MRI of the index patient at the age of 54 years.

<p>A. Sagital T1 weighted MRI of the brain showing atrophy of the cerebellar midline, particularly the vermis superior. B&C. Axial T2 weighted MRI showing linear T2 hypointensities in the pons. C. Axial T2 weighted MRI showing prolongation of T2 signal in the dentate nuclei. D. Sagital T2 weighted MRI showing atrophy of the cord, straight dorsal spine and loss of the dorsal kyphosis.</p

Compound heterozygous mutations found in the proband.

<p>Upper part, IGV-browser screenshots of the mutations found by exome sequencing and corresponding Sanger sequencing results. Lower part. Protein multiple sequence alignments (PMSA) of the corresponding residues generated by MUSCLE v3.6 (NCBI HomoloGene) including genes conserved in bony vertebrates (Euteleostomi). Residues in red are predicted to be affected by the mutations found in the proband.</p

Variant filtration of exome sequencing data from the index case compared with whole genome genotyping data in all three affected siblings.

<p>Only one gene, <i>SACS</i>, harbors variants consistent with autosomal recessive inheritance and shared by all three siblings.</p>*<p>166 variants were not listed in dbSNP.</p

Genetic and molecular findings.

<p>A: coverage analysis of whole-genome sequencing data in patient 3 shows a clear regional drop in sequencing depth (red line) corresponding to the 45,049 bp deletion in the 5’-UTR of the <i>SLC19A3</i> gene. The two arrows mark the position of the break-points. Coverage in the same region of a control is shown in the bottom lane for comparison. The blue diagram depicts the SLC19A3 gene in a 3’-5’ alignment. Vertical rectangulars show the proportional position of the exons. B: Western blot analysis in frontal cortex homogenate of patient 1 using two primary antibodies (left and right panel) shows an increase of SLC19A3 protein immunoreactivity in the patient (P) compared to two age and gender matched controls (C1, C2).</p

Neuroimaging findings.

<p>A: axial T2 weighted MRI of patient 2 taken during an episode with seizures, increasing encephalopathy and exacerbation of the dystonia. Images show bilateral high T2-signal changes and swelling in the putamen and caudate head. In addition there are multiple cortical lesions preferentially occurring in the depths of sulci, which is typical of BBGD. B: axial T2 and coronal T2-FLAIR weighted MRI of patient 1 taken during an episodic exacerbation show a similar pattern with bilateral striatal and multiple cortical lesions. The signal abnormalities regressed completely on later scans (not shown). C (upper lane): FDG-PET scan of the brain of patient 1 taken ~5 years later shows multiple foci of decreased glucose metabolism that correlate to the localization of the transient cortical signal changes on MRI. The striatum shows minimal uptake consistent with severe neuronal loss. A normal scan is shown in the lower lane for comparison.</p

Pedigrees with homozygous c.1528_1529del [p.Met510Valfs*17] <i>GBA2</i> mutation

<p>A) Kindred 66 was originally reported by Skre and Berg [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169309#pone.0169309.ref013" target="_blank">13</a>]. Arrow: probands; Squares: males; circles: females; diagonal line: deceased individual; Black symbols: affected individuals; white symbols: unaffected individuals; w/w: homozygous for the wild-type allele; w/m: heterozygous for the <i>GBA2</i> c.1528_1529del mutation; m/m: homozygous for the <i>GBA2</i> c.1528_1529del mutation. B) Sequencing of the <i>GBA2</i> transcript. Sequencing of the GBA2 transcript using RNA purified from cultured patient fibroblasts showed homozygosity for the 2 bp deletion (c.1528_1529del).</p

Clinical features of patients harbouring the homozygous c.1528_1529del [p.Met510Valfs*17] GBA2 mutation.

<p>Clinical features of patients harbouring the homozygous c.1528_1529del [p.Met510Valfs*17] GBA2 mutation.</p

The <i>TRIT1</i> mutation disrupts modification activity on cytosolic and mitochondrial tRNAs but not enzyme abundance.

<p><b>A</b>) No decrease in the levels of the native TRIT1 protein in patient fibroblasts was observed by immunoblotting (using β-actin as a loading control) <b>B</b>) The isopentenyl modification status of both mitochondrial (mt-) and cytosolic (cy-) tRNAs in patient fibroblasts (lane P) compared to controls (lane C); by this approach a positive signal is due to lack of isopentenyl modification as detected by an anticodon loop (ACL) probe (the bulky modification on the N of adenine blocks base pairing with the probe, such that no signal for cy-tRNA^Ser(UGA) with the ACL probe indicates efficient modification in the control cells <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004424#pgen.1004424-Lamichhane2" target="_blank">[11]</a>); a body probe to a different region of the same tRNA is used as a control for calibration and calculation of steady-state levels. Each panel shows hybridisation of the same blot with a different probe as indicated to the right. The cytosolic tRNA^Ser(UGA) is poorly modified in patient fibroblasts (strong ACL probe signal), but tRNA^Ser(UGA) steady-state levels are unchanged. Mt-tRNA^Ser(UCN) is also poorly modified in patient fibroblasts, although a small pool of mt-tRNA^Ser(UCN) in control fibroblasts lacks the modification. The modification appears to be influential on mt-tRNA^Ser(UCN) stability, as steady-state levels are decreased by 40% in the patient. The non-substrate mt-tRNA^Cys was probed as a control.</p

A <i>TRIT1</i> mutation segregates with disease and disrupts a conserved tRNA-binding basic side-chain.

<p><b>A</b>) Targeted resequencing of <i>TRIT1</i> confirmed that the proband (II-3; arrow), and his clinically affected sister (II–1) are homozygous for the c.968G>A <i>TRIT1</i> mutation, while his unaffected older brother (II–2) and both of his parents (I–1 and I–2) are heterozygous carriers. <b>B</b>) The <i>TRIT1</i> mutation is located in exon 8, whilst there is a putative mitochondrial targeting sequence in exon 1 and a matrin-type zinc finger domain spanning exons 10 and 11. <b>C</b>) The mitochondrial sub-localisation of TRIT1 is demonstrated by sub-fractionation and immunoblotting, using markers for each sub-fraction to confirm there was no contamination: TOMM20 (mitochondrial outer membrane), AIF (mitochondrial intermembrane space), GDH (mitochondrial matrix), NDUFA9 (mitochondrial inner membrane) and eIF4E (cytosol). TRIT1 localized with eIF4E in the cytosol (lane 2) and showed the same profile as GDH (lanes 3–6), but was undetectable in the inner mitochondrial membrane fraction (lane 7). A total of 40 µg protein was loaded for each sample, and all mitochondrial subfractions were prepared from the same mitochondrial lysate. <b>D</b>) Clustal Omega alignment of the TRIT1 protein and known orthologs revealed that the affected amino acid (p.Arg323) is conserved in each species excluding <i>S. pombe, S. cerevisiae</i> and <i>E. coli</i>, where the equivalent amino acid is lysine, which has similar electrochemical properties (:). Asterisks (*) indicate completely conserved residues. <b>E–G</b>) The co-crystal structure of Mod5 bound to a substrate tRNA (based on <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004424#pgen.1004424-Zhou1" target="_blank">[14]</a>) shows the interaction of the tRNA backbone (nucleotides 27–29) with an extended α-helix in which are located multiple basic side chains (indicated in red) of the enzyme including that corresponding to the mutated position (Lys294).</p

Identification of a mitochondrial respiratory chain deficiency and defective mtDNA translation.

<p><b>A</b>) Cytochrome <i>c</i> oxidase (COX) histochemical reactivity revealed a mosaic of COX-deficiency in patient skeletal muscle compared to control. <b>B</b>) The assessment of individual respiratory chain enzyme activities identified a combined OXPHOS deficiency affecting complexes I and IV in skeletal muscle from the proband. The mean activity measured in 25 controls was set at 100%. <b>C</b>) Patient fibroblasts (grey) are less capable of responding to stress in comparison to control fibroblasts (black), as measured by the spare respiratory capacity. *: P<0.05. <b>D</b>) The coupling efficiency of ATP synthesis and respiration, and therefore the level of proton leak, is not decreased in patient fibroblasts (grey) compared to controls (black). The error bars displayed on each graph indicate standard deviation. <b>E</b>) <i>In vitro</i> metabolic labelling of mitochondrial translation in patient fibroblasts (P) showed a generalised decrease in translation activity, with the subunits of Complex I (notably ND5) and Complex IV (notably COXI) most substantially decreased. Even loading was confirmed by Coomassie blue staining (CBS). <b>F</b>) Immunoblotting demonstrated a generalised decrease in the level of individual subunits from respiratory chain complexes I–V (normalised to β-actin) in patient fibroblasts, whilst the mitochondrial marker, TOMM20 was unchanged.</p