Targeted Exome Sequencing Identifies PBX1 as Involved in Monogenic Congenital Anomalies of the Kidney and Urinary Tract

Abstract

Congenital anomalies of the kidney and urinary tract (CAKUT) occur in three to six of 1000 live births, represent about 20% of the prenatally detected anomalies, and constitute the main cause of CKD in children. These disorders are phenotypically and genetically heterogeneous. Monogenic causes of CAKUT in humans and mice have been identified. However, despite high-throughput sequencing studies, the cause of the disease remains unknown in most patients, and several studies support more complex inheritance and the role of environmental factors and/or epigenetics in the pathophysiology of CAKUT. Here, we report the targeted exome sequencing of 330 genes, including genes known to be involved in CAKUT and candidate genes, in a cohort of 204 unrelated patients with CAKUT; 45% of the patients were severe fetal cases. We identified pathogenic mutations in 36 of 204 (17.6%) patients. These mutations included five de novo heterozygous loss of function mutations/deletions in the PBX homeobox 1 gene (PBX1), a gene known to have a crucial role in kidney development. In contrast, the frequency of SOX17 and DSTYK variants recently reported as pathogenic in CAKUT did not indicate causality. These findings suggest that PBX1 is involved in monogenic CAKUT in humans and call into question the role of some gene variants recently reported as pathogenic in CAKUT. Targeted exome sequencing also proved to be an efficient and cost-effective strategy to identify pathogenic mutations and deletions in known CAKUT genes.

Keywords: genetic renal disease; genetics and development; kidney development.

Figure 1.

Identification of de novo PBX1 mutations in three families and deletions in two families. (A) Pedigrees of three families with one affected individual (in black) carrying a de novo point mutations in PBX1. The affected individual in family 1 (K175) was a 21-year-old woman presenting with renal hypoplasia and deafness. The affected individual in family 2 (K179) was a 12-year-old girl presenting with small hyperechogenic kidneys with cysts, developmental delay, growth retardation, and long and narrow face. The affected individual in family 3 (K186) was a male fetus with extremely severe renal hypoplasia. Loss of function mutations (deletion of one base leading to a frameshift, nonsense mutation, or nucleotide change in the consensus acceptor splice site located in 3′ of intron 3) identified in patients K175, K179, and K186, respectively, were validated by Sanger sequencing. Absence of the mutation in the parents showed de novo occurrence in all patients. NT, not tested. (B) Pedigree of family 4 with a 40-year-old woman (K181) affected with small and dysplastic horseshoe kidney and deafness. NGS analysis of patient DNA revealed a reduced number of reads for all of the exons of PBX1 (in red) compared with the mean number of reads for the 37 other DNAs analyzed in the same run (in green), suggesting deletion of one PBX1 allele. Haplotypes of the affected woman and her parents were generated using four known microsatellite markers upstream and downstream of PBX1 (markers A, B, G, and H) as well as four intragenic PBX1 markers (markers C–F). Genomic positions of the microsatellite markers (on Human Genome version GRCh38.p7 from Ensembl) are A (D1S2675): chr1:162,240,203–162,240,364; B (D1S2844): chr1:162,979,036–162,979,218; C (in PBX1 intron 1): chr1:164,562,253–164,562,694; D (in PBX1 intron 2): chr1:164,651,731–164,652,170; E (in PBX1 intron 2): chr1:164,707,219–164,707,656; F (in PBX1 intron 8): chr1:164,833,830–164,834,270; G (D1S2762) chr1:166,986,900–166,987,137; and H (D1S196): chr1:167,635,063–167,635,195. Deletion of a <4-Mb region encompassing PBX1 located on the maternally inherited chromosome was shown by the lack of maternal contribution for markers C–F (genotypes are in Supplemental Table 3). (C) Pedigree of family 5 with a 2-year-old infant boy (K136) presenting with a single hyperechogenic kidney associated with developmental delay, microcephaly, and facial dysmorphism (with long and narrow face as well as abnormal ear lobes). The mother was presenting pyeloureteric junction obstruction. NGS analysis of K136 DNA revealed a reduced number of reads for all of the exons of PBX1 (in red) compared with the mean number of reads for the 38 other DNA analyzed in the same run (in green), suggesting deletion of one PBX1 allele. Validation of this deletion and identification of its extent and de novo occurrence were performed by CGH analysis (Supplemental Figure 1).

Figure 2.

Identification of causative mutations/deletions in 36 of the 204 CAKUT cases. For each gene (ANOS1, CHD7, EYA1, GATA3, HNF1B, KIF14, PAX2, and PBX1), the proportions of patients with mutations and deletions are shown in dark and light colors, respectively. All of the mutations/deletions are heterozygous, except for KIF4, for which they are biallelic.