Mutations in mitochondrial histidyl tRNA synthetase HARS2 cause ovarian dysgenesis and sensorineural hearing loss of Perrault syndrome

Abstract

Perrault syndrome is a genetically heterogeneous recessive disorder characterized by ovarian dysgenesis and sensorineural hearing loss. In a nonconsanguineous family with five affected siblings, linkage analysis and genomic sequencing revealed the genetic basis of Perrault syndrome to be compound heterozygosity for mutations in the mitochondrial histidyl tRNA synthetase HARS2 at two highly conserved amino acids, L200V and V368L. The nucleotide substitution creating HARS2 p.L200V also created an alternate splice leading to deletion of 12 codons from the HARS2 message. Affected family members thus carried three mutant HARS2 transcripts. Aminoacylation activity of HARS2 p.V368L and HARS2 p.L200V was reduced and the deletion mutant was not stably expressed in mammalian mitochondria. In yeast, lethality of deletion of the single essential histydyl tRNA synthetase HTS1 was fully rescued by wild-type HTS1 and by HTS1 p.L198V (orthologous to HARS2 p.L200V), partially rescued by HTS1 p.V381L (orthologous to HARS2 p.V368L), and not rescued by the deletion mutant. In Caenorhabditis elegans, reduced expression by RNAi of the single essential histydyl tRNA synthetase hars-1 severely compromised fertility. Together, these data suggest that Perrault syndrome in this family was caused by reduction of HARS2 activity. These results implicate aberrations of mitochondrial translation in mammalian gonadal dysgenesis. More generally, the relationship between HARS2 and Perrault syndrome illustrates how causality may be demonstrated for extremely rare inherited mutations in essential, highly conserved genes.

Fig. 1.

Identification of mutations in HARS2 in a family with Perrault syndrome. (A) A nonconsanguineous family of French, Irish, and Scottish ancestry, with Perrault syndrome in 5 of 11 siblings. Affected siblings are compound heterozygotes for mutations in HARS2. (B) Progressive hearing loss in the affected individuals, measured by pure tone audiometry as described by Pallister and Opitz (11). (C) Paternal allele chr5:140,075,395C > G, corresponding to HARS2 c.598C > G, and maternal allele chr5:140,076,926, corresponding to HARS2 c.1102G > T. (D) Sequence from cDNA from lymphoblasts of II-1, indicating that HARS2 c.598C > G yields two transcripts, encoding HARS2 p.L200V and HARS2 p.Δ200–211. (E) The paternal allele HARS2 c.598C > G encodes HARS2 p.Δ200–211 and HARS2 p.L200V. The proportion of HARS2 c.598C > G transcripts encoding HARS2 p.Δ200–211 is significantly lower for the unaffected father I-1 than for his affected children II-1, II-4, II-7, and II-8 (P = 0.009). Transcripts were derived from lymphoblast cDNA. P value for the comparison is based on the Z score for significance of a difference between independent proportions.

Fig. 2.

Effects of the mutations on the HARS2 protein. (A) Schematic of the HARS2 protein. (B) Protein sequence alignment of HARS2 orthologs. Mutated amino acids are in red and indicated by arrows; other amino acids critical to the protein structures of Fig. 3 are in boldface type and marked with asterisks. (C) Analysis by Western blot (WB) of cytoplasmic (C) and mitochondrial (M) extracts of 293T cells transfected with HARS and HARS2 constructs demonstrates that HARS localizes to cytoplasm and HARS2 localizes to mitochondria. HARS2 p.Δ200–211 is poorly expressed relative to HARS2 wild type, HARS2 p.L200V, and HARS2 p.V368L. β-Actin and VDAC are cytoplasmic and mitochondrial controls, respectively, for fractionation and loading. (D) Immunoprecipitates (IP) from mitochondrial extracts of 293T cells transfected with pairs of constructs. Coimmunoprecipitation between Myc- and FLAG-tagged HARS2 p.V368L (lane 3) is stronger than between Myc- and FLAG-tagged HARS2 or HARS2 p.L200V (lanes 1 and 2), indicating that HARS2 p.V368L homodimerizes more efficiently than does HARS2 or HARS2 p.L200V. Coimmunoprecipitation between HARS2 p.V368L and either HARS2 or HARS2 p.L200V (lanes 6 and 8) is stronger than between HARS2 and HARS2 p.L200V (lane 5), indicating that dimerization is increased even when only one monomer in the dimer harbors the p.V368L mutation. HARS2 p.Δ200–211 was expressed at very low levels; no dimerization with this mutant protein was detected (lanes 4, 7, 9, and 10). (E) Measurement of the enzymatic activity, using the pyrophosphate exchange assay, of purified wild-type HARS2 (wt), HARS2 p.L200V, and HARS2 p.V368L indicates that activities of both mutant proteins are reduced relative to wild-type protein, with HARS2 p.V368L more severely affected. Results illustrate a representative experiment, performed in triplicate; error bars indicate SD.

Fig. 3.

Structures of homologs of HARS2 at the mutant sites. Ribbon diagrams are shown of histidyl tRNA synthetases from (A) T. thermophilus (PDB 1ADY) and (B) E. coli (PDB 1KMM), each in complex with histidyl-adenylate monophosphate (HAM), which is represented by light gray sticks with element coloring in red (oxygen), blue (nitrogen), and orange (phosphorus). Substitution at either L200 or V368 could result in movement of conserved HAM contact residues. Numbers listed in A and B indicate analogous residues in human/prokaryote proteins. (A) Region surrounding residue L151, analogous to HARS2 p.L200. L200/L151 (magenta) contacts L203/L154 and I390/V312 (pink). The helix containing I390/V312 and the conserved R389/R311 residue is shown in dark gray, with R389/R311, which contacts HAM, shown in dark gray sticks with element coloring as above. For clarity, residues 151–160 have been removed from the view. (B) Region surrounding residue V292, analogous to HARS2 p.V368. V368/V292 (magenta) contacts A137/V89 and A141/I93 (pink) on adjacent turns of the α-helix containing the invariant T133/T85 (dark gray sticks with element coloring), which contacts the HAM. For clarity, residues 293–304 have been removed from the view.

Fig. 4.

Effects on growth in yeast of mutations in HTS1 analogous to mutations in HARS2. HTS1-L198V, corresponding to HARS2 p.L200V, complements loss of HTS1 more effectively than does HTS1-V381L, corresponding to HARS2 p.V368L. HTS1-Δ198–205, corresponding to HARS2 p.Δ200–211, fails to complement loss of HTS1. (A) hts1Δ yeast with a rescuing wild-type copy of HTS1 on a URA3 plasmid were transformed with TRP1 plasmids encoding HTS1 of various genotypes. The resulting strains were streaked on Trp⁻ media containing 5-fluoroorotic acid, to select against the rescuing wild-type HTS1. Growth indicates complementation by wild-type or mutant HTS1 on the TRP plasmid. (B) Growth in liquid culture of the strains from A.

Fig. 5.

RNAi of the C. elegans histidyl tRNA synthetase gene hars-1 reduces fertility. hars-1 RNAi decreased the brood size of wild-type animals more dramatically than it did that of ced-4(n1162) animals, which have a defect in apoptosis. Eggs were collected from wild-type N2 or ced-4(n1162) adults on plates seeded with control or hars-1 RNAi bacteria and allowed to develop to the L4 stage. F₂ progeny of 14 F₁ animals exposed to control RNAi and 48 F₁ animals exposed to hars-1 RNAi were counted. A representative experiment is shown. Error bars indicate SD.