Abstract
ABSTRACTHeterozygous mutations in HNF1B cause the complex syndrome renal cysts and diabetes (RCAD), characterized by developmental abnormalities of the kidneys, genital tracts and pancreas, and a variety of renal, pancreas and liver dysfunctions. The pathogenesis underlying this syndrome remains unclear as mice with heterozygous null mutations have no phenotype, while constitutive/conditional Hnf1b ablation leads to more severe phenotypes. We generated a novel mouse model carrying an identified human mutation at the intron-2 splice donor site. Unlike heterozygous mice previously characterized, mice heterozygous for the splicing mutation exhibited decreased HNF1B protein levels and bilateral renal cysts from embryonic day 15, originated from glomeruli, early proximal tubules (PTs) and intermediate nephron segments, concurrently with delayed PT differentiation, hydronephrosis and rare genital tract anomalies. Consistently, mRNA sequencing showed that most downregulated genes in embryonic kidneys were primarily expressed in early PTs and the loop of Henle and involved in ion/drug transport, organic acid and lipid metabolic processes, while the expression of previously identified targets upon Hnf1b ablation, including cystic disease genes, was weakly or not affected. Postnatal analyses revealed renal abnormalities, ranging from glomerular cysts to hydronephrosis and, rarely, multicystic dysplasia. Urinary proteomics uncovered a particular profile predictive of progressive decline in kidney function and fibrosis, and displayed common features with a recently reported urine proteome in an RCAD pediatric cohort. Altogether, our results show that reduced HNF1B levels lead to developmental disease phenotypes associated with the deregulation of a subset of HNF1B targets. They further suggest that this model represents a unique clinical/pathological viable model of the RCAD disease.
Highlights
The transcription factor HNF1B is an important regulator of early mouse kidney, liver and pancreas organogenesis (Coffinier et al, 2002; De Vas et al, 2015; Gresh et al, 2004; Haumaitre et al, 2005; Heliot et al, 2013; Lokmane et al, 2008; Lokmane et al, 2010; Massa et al, 2013)
To define the consequences of this mutation on Hnf1b mRNA processing, we initially performed semiquantitative RT-PCR using RNA from Hnf1bSp2/+ heterozygotes and wild-type (WT) kidneys and primers located in exon-1 and exon-3
Sequence of the PCR products in Hnf1bSp2/+ mutants indicated the production of the two expected Hnf1b spliced isoforms A and B and four additional novel transcripts, present at low levels, corresponding to the isoforms A and B in which were deleted either the exon 2 or the last 32 base pairs of exon 2 through the activation of a near cryptic splice donor site (Fig. S1B; see Material and Methods)
Summary
The transcription factor HNF1B is an important regulator of early mouse kidney, liver and pancreas organogenesis (Coffinier et al, 2002; De Vas et al, 2015; Gresh et al, 2004; Haumaitre et al, 2005; Heliot et al, 2013; Lokmane et al, 2008; Lokmane et al, 2010; Massa et al, 2013). More than one hundred fifty HNF1B heterozygous mutations have been described, including missense, nonsense, insertion/deletions, frameshift and splice site mutations as well as whole gene deletions (Alvelos et al, 2015; Barbacci et al, 2004; Bellanne-Chantelot et al, 2004; Chen et al, 2010; Edghill et al, 2006; Heidet et al, 2010). These mutations are either familial or de novo (40%). No clear genotype-phenotype correlations were observed neither for the type or location of mutations, and haploinsufficiency has been the main underlying disease proposed mechanism
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