
395_ 501	V, A, R, L	SP8	Wnt, Shh and Bmp signaling	NA	c.1510C>T	het	p.(Arg504Cys)	C. intestinalis	0.988 Del. D.C. 31	rs368503334	0/78/131 992	VUS (PM2, PP3)	Variant inherited from healthy mother
		SP8	Wnt, Shh and Bmp signaling	NA	c.417C>G	het	p.(Phe139Leu)	D. rerio	0.086 Tol. D.C. 17.23	rs779330461	2/218/103 172	VUS (PM2, PP3)	Variant inherited from healthy father
605_ 501	V, A, C, R	ACOT9	Fatty Acyl-CoA Biosynthesis	recessive	c.953G>A	hemi	p.(Arg318Gln)	D. rerio	0.812 Del. D.C. 32	/	/	VUS (PM2, PP3)	Variant inherited from healthy mother
706_ 501	A, R, L	TTLL11	Spindle and cilia microtubules organization	recessive	c.1540-8C>A	het	p.?	/	/	/	/	VUS (PM2)	Variant inherited from healthy mother
		TTLL11	Spindle and cilia microtubules organization	recessive	c.1298C>G	het	p.(Thr433Ser)	D. melanogaster	0.997 Del. D.C. 25.9	rs187536422	0/2/251 496	VUS (PM2, PP3)	Variant inherited from healthy father

Information on phenotype: A, anorectal malformation; C, cardiac malformation; L, limb anomalies; R, renal anomalies; V, vertebral malformation

CADD, Combined Annotated Dependent Depletion; D.C., disease-causing; Del., deleterious; del; deletion; fs, frameshift; gnomAD, Genome Aggregation Database; het, heterozygous; MT, Mutation Taster; NA, not available; PP2, PolyPhen 2; SIFT, Sorting intolerant from tolerant

^AEvolutionary conservation was assessed over 8 species: M. muscularis, Mus musculus; G. gallus, Gallus gallus; X. tropicalis, Xenopus tropicalis; D. rerio, Danio rerio; C. elegans, Caenorhabditis elegans; C. intestinalis, Ciona intestinalis; D. melanogaster, Drosphilia melanogaster; S. cerevisiae, Saccharomyces cerevisiae.

^BVariant frequencies listed for homozygous/ (if applicable) hemizygous/ heterozygous/ total alleles detected in the population.

^CACMG American College of Medical Genetics guidelines classification as pathogenic, likely pathogenic, or variant of uncertain significance (VUS) (Richards et al., 2015).

^*no maternal DNA available; according to frequencies in gnomAD decided to be compound heterozygous.

Exome sequencing and variant calling

Exome sequencing (WES) was performed as previously described (Braun et al., 2016; Warejko et al., 2018). We performed trio analysis in 13 families and duo analysis in 1 family. The remaining 7 affected individuals were evaluated as singlets. The Genomics Platform at the Broad Institute of Harvard and Massachusetts Institute of Technology (Cambridge, MA, USA) performed data processing of FASTQs. The readouts were then filtered for most likely non-impairing variants by assembling the sequences to the human reference genome (Sadowski et al., 2015). Insertions/deletions (indels) and Single Nucleotide Polymorphism (SNPs) were collectively called across all samples using Genome Analysis Toolkit (GATK) HaplotypeCaller. Using the GATK Variant Quality Score Recalibration approach indel and SNP calls were filtered and annotated using Variant Effect Predictor. The data set of the remaining variants was uploaded on to Seqr for further analysis of the WES output. In order to include only rare alleles (minor allele frequency <1%) variant filtering based on allele frequency was performed using population databases (1000-genomes, exome variant server (EVS) and genome aggregation database (gnomAD)) as they were unlikely to be deleterious. Intronic and synonymous variants were excluded unless they were located within splice site regions. Non-synonymous and splice site variants were then further analyzed. Remaining variants were confirmed by Sanger sequencing. For the evaluation of singlets, we hypothesized a recessive cause and analyzed for homozygous variants. For duo analysis, we hypothesized a recessive cause and analyzed the homozygous variants and compound heterozygous variants. For the evaluation of trios, we could further query for de novo mutations.

The potential deleterious impact of the remaining variants on the protein function was assessed by considering evolutionary conservation. Evolutionary conservation among orthologues was assessed across the following 8 species using ENSEMBL Genome Browser: Mus musculus (M. muscularis), Gallus gallus (G. gallus), Xenopus tropicalis (X. tropicalis), Danio rerio (D. rerio), Caenorhabditis elegans (C. elegans), Ciona intestinalis (C. intestinalis), Drosphilia melanogaster (D. melanogaster), Saccharomyces cerevisiae (S. cerevisiae). The online tool Clustal Omega was used for assembling the variants according to phylogeny. Additionally, variants were rated by the in silico prediction tools PolyPhen-2 (PP2), Sorting intolerant from tolerant (SIFT), and MutationTaster (MT). For Combined Annotation Dependent Depletion (CADD) a suggested cutoff on deleteriousness was put at 15 as suggested on the corresponding website. Phenotypic and functional aspects of each variant were discussed and reviewed in a minimum of a 5-member nephro-genetic panel for each of the 21 individuals and their families before final candidate gene decisions were made. The website GeneMatcher enabled the connection between researchers and providers caring for the family GE_052 (Sobreira et al., 2015; Sobreira et al., 2015).

RESULTS

Exome analysis

In total, we identified potentially pathogenic variants in 6 of the 21 VATER/VACTERL families (29%; B9D1, FREM1, ZNF157, SP8, ACOT9 and TTLL11) (Fig. 1). Two individuals carried variants in genes known to cause a syndrome that can display similar phenotypic features as VATER/VACTERL (10%; B9D1 and FREM1) (Table 1 and Fig. 1). Four genes are putative new candidates for causing VATER/VACTERL (19%; ZNF157, SP8, ACOT9 and TTLL11) (Table 1 and Fig. 1). In 15 of 21 families (71%), we were unable to identify a potentially pathogenic variant in a gene per family. Detailed clinical features of all individuals included in this study are outlined in Table S1. Prior to this WES study the 21 individuals underwent array-based molecular karyotyping (Zhang et al., 2017). No disease-causing copy number variations were identified. Through the use of the online tool GeneMatcher we facilitated the discovery of another male individual displaying VATER/VACTERL features carrying a variant in ZNF157 (Sobreira et al., 2015; Sobreira et al., 2015).

Figure 1. Number and percentage of 21 VATER/VACTERL families in whom a potential causative variant was detected by WES.

Purple color denotes the fraction of families with a variant in a causative gene assumed to phenocopy the VATER/VACTERL phenotype. Red denotes a variant in a potential novel monogenic candidate gene resulting from WES was identified. Yellow denotes the fraction of families if no variant could be derived from WES.

(A) In 2 of 21 families with a VATER/VACTERL phenotype (10%), variants were detected in genes (B9D1 and FREM1) phenocopying the VATER/VACTERL features (purple).

(B) In 4 of 21 families (19%), a single potential novel candidate gene (ZNF157, SP8, ACOT9, and TTLL11) for VATER/VACTERL could be identified per family (red).

(C) In 15 of 21 families (71%), no variant was detected (yellow).

Identified variants

In 6 of 21 families (29%), we identified biallelic and X-chromosomal hemizygous potentially disease-causing variants (Table 1 and Fig. 1).

In individual 381_501, who presented with anal atresia with vestibular fistula (A), ventricular septal defect (C) and right renal agenesis (R), we detected compound heterozygous variants in B9D1 (OMIM #614209), previously implicated in Meckel syndrome, which resembles the VATER/VACTERL malformation spectrum. The first detected missense variant in B9D1 was inherited from the healthy father (c.324G>A; p.(Val89Met); ENST00000477683, Table 1). The amino acid valine is highly conserved amongst vertebrates to D. rerio (Table 1). The variant has been reported twice heterozygously in 251,074 alleles in the gnomAD database (rs777500443). Four in silico prediction programs rated the amino acid change as potentially disease-causing (Table 1). The variant was classified as variant of uncertain significance (VUS) by the guidelines of the American College of Medical Genetics (ACMG) (Table 1) (Richards et al., 2015). The second identified frameshift variant in B9D1 (c.71_72del; p.(Pro24fs); ENST00000477683, Table 1) has been reported two times heterozygously in 150,750 alleles in the gnomAD database (rs748661746). According to the ACMG guidelines we rated this variant as likely pathogenic (Richards et al., 2015). As these variants were detected by performing duo analysis and no maternal DNA was available for genetic testing, we considered these biallelic variants to be potentially compound heterozygous according to the frequencies in gnomAD (Table 1).

Individual 794_501 carried compound heterozygous variants in FREM1 (Table 1). Homozygous variants in FREM1 are known to cause the closely to the Fraser-related Manitoba oculotrichoanal syndrome (OMIM #248450). Individual 794_501 presented with ARM with vestibular fistula (A), left renal dysplasia (R) and wedge-shaped vertebra (V). Additionally, 794_501 showed a bicornuate uterus and vaginal stenosis (Table S1). The first identified missense variant in FREM1 was inherited from the healthy father (c.1408C>T; p.(Leu470Phe); ENST00000422223, Table 1). The amino acid is conserved to M. musculus and no heterozygous carriers in 245,792 alleles in gnomAD have been reported so far (rs1418448401). Three of four in silico prediction programs rate this amino acid change to be deleterious and valuated by the ACMG guidelines as VUS (Table 1) (Richards et al., 2015). The second identified missense variant was inherited from the healthy mother (c.56C>T; p.(Ala19Val); ENST00000422223, Table 1). Six heterozygous carriers in 232,056 alleles in gnomAD have been reported (rs368794455). The amino acid was not conserved amongst vertebrates and four in silico prediction programs rate the amino acid change as benign (Table 1). According to the ACMG guidelines we rated the variant as VUS (Richards et al., 2015).

In individual 358_501, we detected a hemizygous novel missense variant in ZNF157 (c.764T>C; p.(Phe255Ser); ENST00000377073, Table 1), which was inherited from a healthy mother. Individual 358_501 displayed two CFs, namely caudal regression (V) and a horseshoe kidney (R). Additively, 358_501 presented with hypospadia (Table S1). The amino acid is conserved to X. tropicalis. Two of four in silico prediction programs predict the amino acid change to be deleterious (Table 1). We classified this variant as VUS according to the standards of the ACMG (Richards et al., 2015). Through the online tool GeneMatcher we identified individual GE_052, who also carried a novel missense variant in ZNF157 (c.667T>G; p.(Cys223Gly); ENST00000377073, Table 1), which was inherited from a healthy mother. Individual GE_052 presented with cervical spine malformation and loss of lumbar lordosis (V) next to a right kidney hypoplasia and vesicoureteral reflux (R) (Table 1). In addition, individual GE_052 showed global developmental delay, dysmorphic facial features and bone abnormalities (Table S1). The amino acid is conserved amongst vertebrates and four in silico prediction programs rate the amino acid change as deleterious. Therefore, we assessed the variant as VUS (Richards et al., 2015).

In individual 395_501, who presented with fused vertebrae (V), anal atresia with recto-perineal fistula (A), horseshoe kidney (R) and a supernumerary right thumb (L), we found compound heterozygous variants in a potential new candidate gene SP8 (Table 1). The first identified missense variant in SP8 was inherited from the healthy mother (c.1510C>T; p.(Arg504Cys); ENST00000361443, Table 1) and predicted to be deleterious by four in silico prediction programs. The amino acid arginine is highly conserved to C. intestinalis and 78 heterozygous carriers in 131 992 alleles in gnomAD have been reported (rs368503334). According to the ACMG standards we rated this variant as VUS (Richards et al., 2015). The second missense variant was inherited from a healthy father (c.417C>G; p.(Phe139Leu); ENST00000361443, Table 1) and the amino acid change is predicted to be deleterious by only two of four in silico prediction programs. The amino acid is conserved through vertebrates to D. rerio. Two homozygous and 216 heterozygous carriers in 103,172 alleles have been reported in gnomAD (rs779330461). We classified this variant as VUS according to the ACMG guidelines (Richards et al., 2015).

Individual 605_501 carried a novel hemizygous missense variant in ACOT9 (c.953G>A; p.(Arg318Gln); ENST00000379303, Table 1), which was inherited from an unaffected mother. Individual 605_501 presented with sacral hypoplasia (V), anal atresia with recto-prostatic fistula (A), atrial septal defect (C) and left renal dysplasia (R). Additionally, 605_501 showed a left ureterocele and a femoral hemangioma (Table S1). The amino acid arginine is well conserved through vertebrates to D. rerio and four in silico prediction programs rate this variant as deleterious (Table 1). According to the ACMG guidelines we classified this variant as VUS (Richards et al., 2015).

In individual 706_501, we detected compound heterozygous variants in TTLL11 (Table 1). Individual 706_501 presented with ARM (A), right renal agenesis (R) and hypoplasia of digit V (L). Additionally, 706_501 displayed a bicornuate uterus, syringomyelia and tethered cord (Table S1). The first identified variant (c.1540-8C>A; p.?; ENST00000321582, Table 1) is an extended acceptor splice site variant, which has not been reported in the database gnomAD before. The variant was inherited from a healthy mother and classified as VUS according to the ACMG standards (Richards et al., 2015). The second identified missense variant (c.1298C>G; p.(Thr433Ser); ENST00000321582, Table 1) was inherited from a healthy father. The amino acid is highly conserved to D. melanogaster and the amino acid change is predicted to be deleterious by four in silico prediction programs (Table 1). Two heterozygous carriers in 251,496 alleles have been reported in gnomAD (rs187536422). We considered this variant to be a VUS according to the guidelines of the ACMG (Richards et al., 2015).

No potentially pathogenic variants were detected in any affected individual following a dominant or de novo inheritance pattern.

DISCUSSION

In 6 of 21 families (29%), WES was able to detect potentially causative variants (Table 1). We demonstrate here the utility of WES for the identification of genetic causes in families with VATER/VACTERL (Table 1 and Fig. 1).

In individual 381_501 and 794_501 variants in genes were identified that, if mutated, could lead to conditions that may phenocopy the VATER/VACTERL phenotype: In individual 381_501, we identified compound heterozygous variants in B9D1 (OMIM #614209), previously implicated in Meckel syndrome. As individuals with primary cilia defects may present clinically with congenital heart defects (C), congenital malformations of the kidney (R), and limb (L) anomalies, ciliary diseases can present with multiple malformations similar to the VATER/VACTERL spectrum (Bujakowska et al., 2015). The finding of biallelic variants in B9D1 could therefore implicate a potential ciliary pathogenesis for a VATER/VACTERL or VATER/VACTERL-like phenotype.

Compound heterozygous variants were detected in a syndromic CAKUT gene (FREM1) in individual 794_501 (Table 1), known to cause the closely to the Fraser-related Manitoba oculotrichoanal syndrome (OMIM #248450). The Fraser proteins encoded by the genes FREM1, FREM2, and FRAS1 form a tertiary protein complex (Nathanson et al., 2013). Pathogenic variants that lead to a loss of function of any of these Fraser-proteins disrupt the tertiary structure of the formed protein complex (McGregor et al., 2003). This gives rise to the multiorgan developmental anomalies of Fraser syndrome observed in mice and humans (McGregor et al., 2003). Individuals may present with anal (A) and renal (R) anomalies, which again overlaps with several features of the VATER/VACTERL phenotype. As these features resemble the VATER/VACTERL phenotype this respectively might explain the individual’s phenotype (Nathanson et al., 2013). Interestingly, individual 794_501 presented with a bicornuate uterus, a clinical feature of the Fraser syndrome (Slavotinek et al., 2002).

Individuals 358_501 and GE_052 carried hemizygous novel variants in ZNF157 (Table 1). ZNF157 contains zinc finger domains and linking motifs similar to those of proteins of the Kruppel family (Derry et al., 2012). In the literature, there is no link between the pathogenesis of VATER/VACTERL and ZNF157 so far. But interestingly, two individuals could be identified who present with vertebral (V) and renal (R) anomalies. Therefore, a potential role of ZNF157 in the pathogenesis of the individuals VATER/VACTERL phenotype seems likely.

Compound heterozygous variants in SP8 were identified in individual 395_501. A disease for individuals with biallelic variants in SP8 has not been defined yet. Interestingly, recessive mouse models for Sp8 describe mice with vertebral (V), anal (A) and limb (L) malformations which present three of the VATER/VACTERL component features (Lin et al., 2012). Therefore, the variants in the new candidate gene SP8 might explain the individual's phenotype.

In individual 605_501, we found a novel variant in ACOT9. ACOT9 is an Acyl-CoA thioesterase, a protein of unknown function. We hypothesized that ACOT9 could be involved in the cholesterol metabolism. Cholesterol is known to play an essential role during fetal development (Ofori et al., 2007). Statins reduce the production of cholesterol and are therefore contraindicated in pregnancy due to their potential teratogenic effect (Ofori et al., 2007). Newborns present with congenital anomalies such as vertebral defects (V), cardiac (C) and limb anomalies (L), resembling the VATER/VACTERL phenotypic spectrum (Ofori et al., 2007). Moreover, Shi et al. (2017) demonstrated that a lack of NAD in humans impairs DNA repair mechanisms which can result in multiple congenital malformations defining the VATER/VACTERL association. A potential link between the cholesterol pathway, the NAD metabolism and development of congenital malformations resembling the VATER/VACTERL association seems possible.

Individual 706_501 carried compound heterozygous variants in TTLL11 (Table 1). In the literature, there is no link between TTLL11 and the pathogenesis of VATER/VACTERL so far. Interestingly, TTLL11 seems to play a role in the organization of cilia microtubules which again suggests a ciliary pathogenesis (Fullston et al., 2011). As no homozygous carrier was reported in gnomAD for any of these variants, a potential role in the pathogenesis of the individual’s VATER/VACTERL phenotype seems likely.

Exome analysis identified biallelic and X-chromosomal hemizygous potentially pathogenic variants in six individuals, but no potentially pathogenic variants were detected following a dominant or de novo inheritance pattern. The involvement of genetic factors has been suggested by familial occurrence, the increased prevalence of component features among first-degree relatives of affected individuals and high concordance rates among monozygotic twins (Reutter et al., 2016). Therefore, heredity would be conceivable, but none of the parents on which clinical information were available were described as being affected. A dominant inheritance seemed therefore rather unlikely. The occurrence of de novo variants causing VATER/VACTERL phenotypes must nevertheless be considered.

To decide whether variants in these newly identified candidate genes are indeed disease-causing additional genetic and experimental testing will be needed. The work presented here suggests that panel diagnostics may not be sufficient to clarify complex phenotypes genetically, as they limit the range of phenotypes in advance and thus phenocopies may not be found. We therefore suggest that in the case of complex phenotypes, in medical genetics the analysis of the complete exome should be performed within the framework of molecular genetic diagnostics.

Congenital renal anomalies in a cohort with VATER/VACTERL or VATER/VACTERL-like phenotype

In this study, we applied WES to a cohort of individuals with VATER/VACTERL association that all presented with CAKUT. Epidemiological observations show that renal anomalies are by far the most frequent CF among individuals with VATER/VACTERL syndrome (Cuschieri et al., 2002). The variety of monogenic genes that have been discovered so far suggests that the VATER/VACTERL association may be genetically heterogeneous, but all of the identified human disease genes are linked to renal phenotypes. These findings propose that finding potential novel genetic causes by WES in individuals with VATER/VACTERL syndrome might allow new insights into the genetics and pathogenesis of congenital renal anomalies.

CONCLUSION

In total, through WES we detected potentially causative variants in 6 of 21 families (29%). Through GeneMatcher one more family was identified with a novel variant in ZNF157. This study established that WES can identify potentially pathogenic variants in potential novel candidate genes in 19% of families with a renal VATER/VACTERL or VATER/VACTERL-like phenotype. As CAKUT is the most frequent CF in individuals with VATER/VACTERL syndrome and all identified disease genes so far are linked to a renal phenotype, finding novel potential candidate genes for VATER/VACTERL can help to provide new insights into the genetics and pathogenesis of CAKUT.

Supplementary Material

tS1

Table S1 Detailed clinical information on individuals with VATER/VACTERL syndrome

This table includes clinical information on the solved and unsolved individuals by WES and the individual detected by GeneMatcher.