Clinical and molecular characterization of cystinuria in a French cohort: relevance of assessing large-scale rearrangements and splicing variants

Abstract

Background: Cystinuria is an autosomal recessive disorder of dibasic amino acid transport in the kidney and the intestine leading to increased urinary cystine excretion and nephrolithiasis. Two genes, SLC3A1 and SLC7A9, coding respectively for rBAT and b0,+AT, account for the genetic basis of cystinuria.

Methods: This study reports the clinical and molecular characterization of a French cohort including 112 cystinuria patients and 25 relatives from 99 families. Molecular screening was performed using sequencing and Quantitative Multiplex PCR of Short Fluorescent Fragments analyses. Functional minigene-based assays have been used to characterize splicing variants.

Results: Eighty-eight pathogenic nucleotide changes were identified in SLC3A1 (63) and SLC7A9 (25) genes, of which 42 were novel. Interestingly, 17% (15/88) and 11% (10/88) of the total number of variants correspond, respectively, to large-scale rearrangements and splicing mutations. Functional minigene-based assays were performed for six variants located outside the most conserved sequences of the splice sites; three variants affect splice sites, while three others modify exonic splicing regulatory elements (ESR), in good agreement with a new in silico prediction based on ΔtESRseq values.

Conclusion: This report expands the spectrum of SLC3A1 and SLC7A9 variants and supports that digenic inheritance is unlikely. Furthermore, it highlights the relevance of assessing large-scale rearrangements and splicing mutations to fully characterize cystinuria patients at the molecular level.

Keywords: Cystinuria; SLC3A1; SLC7A9; exonic splicing regulatory elements; large‐scale rearrangements; splicing mutations.

Figure 1

Effect on splicing of selected variants located in SLC3A1 and SLC7A9 genes, assessed using a functional minigene assay. (A) Schematic representation of the pCAS2‐SLC3A1/SLC7A9 minigene used in the splicing reporter assay. Boxes indicate exons, whereas lines in between represent introns. The minigenes were generated by inserting a genomic fragment, containing the SLC3A1 or SLC7A9 exon of interest (gray box), as well as part of the upstream and downstream flanking intronic sequences (thick lines), into the intron of the minigene using the BamHI and MluI restriction sites. Expression of the minigenes is driven by the CMV promoter. Arrows above the minigene exons A and B (white boxes) indicate the positions of primers used in RT‐PCR analysis. (B–F) Analysis of the splicing pattern of the wild‐type and mutant pCAS2‐SLC3A1/SLC7A9 minigenes for the selected variants. Wild‐type (WT) and mutant pCAS‐2 SLC3A1/SLC7A9 constructs were transiently expressed in HeLa cells by transfection. The splicing patterns of the minigene transcripts were then analyzed by RT‐PCR as described under Materials and Methods. The image shows the electrophoresis on a 2% agarose ethidium bromide‐stained gel of the RT‐PCR products obtained for each minigene. The identities of the RT‐PCR products, with the inclusion (+) or the skipping (Δ) of the exon are indicated on the right.

Figure 2

Schematic representation of large‐scale rearrangements in SLC3A1,PREPL,CAMKMT, and SLC7A9 genes. Genes are written on top of the arrows indicating the direction of the transcription. The exons are indicated (squares). (A) Schematic representation of 11 deletions in the 2p21 locus. (B) Schematic representation of 11 deletions in the SLC3A1 gene. (C) Schematic representation of two deletions in the SLC7A9 gene.

Figure 3

Genealogic trees of four families. (A) Family ET. A1 allele: c.163C>T – p.(Gln55*); A2 allele= c.(891+1_892‐1)_(1617+1_1618‐1)dup – p.(?); B1 allele= c.544G>A – p.(Ala182Thr). (B) Family MA. A1 allele: c.647C>T – p.(Thr216Met); A2 allele: c.(765+1_766‐1)_(1011+1_1012‐1)dup ‐ p.(Asp338Leufs*80); B1 allele: c.26G>A – p.(Arg9Gln). This missense variant is not predicted to be pathogenic (Table S3). (C) Family GA. A1 allele: c.566C>T – p.(Thr189Met); B1 allele: c.614dup – p.(Asn206Glufs*3). (D) Family COL. A1 allele: c.1500+1G>T – p.(?); A2 allele: SLC3A1; NM_000341.3 :c.(?_1)_CAMKMT; NM_024766.4 c.(311+1_?)del ‐ p.(?); A3 allele: c.1134C>A ‐ p.(Tyr378*). A denotes SLC3A1 NM_000341; B denotes SLC7A9 NM_014270.

Figure 4

Genotype distribution of 99 patients. Seventy‐four patients with genotype A (70 AA, 3 AAA, 1 AAB), 22 with genotype B, 1 A0 and 2 B0 genotypes.