A Novel Gene, A New Syndrome from Hacettepe University to The Medical Literature
18.03.2022 / Publications

A new syndrome in the world has been identified with the signature of Pediatric Nephrology Unit of the Hacettepe University as a consequence of more than 5 years of an intense study.

With leadership of Professor Fatih Ozaltin, M.D., Faculty Member of Pediatric Nephrology Unit, Hacettepe University and Head of the Nephrogenetics Laboratory,  a new syndrome was identified with national and international collaboration after comprehensive genetic studies done in patients seen for the first time in Pediatric Nephrology Unit of İstanbul University-Cerrahpasa. Affected individuals shared facial anomalies, abnormal dentition, deep and shrill voice, brain abnormalities demonstrated in magnetic resonance imaging studies and atypical hemolytic uremic syndrome progressing to end stage kidney disease.  Affected individuals were performed whole exome sequencing which uncovered intronic variation in the gene TSEN2 encoding transfer RNA splicing endonuclease-2. Segregation of this variation with the disease was demonstrated in the families. After identification of these 2 index families, 2 affected individuals with similar phenotype from 2 consanguineous families were identified in Hacettepe University Pediatric Nephrology Unit; reaching totally 6 affected individuals from 4 families from the Southeastern Anatolia. Although the families stated that they did now each other, we searched relatedness between 2 index families using exome data and found that these 2 index families were related even if they were not aware of this given relatedness analysis and haplotype analysis in which a 4.2 Mb region including TSEN2 was shared. In this way, we documented that identified variation in TSEN2 is a founder variation at that geographical region (Figure 1).  

Figure 1. A) Relatedness analysis of the families




Figure 1. B) Haplotype analysis

 

Functional and bioinformatic analyses predicted that the variation might impair tRNA biology.

The human genome contains approximately 500 tRNA genes, which are transcribed as pre-tRNAs that undergo complex modification before becoming mature tRNAs, and 32 of these contain a short intron located within the anticodon that is cleaved out by the tRNA splicing endonuclease complex (TSEN complex). TSEN2 encodes one of the two catalytic subunits of this complex. Mutant TSEN2 gives rise to defective TSEN catalytic subunit thereby defective TSEN complex. With this hypothesis, when we searched how the variant identified in the patients affects TSEN2 structure, we demonstrated 3 different transcripts through cDNA sequencing and mini-gene experiments: 1) Normal transcript, 2) A transcript in which exon 9 is spliced to exon 11 that results in the skipping of exon 10, which is highly conserved in the evolution and 3) A splicing variant in which exon 9 is spliced to the newly created alternative splice site that gives rise to a TSEN2 transcript with an additional in-frame codon (CAG) between exon 9 and exon 10 that results in the inclusion of two extra amino acids (Figure 2A).


Figure 2. A) cDNA sequence electrogram

 

Figure 2. B) Schematic representation of minigene experiments deneyinin şematik gösterimi

 

While normal transcript was needed for the survival of the patients, anormal transcripts were considered to be responsible for the syndromic features described in the patients.  To confirm this hypothesis, bulk RNA sequencing was performed and was compared with age matched healthy individuals. With this approach, while up- and down-regulated pathways were described, 5 abnormal tRNA transcripts resulted from abnormal TSEN complex activity were also identified at the same time, confirming the fact that mutation itself impair TSEN activity and thereby tRNA modification required for becoming mature and functional tRNAs. Pathway analyses showed up-and down-regulated pathways including spliceosome, ribosome, synaptic vesicle cycle, glutamergic synapses, Hippo signaling pathways as most significantly enriched terms with high-fold value and lowest p-values (Figure 3). As these pathways have been shown previously to be relevant in development of brain, face, teeth as well as homeostasis of vascular endothelial cells, we concluded that the phenotypic features in our patients were related to dysregulation of these pathways. 

To further confirm that defined mutation was responsible for the phenotype, we generated a zebrafish model which showed equivalent features with the human phenotype (Figure 4).  Taken together, our findings clearly proved that identified intronic mutation indeed caused the syndromic features in the patients by disrupting tRNA biology in the human body.




Figure 3. A) Down-regulated pathways                                                  B) Up-regulated pathways

 



Figure 4. Zebrafish model. Normal zebrafish (left column), Zebrafish without exon 10 of TSEN2 (right column)

 

Finally, we proposed to name this new syndrome as TRACK syndrome (TSEN2 Related Atypical hemolytic uremic syndrome, Craniofacial malformations, Kidney failure) and registered in the medical literature. While advanced studies to enlighten the pathogenesis are still under way, it is expected that our findings will provide better understanding regarding tRNA biology.

This study has been published in March 2022 issue of Clinical Genetics journal, one of the prestigious journals of Genetics Society (Clin Genet 2022; 101 (3): 346-358).