Novel SLC7A7 large rearrangements in lysinuric protein intolerance patients involving the same AluY repeat

Abstract

Lysinuric protein intolerance (LPI) is a rare autosomal inherited disease caused by defective cationic aminoacid transport 4F2hc/y(+)LAT-1 at the basolateral membrane of epithelial cells in the intestine and kidney. LPI is a multisystemic disease with a variety of clinical symptoms such as hepatosplenomegaly, osteoporosis, hypotonia, developmental delay, pulmonary insufficiency or end-stage renal disease. The SLC7A7 gene, which encodes the y(+)LAT-1 protein, is mutated in LPI patients. Mutation analysis of the promoter localized in intron 1 and all exons of the SLC7A7 gene was performed in 11 patients from 9 unrelated LPI families. Point mutation screening was performed by exon direct sequencing and a new multiplex ligation probe amplification (MLPA) assay was set up for large rearrangement analysis. Eleven SLC7A7-specific mutations were identified, seven of them were novel: p.L124P, p.C425R, p.R468X, p.Y274fsX21, c.625+1G>C, DelE4-E11 and DelE6-E11. The novel large deletions originated by the recombination of Alu repeats at introns 3 and 5, respectively, with the same AluY sequence localized at the SLC7A7 3' region. The novel MLPA assay is robust and valuable for LPI molecular diagnosis. Our results suggest that genomic rearrangements of SLC7A7 play a more important role in LPI than has been reported, increasing the detection rate from 5.1 to 21.4%. Moreover, the 3' region AluY repeat could be a recombination hot spot as it is involved in 38% of all SLC7A7 rearranged chromosomes described so far.

Figure 1

New SLC7A7 point mutations in patients with LPI and segregation analysis of all the mutations detected in LPI families. (a) Electropherogram sections showing the new point mutations: p.L124P, p.C425R, p.R468X, c.625+1G>C and c.820dupT. (b) Scheme of the predicted mutant proteins in the topology model of human y⁺LAT-1 of 12 transmembrane domains. All the novel mutations but two encode truncated proteins, if translated. Interestingly, c.625+1G>C and DelE4-E11 mutations would encode nearly the same protein lacking the last nine transmembrane domains, but the differences at the mRNA level (presence or absence of polyA signal, 3′-UTR and so on) could give differences in its stability and translation. Notice that two of the frameshift mutations, Del E4-E11 and c.820dupT, have addition of missense residues (white circles). (c) Segregation of all the mutations found in 11 patients with LPI by direct sequencing. Parents of patient nos. 1 and 7 are closely consanguineous and patient nos. 6 and 7 have the same mother and their fathers are brothers. In families 4 and 6, the segregation could not be performed because there was no DNA available from the relatives, but normal MLPA pattern of these patients (Supplementary Table 1) confirmed the homozygosity of these patients. Square symbols are men and circles are women. The filled symbols are affected individuals and the half-filled symbols are obligated carriers. B, brother; F, father; M, mother; P, patient; +, wild-type allele.

Figure 2

MLPA patterns of families 3 and 5. Samples were subjected to MLPA with 11 specific oligonucleotide probes for SLC7A7 exon sequences. Three control probes for genes located on different chromosomes were also added in the assay as external controls. Each individual region's peak height is normalized by dividing it by the sum of control gene peak heights (WBSCR1, HIRA, ESR1). Each normalized peak height is then divided by the corresponding average normalized peak height from control samples to obtain the RPH. (a) Normal SLC7A7 MLPA pattern obtained from a control individual. (b) Deletion of the two-thirds of the SLC7A7 gene detected by MLPA in family no. 3. The father and the patient show an abnormal pattern. MLPA peak pattern from the mother is the same as that of the control. (c) Deletion of the last six exons of SLC7A7 gene detected by MLPA in family no. 5. Both parents carry the deletion in heterozygosis and the patient is a homozygote.

Figure 3

Scheme of the two large deletions in SLC7A7 originated by the crossing over between two direct Alu repeats, found in LPI patient nos. 3 and 5. (a) Genomic scheme of the SLC7A7 gene and the 12 kb deletion (DelE4-E11) found in patient no. 3, with a breakpoint at 4294 bp upstream exon 4 and 1028 bp downstream exon 11 (c.500−4294_1908+1028del12136). Notice that the breakpoint could be generated in any of the nucleotides in the box, as this 20 nt sequence is the same in both the Alu repeats involved in this genomic rearrangement. The mutation nomenclature localizes the breakpoint at the 3′ end of the 20 nt sequence. (b) The 4.6 kb deletion (DelE6-E11) found in patient no. 5, had the breakpoint at 848 bp upstream of exon 6 and 718 bp downstream of exon 11 (c.771−848_1908+718del4647). In DelE4-E11, the breakpoint was at the 3′ end of AluY, whereas in DelE6-E11, the breakpoint was located at the first nucleotide of the AluY sequence.