Novel genomic and transcriptomic tools to study human congenital anomalies of the kidney and urinary tract

CAKUT is a collection of congenital abnormalities that deform the urinary system and lead to organ failure. In fact, CAKUT accounts for about half of the cases of end-stage renal disease requiring dialysis or transplantation in children. CAKUT pathogenesis results in abnormal growth, differentiation, and connectivity between different organs and tissues that comprise the urinary system. The causes of CAKUT have long been thought to be the sole result of misalignment of ureteric bud and metanephric mesenchyme, but the advent of human genetics has provided a glimpse into the myriad of genes that may direct the CAKUT phenotype; these genes are found to be expressed throughout the urinary system and the developmental timeline. Our genetic knowledge to date is incomplete because the genetic diagnosis often derives from simple searches of genetic mutations, it does not ascertain the damaged embryonic tissue itself, and it has profound limitations to connect the genome with the cellular readout. We believe we have developed novel approaches and tools that can solve these challenges by: 1) using whole-genome sequencing, rather than exome sequencing or candidate approaches, of parents and fetuses with CAKUT to comprehensively capture all sources of genetic variation; 2) evaluating the genome of families suffering termination of pregnancy rather than live births because early CAKUT manifestations are likely referable to rare mutations with large effect size that are more amenable to functional interpretation; 3) accessing the affected tissues themselves so that the consequences of the genetic abnormality can be determined by an investigation of the transcriptome and its sequelae. In fact, given the complexity of the cell types in the kidney, ureter, bladder, and lower urinary tract, often manifesting gradients of gene expression, we have worked out the conditions to derive the transcriptome from individual cells from frozen tissues. This involves the entirely novel technique of single nuclear sequencing and downstream informatics. In sum, we are now in a position to understand the subtypes of CAKUT by pairing a specific mutation, with a specific cellular or tissue lesion, with specific transcriptomic readouts. These efforts, consequently, bridge the gap between human phenotype, human genotype, and the defective developmental program leading to CAKUT and organ failure.