The expanding spectrum of PRPS1-associated phenotypes: three novel mutations segregating with X-linked hearing loss and mild peripheral neuropathy

Abstract

Next-generation sequencing is currently the technology of choice for gene/mutation discovery in genetically-heterogeneous disorders, such as inherited sensorineural hearing loss (HL). Whole-exome sequencing of a single Italian proband affected by non-syndromic HL identified a novel missense variant within the PRPS1 gene (NM_002764.3:c.337G>T (p.A113S)) segregating with post-lingual, bilateral, progressive deafness in the proband's family. Defects in this gene, encoding the phosphoribosyl pyrophosphate synthetase 1 (PRS-I) enzyme, determine either X-linked syndromic conditions associated with hearing impairment (eg, Arts syndrome and Charcot-Marie-Tooth neuropathy type X-5) or non-syndromic HL (DFNX1). A subsequent screening of the entire PRPS1 gene in 16 unrelated probands from X-linked deaf families led to the discovery of two additional missense variants (c.343A>G (p.M115V) and c.925G>T (p.V309F)) segregating with hearing impairment, and associated with mildly-symptomatic peripheral neuropathy. All three variants result in a marked reduction (>60%) of the PRS-I activity in the patients' erythrocytes, with c.343A>G (p.M115V) and c.925G>T (p.V309F) affecting more severely the enzyme function. Our data significantly expand the current spectrum of pathogenic variants in PRPS1, confirming that they are associated with a continuum disease spectrum, thus stressing the importance of functional studies and detailed clinical investigations for genotype-phenotype correlation.

Figures

Figure 1

Identification of the novel PRPS1 variant c.337G>T (p.A113S) segregating with NSHL by whole-exome sequencing. (a) Pedigree of Family 1 and audiograms of both affected males and one carrier female (average HL for the right and left ears is shown). This family, which showed a clear recessive inheritance pattern of the disease, was selected for targeted capture and exome sequencing (the analyzed subject is pointed by an arrow). The genotype of each individual is indicated below the corresponding symbols. na: not analyzed. (b) IGV (Integrative Genome Viewer) screenshot showing sequencing reads that support the mutant allele (the T nucleotide is reported) identified in the II2 proband. The identified G>T transversion maps within PRPS1 exon 3 and results in the A113S amino-acid change. The mean coverage of the region here shown is 31.

Figure 2

Identification of novel missense variants (c.343A>G (p.M115V) and c.925G>T (p.V309F)) in PRPS1 in two additional families with X-linked deafness. Pedigree of Families 2 (a) and 3 (b) and the corresponding audiograms, showing the average HL for the right and left ears. The genotype of each individual is indicated below the corresponding symbol. Audiological and neurological phenotypes are indicated separately using different shades of gray. All female carriers had significantly milder phenotypes compared with affected males of the same family. The probands are pointed by an arrow. na: not analyzed.

Figure 3

Functional characterization of the identified PRPS1 variants. (a) PRS-I activity in erythrocytes was evaluated by measuring the accumulation of AMP by HPLC in five affected males, four female carriers, as well as five healthy control individuals (wt). The diagram shows the correlation between ribose-5-phosphate-dependent AMP generation (y axis) and the reaction incubation time (x axis). Error bars represent the standard error of the mean (SEM) for genotypes represented by >1 individual (wt, n=5; M115V/-, n=2; M115V/wt, n=3; A113S/-, n=2), whereas they indicate the standard deviation (SD) for genotypes represented by a single individual. (b) Summary of the functional data obtained from PRPP synthetase activity assay. The enzyme activity is expressed as nmoles of AMP per milligram of Hb per hour. SD was calculated from three independent experiments performed in triplicate. The percentage was calculated as the ratio between the individual PRS-I activity and the mean of the PRS-I activity of the unaffected male and wt controls (range of PRS-I activity in controls: 15.43–26.44 nmol/mg/h).

Figure 4

Structural analysis of the newly identified PRS-I missense variants. On the right: Molecular surface of the three-dimensional structure of the PRS-I enzyme in its physiologic assembly, with the six different subunits (a to f) indicated by separated colors. The two monomers forming a dimer are highlighted with different shades of the same color. The alpha-helices hosting A113S and M115V substitutions are shown as ribbons. Residue Val309 is shown by spheres. The allosteric sites of subunit a (dark green) are highlighted with a darker surface. The mutated residues discussed in the panels are from subunit a. The predicted ddG of the hexamer structure is 9.88, 15.57, and 26.53 kcal/mol in the presence of A113S, M115V, and V309F, respectively. (A, B) Structural analysis of the c.337G>T (p.A113S) variant. Left: Ala113 is completely buried in the hydrophobic core of the corresponding subunit, interacting with Phe92 (belonging to the flexible loop), Phe137, and Phe138. Right: Ser113 differs from Alanine in that one of the methylenic hydrogens is replaced by a hydroxyl group, through which this amino acid makes a new hydrogen bond. The larger and polar (hydrophilic) side chain of Ser113 makes this region to rearrange (highlight in yellow). (C, D) Structural analysis of the c.343A>G (p.M115V) variant. Left: Met115 is completely buried at the trimer interface, making hydrophobic interactions with the surrounding amino acids. Two Methionine 115 side chains from two different PRS-I dimers participate in a hydrophobic interaction at the interface. Right: Val115 is a beta-branched residue, introducing a completely different steric hindrance, making this region to rearrange (highlight in yellow). (E, F) Structural analysis of the c.925G>T (p.V309F) variant. Left: Val309 is part of the allosteric site I and is located at the trimer interface near the center of the hexamer. Right: Phe309 is bulkier than Val and surrounding residues are predicted to move to avoid Van der Waals crashes. Residues of allosteric site I are indicated with spheres. A sulphate ion present in the crystal structure is also shown that occupies the position of the b-phosphate of ADP; indeed an activator phosphate and an inhibitor ADP compete for binding at the allosteric regulatory site. In all panels, the mutated residue is shown by sticks: C atoms are colored magenta, O red, S yellow, and H white. Residues predicted by FoldX to be perturbed are colored in yellow.