A Mild Form of SLC29A3 Disorder: A Frameshift Deletion Leads to the Paradoxical Translation of an Otherwise Noncoding mRNA Splice Variant

We investigated two siblings with granulomatous histiocytosis prominent in the nasal area, mimicking rhinoscleroma and Rosai-Dorfman syndrome. Genome-wide linkage analysis and whole-exome sequencing identified a homozygous frameshift deletion in SLC29A3, which encodes human equilibrative nucleoside transporter-3 (hENT3). Germline mutations in SLC29A3 have been reported in rare patients with a wide range of overlapping clinical features and inherited disorders including H syndrome, pigmented hypertrichosis with insulin-dependent diabetes, and Faisalabad histiocytosis. With the exception of insulin-dependent diabetes and mild finger and toe contractures in one sibling, the two patients with nasal granulomatous histiocytosis studied here displayed none of the many SLC29A3-associated phenotypes. This mild clinical phenotype probably results from a remarkable genetic mechanism. The SLC29A3 frameshift deletion prevents the expression of the normally coding transcripts. It instead leads to the translation, expression, and function of an otherwise noncoding, out-of-frame mRNA splice variant lacking exon 3 that is eliminated by nonsense-mediated mRNA decay (NMD) in healthy individuals. The mutated isoform differs from the wild-type hENT3 by the modification of 20 residues in exon 2 and the removal of another 28 amino acids in exon 3, which include the second transmembrane domain. As a result, this new isoform displays some functional activity. This mechanism probably accounts for the narrow and mild clinical phenotype of the patients. This study highlights the ‘rescue’ role played by a normally noncoding mRNA splice variant of SLC29A3, uncovering a new mechanism by which frameshift mutations can be hypomorphic

A mild form of SLC29A3 disorder: a frameshift deletion leads to the paradoxical translation of an otherwise noncoding mRNA splice variant

We investigated two siblings with granulomatous histiocytosis prominent in the nasal area, mimicking rhinoscleroma and Rosai-Dorfman syndrome. Genome-wide linkage analysis and whole-exome sequencing identified a homozygous frameshift deletion in SLC29A3, which encodes human equilibrative nucleoside transporter-3 (hENT3). Germline mutations in SLC29A3 have been reported in rare patients with a wide range of overlapping clinical features and inherited disorders including H syndrome, pigmented hypertrichosis with insulin-dependent diabetes, and Faisalabad histiocytosis. With the exception of insulin-dependent diabetes and mild finger and toe contractures in one sibling, the two patients with nasal granulomatous histiocytosis studied here displayed none of the many SLC29A3-associated phenotypes. This mild clinical phenotype probably results from a remarkable genetic mechanism. The SLC29A3 frameshift deletion prevents the expression of the normally coding transcripts. It instead leads to the translation, expression, and function of an otherwise noncoding, out-of-frame mRNA splice variant lacking exon 3 that is eliminated by nonsense-mediated mRNA decay (NMD) in healthy individuals. The mutated isoform differs from the wild-type hENT3 by the modification of 20 residues in exon 2 and the removal of another 28 amino acids in exon 3, which include the second transmembrane domain. As a result, this new isoform displays some functional activity. This mechanism probably accounts for the narrow and mild clinical phenotype of the patients. This study highlights the"rescue" role played by a normally noncoding mRNA splice variant of SLC29A3, uncovering a new mechanism by which frameshift mutations can be hypomorphic

Identification of a frameshift deletion in <i>SLC29A3</i>.

<p>(A) Pedigree of the family. The <i>SLC29A3</i> genotypes of the patients and the family members from whom DNA was available for sequence analysis are listed under their symbols. Genotyping was carried out twice. (B) Illumina sequencing reads displayed for patient P1. Reads overlapping the mutation in exon 2 of <i>SLC29A3</i> (bp position g.73,082,741–g.73,082,765; hg19; NCBI 37) show the homozygous deletion of one A leading to a frameshift (c.243delA). (C) Diagram of <i>SLC29A3.</i> Introns are represented by a straight line. 5′-UTR and 3′-UTR are represented by open rectangles. The coding region is represented by closed rectangles. Previously reported mutations are indicated at the corresponding locations. The mutation identified in P1 and P2 is shown in a red rectangle. (D–E) Abnormal expression of <i>SLC29A3</i> transcript variants 1, 2 and 3 in the patients' EBV-B cells. (D) <i>SLC29A3</i> exons 2–4 were amplified from cDNA obtained from the EBV-B cells of two controls (Ctrl 1 and Ctrl 2) and two patients (P1 and P2) and were ligated to the pCR2.1 vector. Clones containing <i>SLC29A3</i> transcripts were sequenced, and the frequency of each variant was calculated by dividing the number of clones containing the particular transcript by the total number of sequenced clones. For Ctrl1: variants 1 and 2: 65/81 and variant 3: 16/81. For Ctrl2: variants 1 and 2: 37/44 and variant 3: 7/44. For P1: variants 1 and 2: 31/85 and variant 3: 54/85. For P2: variants 1 and 2: 20/76 and variant 3: 56/76. Each variant is represented by a diagram, with numbers indicating the number of the exon. The red vertical line indicates the position of the c.243delA mutation. The closed rectangle below each transcript variant represents the corresponding translation products. Gray boxes indicate amino acids modified with respect to the WT form. (E) Levels of <i>SLC29A3</i> mRNA (variants 1 and 3 combined) were assessed by Q-PCR on EBV-B cells from four healthy controls (Ctrls), two parents (Het.), and the two patients. Threshold cycles (Ct) for <i>SLC29A3</i>, normalized with respect to those of GUS (ΔCt), are plotted as 2^−(ΔCt). Each dot represents the mean of three independent experiments for each individual. The horizontal bars indicate the mean for all individuals sharing the same genotype.</p

Clinical phenotype of the patients and patients already reported in the literature with SLC29A3 mutations.

<p>Clinical phenotype of the patients and patients already reported in the literature with SLC29A3 mutations.</p

Expression and characteristics of hENT3-variant3-81fs.

<p>(A) Scheme of hENT3-variant1-WT based on the protein structure predicted by SVMtm or TMpred programs. Transmembrane domains are represented by long rectangles. The gray and red boxes highlight the amino acids that are not identical in hENT3-variant1-WT and hENT3-variant3-81fs. In the gray box: amino acids 81–100 that are replaced by 20 other amino acids in hENT3-variant3-81fs. In the red box: amino acids 101–128 that are deleted in hENT3-variant3-81fs. (B) Levels of hENT3 proteins were assessed by immunoblotting with an anti-V5-tag antibody. The V5 tag was located at the C-terminus of hENT3. (C) The graph shows the relative <i>SLC29A3</i> mRNA levels measured by Q-PCR for the same experiment with the V5-tagged constructs. Threshold cycles (Ct) for SLC29A3, normalized with respect to those of GUS (ΔCt), are plotted as 2^−(ΔCt). Immunoblotting and Q-PCR results representative of three independent experiments are shown. (D) Adenosine transport activity. Uptake of [³H]adenosine (0.026 µM) in <i>Xenopus</i> oocytes 48 h after the injection of Δ36hENT3-variant1-WT or other variants with or without the 81fs mutation as well as one H (349fs) and one PHID syndrome (314fs) mutant cRNAs. pH-dependent uptake at pH 5.5. The data shown are the means ± SEM from three independent experiments. ^*, <i>p</i><0.05, ^***, <i>p</i><0.001.</p

Whole-exome analysis results of P1.

<p>Coding variants include missense, nonsense, frameshift, in-frame deletions and insertions and readthrough variants.</p><p>Splice variants include all variants within 8 bp in the intron side, or 3 bp in the exon side of a splice junction.</p>a<p>: Both homozygous and heterozygous variations are included.</p>b<p>: Position coordinates for the markers correspond to the hg19, NCBI build 37.</p

Histology of nasal (A, B, F) and skin (C, D, E) biopsy specimens from P1 (A–E) and P2 (F).

<p>Most biopsy specimens contained vacuolated histiocytes suggestive of Mikulicz cells (A, E). Some specimens also contained large S100 protein-positive histiocytes enclosing lymphocytes (emperipolesis) (B, C, D, F). H&E staining (A, C, E) and immunohistochemical staining for S100 protein (B, D, F). Original magnification ×200 (A) and ×400 (B–F).</p