Novel gene PUS 3 c . A 212 G mutation in Ukrainian family with intellectual disability

Aim. To evaluate a possible role of a novel c.A212G substitution in the PUS3 gene at intellectual disability (ID). Methods. The observed group consisted of the ID Ukrainian family members (parents and two affected children) and the control group – of 300 healthy individuals from general population of Ukraine. Sanger sequencing of the PUS3 gene exon 1 was performed for the family members. Polymorphic variants of c.A212G were analyzed using ARMS PCR. The homology models of wild type and p.Y71C mutant catalytic domains of human Pus3 were generated using the crystal structure of the human Pus1 catalytic domain (PDB ID: 4NZ6) as a template. Results. It was shown that the father of the affected siblings was the c.A212G substitution heterozygous carrier whereas the mother was a wild type allele homozygote, and the exom sequencing result was confi rmed – the affected children are 212G homozygotes. We supposed de novo mutation in the maternal germ line. A low frequency of 212G allele (0.0017) was shown in the population of Ukraine. Homology modelling of the wild type and p.Y71C mutant catalytic domain of human Pus3 revealed that substitution p.Y71C is located in close proximity to its active site. Conclusions. The absence of hypoproteinemia in our patients, homozygous for the 212C allele allows us to assume that the mutation c.A212G PUS3 is rather neutral and cannot be the major cause of ID. However, considering a low frequency of the 212G allele in the population and close localization of p.Y71C substitution to the active site of hPus3 we cannot exclude that the c.A212G mutation in PUS3 may be a modifi er for some pathologies including syndromic ID.


Introduction
In the frame of CHERISH project (no.223692) devoted to the genetic basis of intellectual disability, the next generation exome sequencing was conducted for two affected children (proband and his younger sibling) from the Ukrainian family (UKR 094) with healthy non-consanguineous parents.The proband (UKR 263) is a 12-year-old boy with non-syndromic ID (IQ 43), hypermobility of joints, hyperactivity and mild dysmorphic features.His brother (UKR 264) is a 4-year-old boy with non-syndromic ID, epilepsy, hypermobility of joints and mild dysmorphic features.The biochemical analysis of blood revealed no deviations from the normal ranges for the main groups of blood plasma proteins, aminoacids and acylcarnitines examined by TANDEM MS in both probands.The previous extensive genetic investigations (karyotype analysis and array-CGH analysis (400K resolution) have not found out any abnormalities as well.
The exome analysis revealed several variants in either homozygous or compound heterozygous state in few genes: EPHA1 [1], PUS3 and ZNF527.
Among these candidates, we decided to concentrate on the PUS3 gene, where novel homozygous SNP c.A212G (NM_031307:c.A212G:p.Y71C) was detected in both ID-patients.The PUS3 gene is located at chromosome 11q24.2and conserved from Es cherichia coli to human [2].
The human pseudouridine synthase 3 (hPus3) is a member of the tRNA pseudouridine synthase truA family and involved in the formation of pseudouridine (Ψ) at position 39 in the anticodon stem and loop (ASL) of many transfer RNAs [3,4].Ψ is found in almost all tRNAs, Ψ's at positions 38-40 in the Anticodon Stem Loop of tRNAs play an important role in maintaining translational effi ciency and accuracy [5].Pseudouridine appears to be necessary for the correct codon-anticodon interactions [6,7] and to prevent mischarging of the tRNA [8].It was shown that the mouse Pus3 (mPus3) can also serve as a nuclear receptors (NR) coactivator (as well as mPus1 known to form pseudouridine at positions 27, 28, 34, and 36 in tRNAs), except that it does not enhance the sex steroid receptor activity [4].A mutation in the PUS1 gene (another truA family member) has been linked to the mitochondrial myopathy and sideroblastic anemia [4,[9][10][11].
To evaluate a possible involvement of the PUS3 gene c.A212G mutation in intellectual disability, as the fi rst step, we analyzed this mutation in the healthy parents of the affected children and in general population of Ukraine and modeled 3D structure of the hPus3 catalytic domain to defi ne if the change in p.Y71C position infl uences the 3D structure of human Pus3 protein.

Materials and Methods
The observed group consisted of 300 adult (25-30 years-old) individuals including 164 (54.6 %) males and 136 (45.3 %) females.This group is representative for the estimation of DNA polymorphism frequency in autosomal genes [12,13].
The DNA-samples were extracted from peripheral blood leucocytes of unrelated volunteer donors from different regions of Ukraine by the standard phe nolchloroform method.Informed consents were obtained from all the individuals participating in our study.
The polymorphic variants c.A212G found through exome sequencing in the affected children (from UKR 094 family) were analyzed in the DNA samples of their parents by Sanger sequencing.This analysis was performed by the standard dideoxynucleotide chain-termination method using [ 35 S]-dATP or [ 35 S]-dCTP (ICN), the Sequenase version 2.0 DNA sequencing kit (USB), «ABI Prism Big Dye Terminator Cycle Sequincing Ready Reaction Kits» and ABI Prism 3110 Genetic Analyser (Applied Biosystems Ana lysis of the c.A212G substitution was performed by ARMS (amplifi cation refractory mutation system).The primers were designed using the web-based PRIMER 3.0 program (http://workbench.sdsc.edu)and the «BLAST» (http://www.ncbi.nlm.nih.gov/blast) (Table 1).
Amplifi cation of the allele 212A is accomplished using a complementary primer «wild type» paired with a «forward» primer (Table 1).On the other hand, the 212G allele will be amplifi ed if the 3' residue of primer is complementary to the «mutant» se- The PCR product fl anked by «forward» and «reverse» primers (320 bp) will be amplifi ed in all reactions (Fig. 1).Thereby three different patterns could be observed for the c.A212G variant after the amplifi cation: 320 bp and 193 bands (for A/A); 320 bp, 193 bp and 172 bp bands (for A/G); 320 bp and 172 bp bands (for G/ G) (Fig. 3).
The PCR amplifi cation was performed in one tube in a fi nal volume of 25 l containing 1 PCR buffer, 1.5 mM MgCl 2 , 200 M of each dNTP, 1 M of each primer, 0.2 units of Taq-DNA polymerase and 200 ng of the DNA template.The cycling conditions were as follows: initial denaturation at 94 C for 5 min, 30 cycles consisting of denaturation at 94 C for 30s, annealing at 64 C for 30s, extension at 72 C for 30s, and a fi nal elon- In silico modeling.The homology models of wild type and mutant (p.Y71C) catalytic domains of human Pus3 were generated by Swiss Model server (http://swissmodel.expasy.org)using as a template a crystal structure of the human (mitochondrial) Pus1 catalytic domain (PDB ID: 4NZ6).

Results and Discussion
The Sanger sequencing of the gene PUS3 exon 1 revealed that probands UKR 263 and UKR 264 are homozygotes for the c.A212G substitution that confi rms the exome sequencing results.In turn, the father (UKR 266) of the affected siblings is a heterozygous carrier for the c.A212G substitution and, surprisingly, the mother (UKR 265) is a wild type allele homozygote (Fig. 2).
The possible explanation of such results is the de novo mutation in the maternal germ line.
Of the 300 analyzed samples, we found one hete rozygous carrier for the c.A212G mutation only.Thus, the genotypes distribution was as follows: A/A -99.7 %, A/G -0.3 % and GG -0 %.The observed Pseudouridylate synthase 3 is a 481 amino acid (aa) protein that belongs to the highly conserved tRNA pseudouridine synthase truA family and mostly is localized to cytoplasm and nucleus [3,4,14].The members of this family have been shown to modify multi ple positions in cytoplasmic and mitochondrial tRNAs [11,15], as well as non-tRNA substrates: U2 snRNA [16] and SRA [17].The TruA family includes Pus1 from E. coli (also called truA or hisT) which modifi es positions 38, 39, and 40 in the ASL of bacterial tRNAs [18].Other members are Pus3 from mouse [1]; Pus3 from yeast (also known as Deg1) -modifi es positions 38 and 39 [14] and human mitochondrial Pus1 modifi es positions 27, 28, 34, and 36 in tRNAs in vitro and in vivo [19,20].The atomic models for various members of the PUS families (TruA, TruB, TruD, RluA, RsuA, and Pus10) have been solved and have shown a conserved catalytic core that presents a high degree of structural similarity and is the most stable region of the protein.[21][22][23][24][25][26][27][28].
The c.A212G mutation results in substitution of aromatic Tyrosine 71 to sulfur-containing Cysteine located in the catalytic domain of human Pus3.The analyses of site orthologs, using the NCBI Homo lo-Gene database, revealed that the Tyr71 amino acid position is conserved from Escherichia coli to human, indicating that there may be an evolutionarily conserved function (Fig. 3).
To defi ne if the change in p.Y71C position infl uences the 3D structure of human Pus3 protein, we modeled the hPus3 catalytic domain 3D structure using the crystal structures of the human (mitochondrial) Pus1 catalytic domain as a template (Fig. 4, C and D).The mitochondrial hPus1, as well as hPus3, belongs to the tRNA pseudouridine synthase TruA family and is mostly localized in the mitochondria [16,17,29].
From the crystal structure of the bacterial TruA (Fig. 4, A) it is known that the active site of the TruA family members is populated by four strictly conserved amino acids including a catalytic aspartate (D60 in TruA), two arginines (R58 and R205), and a tyrosine (Y118) [22].These residues correspond to D146, R144, R295, Y201 in hPus1 (Fig. 4, B) and D118, R116, R280 and Y195 in hPus3 (see Fig. 4, C and 4, D) [22,27,28,30].Tyrosine 71 of hPus3, corresponding to Y18 in bacterial TruA and Y92 -in hPus1, is another strictly conserved amino acid (Fig. 3).As can be seen from Fig. 4, C and 4, D, the substitution of highly conserved aromatic Tyrosine 71 to sulfur-containing Cysteine is located in close proximity to the active site of hPus3 and may directly cause This is true considering the fact that Sibert et al., interpreting the site-directed mutagenesis experiments with hPus1, indicate that Y173 (corresponds to Y195 in hPus3), and R267 (corresponds to R280 in hPus3) known to compose the active site of TruA of the enzyme near a critical aspartate (position 118), do not play any essential role in the catalysis [30].They changed Tyr201 and Arg295 to several other amino acids and found that many variants had signifi cant activity [30].
However, Ψ's at positions 38-40 in the Anticodon Stem Loop of tRNAs plays an important role in maintaining translational effi ciency and accuracy.These modifi cations of uridines were shown to increase the thermal stability of the ASL, which could affect the anticodon-codon interaction or conformational changes of the tRNA during translation and spliceosome assembly [5,32].Thus, the PUS3 mutations, that result in loss of pseudouridine in a wide range of tRNAs, may affect the protein synthesis.
The strong relationship between the reduced growth rate of E. coli or S. typhimurium and the absence of pseudouridines 38-40 in anticodon stemloop of several tRNAs was reported more than two decades ago [33].The HisT mutant E. coli strains display a 20-25 % reduction in the rate of polypeptide chain elongation and exhibit pleiotropic abnormalities in the cell division processes, resulting in an increase in doubling time of more than 30 % [34].When the PUS3 gene was disrupted in yeast, it was not lethal, but the growth rate of the yeast considerably reduced, especially at 37 C [14].Since there is an effect on the growth of prokaryotes and yeast when the pseudouridine 38-40 synthase activity is deleted, what might be the effect of the PUS3 gene missense mutations in humans?
The physiological importance of the appropriate pseudouridine synthase activity is illustrated by the disorders such as DKC (dyskeratosis congenital) and MLASA (mitochondrial myopathy and sideroblastic anemia) [9][10][11]35].A missense mutation in the human PUS1 gene affecting a highly conserved amino acid (Arg144-to-Trp mutation in the active site of the enzyme and a mutation of Glu220, which leads to C-terminally truncated protein) has been associated with mitochondrial myopathy and sideroblastic anemia, a rare autosomal recessive disorder of oxidative phosphorylation and iron metabolism [9][10][11].The X-linked form of the only other known human disease of pseudouridylation dyskeratosis congenita is caused by the mutations in the gene encoding dyskerin [35].
The previously mentioned PUS1 mutations result in the loss of pseudouridine in some tRNAs that may affect protein synthesis [9][10][11].Furthermore, the mammalian pseudouridine synthase 1 (Pus1) was reported to modulate the class I and class II nuclear receptor responses through its ability to modify the Steroid receptor RNA Activator (SRA) [4,17,28] and it was suggested that other abnormalities in these MLASA patients, such as facial dysmorphisms, may be due to -------SKRKIVLLMAYSGKGYHGMQRNV the loss of this activity of Pus1 [10].Recently it has been shown that Pus3 (as well as Pus1) acts as a regulator of the nuclear receptors activity [4].Therefore, it cannot be excluded that some symptoms observed in the affected children from Ukrainian family (UKR 094), such as facial dysmorphisms, may be the consequence of defective hSRA-NR signaling.
However, in some known human pseudouridylation diseases, the loss of pseudouridine that results in a decrease of the protein synthesis effi ciency, has a pleiotropic effect which causes syndromic pathologies.The similar pleiotropic effect may take place in case of the c.A212G mutation in the PUS3 gene indentifi ed in this study.Nevertheless, we did not observe syndromic intellectual disability in the affected children from Ukrainian family (UKR 094).Furthermore, in our patients we did not detect any evi-dence of hypoproteinemia, which is a common indicator for both MLASA and DKC and may be an evidence of the defi ciency in protein synthesis.
Thus, we assume that the c.A212G PUS3 mutation is rather neutral and cannot be the major cause of intellectual disability.However, considering a low frequency of the 212G allele (0.0017) in the population of Ukraine and the location of p.Y71C substitution in close proximity to the active site of hPus3 protein we cannot exclude that the c.A212G mutation in the PUS3 gene may still be considered as a modifying factor for some pathologies including syndromic intellectual disability.
Further studies will be carried out to evaluate the infl uence of the c.A212G PUS3 gene mutation on the pseudouridylate synthase effi ciency and its involvement in the ID development.

Fig. 3 .
Fig. 3. Pseudouridylate synthase 3 (481 aa) conservation analysis.Conserved amino acid positions are shown in grey boxes.Protein alignment showed conservation of residue Tyr71 (shown in black boxes) from Escherichia coli to human.Tyrosine 71 of hPus3 corresponds to Y92 in hPus1 (shown in black boxes)

Fig. 4 .D
Fig. 4. Overall views of the crystal structure of bacterial TruA (PDB ID: 2NQP) (A), hPus1 (PDB ID: 4NZ6) (B) and homology models of wild type (C) and p.Y71C mutant (D) hPus3 catalytic domain.Crystal structure of pseudoudirinde synthase TruA monomer in complex with leucyl tRNA (A) -uracil 39 shown in ball and stick model and colored in black, the catalytic amino acid residues D146, R144, R295 and Y201 are shown as sticks and colored in green, Y92 shown in stick model and colored in blue.The hPus1 monomer (B) -catalytic amino acid residues D60, R58, R205 and Y118 are shown as sticks and colored in green, Y18 shown in stick model and colored in blue.Homology models of wild-type (C) and p.Y71C mutant (D) catalytic domain of human Pus3 generated by Swiss Model server.The images were created by ViewerLite v.4.2 with Y71 mutation sites shown in blue, residues D118, R116, R280 and Y195 are shown in green[28,31]