Mutations in SLC29A3, encoding an equilibrative nucleoside transporter ENT3, cause a familial histiocytosis syndrome (Faisalabad histiocytosis) and familial Rosai-Dorfman disease

Abstract

The histiocytoses are a heterogeneous group of disorders characterised by an excessive number of histiocytes. In most cases the pathophysiology is unclear and treatment is nonspecific. Faisalabad histiocytosis (FHC) (MIM 602782) has been classed as an autosomal recessively inherited form of histiocytosis with similarities to Rosai-Dorfman disease (RDD) (also known as sinus histiocytosis with massive lymphadenopathy (SHML)). To elucidate the molecular basis of FHC, we performed autozygosity mapping studies in a large consanguineous family and identified a novel locus at chromosome 10q22.1. Mutation analysis of candidate genes within the target interval identified biallelic germline mutations in SLC29A3 in the FHC kindred and in two families reported to have familial RDD. Analysis of SLC29A3 expression during mouse embryogenesis revealed widespread expression by e14.5 with prominent expression in the central nervous system, eye, inner ear, and epithelial tissues including the gastrointestinal tract. SLC29A3 encodes an intracellular equilibrative nucleoside transporter (hENT3) with affinity for adenosine. Recently germline mutations in SLC29A3 were also described in two rare autosomal recessive disorders with overlapping phenotypes: (a) H syndrome (MIM 612391) that is characterised by cutaneous hyperpigmentation and hypertrichosis, hepatomegaly, heart anomalies, hearing loss, and hypogonadism; and (b) PHID (pigmented hypertrichosis with insulin-dependent diabetes mellitus) syndrome. Our findings suggest that a variety of clinical diagnoses (H and PHID syndromes, FHC, and familial RDD) can be included in a new diagnostic category of SLC29A3 spectrum disorder.

Figure 1. Schematic showing the minimal candidate interval on chromosome 10q22.1, positions of candidate genes taken from the Ensembl genome browser (Build 49), genomic organization of SLC29A3, and positions of mutations found in the 3 histiocytosis syndrome families.

Figure 2. Loss of expression of mutant allele due to IVS2+1 G>A splice-site mutation identified in family 1.

PCR and sequencing analysis from the genomic DNA of a mutation carrier from family 1 shows heterozygous state of SNP rs2277257 (G/A). Subsequent RT–PCR and sequencing analysis of SLC29A3 transcript shows lack of the ‘G’ allele and therefore loss of mRNA expression.

Figure 3. Colony formation assays of SLC29A3.

HEK293 cells were transfected with empty vector, SLC29A3, SLC29A3 ^F103X and SLC29A3 ^G437R. Each experiment was done in triplicate. The mean number of colonies counted in the empty vector plates was taken as 100%. Values are mean ± SEM from 3 controls and 3 samples. * P = 0.005 between wildtype SLC29A3 vs. empty vector; ** P = 0.0058 and *** P = 0.0078 between SLC29A3 ^F103X and SLC29A3 ^G437R mutants resp. vs. wildtype SLC29A3.

Figure 4. Knockdown of SLC29A3 expression by siRNA enhanced proliferation of HeLa cells.

(A) Effect of SLC29A3 siRNA on levels of SLC29A3 mRNA assessed by real-time quantitative PCR (qRT–PCR). HeLa cells were treated with control or SLC29A3- 120032 or SLC29A3-26642 siRNA for 72 hours. Cells were harvested and total RNA was extracted. SLC29A3 and β-actin or SLC29A3 and β-2-microglobulin mRNA (was examined by qRT-PCR. Values are mean ± SEM from 3 controls and 3 samples. * P<0.001 between HeLa cells treated with SLC29A3-siRNA versus luciferase siRNA control sequence. (B) SLC29A3 knockdown elevates proliferation in HeLa cells. HeLa cells were transfected with siRNA designed to target SLC29A3 (siRNA-SLC29A3) or control siRNA. Cell proliferation assays were performed 72 hours after siRNA transfection. Results areexpressed in fluorescence at 550 nm using 580 nm as a reference wavelength(fluorescence is directly proportional to the number of living cells). This figure represents 3 experiments.* P<0.05.

Figure 5. Expression of SLC29A3 mRNA in the mouse embryo.

Sagittal sections from e14.5 embryos were processed for in situ hybridisation followed by imaging of representative organs (hybridization signal corresponds to blue and red/pink is a general counterstain). While there is a level ubiquitous expression throughout the embryo, several areas within the central and peripheral nervous system showed increased expression levels particularly in the dorsal spinal cord (meninges and skin to the top) (A), dorsal anterior forebrain (B), dorsal posterior midbrain (C), dorsal root ganglia (D), trigeminal ganglion (E), dorsal root ganglia (E), eye and anterior lens surface (F), ear (G), choroid plexus (H), and olfactory epithelium (I). Within trunk organs localised expression is seen in the developing lung bronchioles (J), glomeruli of the kidney cortex (L), early pancreatic primordial (M), gonads (N), thymus (O), internal mucosa of the gut (P) and stomach (Q), and lower level is the liver (K). Increased SLC29A3 expression is also seen in the outer epidermal layer of the developing posterior ventral trunk skin (R). At e14.5 trunk hair follicles are not well-developed compared, however, snout vibrissae (S,T) show elevated expression levels (a glancing more longitudinal section of an individual vibrassa can be seen in T). Scale bar in T is 100 um and applies to all images.